Trophic spectrum of the yellowtail surgeonfish Prionurus punctatus on the rocky reef of Los Frailes, Baja California Sur,

Mexico ABSTRACT The trophic spectrum of the yellowtail surgeonfish Prionurus punctatus was determined from the stomach contents of 120 individuals, caught monthly from November 2004 to October 2005 in the Los Frailes reef, B.C.S., Mexico. Of all organisms caught, 86 (72%) had food in their stomachs, and 34 (28%) were empty. The frequency of occurrence and gravimetric methods were used in the combined index of relative importance (IRI) in order to characterize the general trophic spectrum by sex, by size and by season. Unlike other species of this family, P. punctatus feeds mainly on macroalgae. According to the IRI, the diet was composed of Gracilaria spp. (15.24%), Hypnea musciformis (8.58%), Jania mexicana (5.79%), Sphacelaria spp. (4%), and Dictyota flabellata (3.7%). Non-identified organic matter (NIOM) had the highest value (45.92%), suggesting an extended period of digestion. According to the diet similarity index, there were no differences by sex (R = 0.01, p = 33.41), by season (R = 0.01, p = 32.1), or size (R = 0.14, p = 3.1). The Levins index showed a low diet width (Bi = 0.13). We conclude that the yellowtail surgeonfish is a specialist herbivore that feeds in schools made up of different sizes and sexes. INTRODUCTION In the eastern tropical Pacific (ETP), the most frequent herbivorous fishes on reefs are the members of the Acanthuridae family, commonly known as surgeonfish. There are 12 known species of acanthurids, grouped in four genera, mostly from the Indo-Pacific (Robertson et al., 2004). Of these, two species from the Prionurus genus are considered

native to Mexico: the razor surgeonfish P. laticlavius and the yellowtail surgeonfish P. punctatus. Both species are distributed in the southern Gulf of California (Robertson & Allen, 2002). The yellowtail surgeonfish forms resident schools with diurnal habits at 0 to 30 m depths, comprising dozens to several hundred individuals, and is characterized by not having a stomach (agastric). It is considered one of the most abundant algae consumers (Montgomery et al., 1980a; Thomson et al., 2000). There currently exits an important fishing pressure on the yellowtail surgeonfish, because its meat is of excellent quality and has good market acceptance. It is also used as an ornamental fish, due to its great beauty (Pia, 1998). However, this species has not been well studied, and information on its basic biology is scarce; particularly on its trophic biology, on which there are only studies by Montgomery et al.(1980a) and Ortiz-Aguirre (2010), who report that this species is a generalist that feeds on a great variety of macroalgal species (Dictyota spp., Padina spp., Sargassum spp. and Liagora spp.). The present study analyzed the trophic spectrum of the yellowtail surgeonfish, in order to evaluate possible variations of frequency and biomass of algae ingested during an annual cycle in the Los Frailes reef, which is part of the Cabo Pulmo Marine Park, BCS, Mexico. MATERIALS AND METHODS Monthly sampling trips were carried out from November 2004 to October 2005 in the rocky reef of Los Frailes, on the southern end of the Baja California Peninsula (between 235N and 109 5W). The reef measures 0.070 km2; different sized rock accumulations can be observed, made of pebbles 0.3 m to over 3m in length, with sand patches and few coral

Comment [L1]: supongo que quiere decir que son nativas de Mexico?

formations. Water in the area is clear and precipitation is scarce; there is a rainy season from July to September (Moreno-Snchez, 2009). Organisms were captured while scuba diving using a pole spear between 10:00 am and 16:00 p.m., when lighting is best. At each sampling location water temperature was recorded. For each organism total length (TL), weight (W) and sex were recorded, and the digestive tract was removed. The digestive tract contents were kept in previously marked plastic bags and frozen for later analysis at the Fish Ecology Laboratory of the Interdisciplinary Centre for Marine Sciences of the Instituto Politcnico Nacional (CICIMAR-IPN). For the stomach contents analysis the different items (food components) were separated according to taxonomic group, and identified to the lowest taxonomic level depending on the digestion state. For taxonomic algae identification the keys of Dawson (1961, 1962, 1963, 1966a and 1966b), Abbott & Hollenberg (1976) and Dawes (1986) were used. Once the taxonomic work was completed, stomach contents were analyzed quantitatively, for which the following were used: The percent frequency of occurrence (%FO), which is the frequency of occurrence of items expressed as a percentage of the total number of stomachs with food.  No. of stomachs including a item No. of stomachs with food

%FO

The gravimetric percent composition (%W), which is the wet weight of each item expressed as a percentage of the total wet weight of stomachs containing food:

%W

Weight of item Total weight of items

It is worth noting that due to the nature of the feeding habits of this species (algae grazers) it was not possible to employ a numeric method to quantify units. In order to evaluate in an integral manner the importance of each food type in the species diet, we used the Index of Relative Importance (IRI) of Pinkas et al. (1971), modified by Ojeda & Muoz 1999: IRI = % FO %W We calculated the diet width (Bi) with the standardized Levins index (Hurlbert 1978), using the absolute values obtained with the index of relative importance (IRI). The Levins index takes values from 0 to 1. When Bi values are under 0.6, a predator is considered a specialist, which indicates that it uses a low number of resources and presents a preference for certain prey. When values are close to one (0.6) the spectrum is of a generalist, meaning that the species uses all resources without selection. This index was calculated using the following formula: Where Bi = niche breadth, number of items. We determined the total sexual proportion; the statistical significance was tested with a Chi-squared analysis, setting as null hypothesis a sexual proportion of one female per male (H0 = 1:1). The observed value was compared to the theoretical chi-square value with a 95% confidence interval (Zar, 1996). = Proportion of the jth item in predator is diet, n = total

According to Trujillo-Milln (2009), the size at first maturity of yellowtail surgeonfish P. punctatus is 14 cm total length (TL); therefore samples obtained in the present study corresponded to adult organisms with a size range between 18 and 43 cm TL. We therefore grouped all organisms in three categories for size analysis: small size (18 to 27 cm), medium size (28 to 37 cm) and large size (38 to 47 cm). To determine whether there were differences in diet between genders, sizes or seasons, similarities were analyzed using permutation-randomization methods in the similarity matrix (ANOSIM, PRIMER 6 v. 6.1.1). The global rank similarity R (0 R 1) is a useful comparative measure of the degree of separation. When R is close to zero, Ho is true, i.e., there is no separation between groups (Clark & Warwick 2001). Finally, we determined the weight-length (W-L) relationship in yellowtail surgeonfish using the allometric equation W = aL b, where W is the fish weight (g), L is total length (cm), and a and b are the two model parameters. Analyses were done using the program STATISTICA 7. RESULTS A total of 120 yellowtail surgeonfish P. punctatus were caught, of which 86 (72%) had stomachs with food and 34 (28%) were empty. A total of 22 algae species (14 Rhodophyta, 6 Phaeophyta, 2 Chlorophyta) were identified, as well as non-identified organic matter (NIOM) and a great quantity of sand, which was not quantified as it is not a component of the fishs diet (Table 1; Fig. 1). Food components had a total biomass of 4118.71 g; red algae (Rhodophyta) represented 43.67%, brown algae (Phaeophyta) 18.71%, green algae (Chlorophyta) 0.77%, and NOIM 36.87%. According to the gravimetric method, the most

important algae were Gracilaria spp. (13%), Hypnea musciformis (8.49%), Dictyota flabellata (4.89%), Jania mexicana (4.82%) and Sphacelaria spp. (4%). NIOM had the highest biomass value at 36.87% (Table 1; Fig. 1). According to the frequency of occurrence, the most important algae were: Jania mexicana (95.35%), Gracilaria spp. (93.02%), Hypnea musciformis (80.23%), Sphacelaria spp. (77.91%) and Dictyota flabellata (62.79%). It is worth noting that NIOM had the highest value and occurred in almost all stomachs (98.84%) (Table 1; Fig. 1). Incorporating the percent values of the previous methods into the index of relative importance (IRI), and taking only values that comprised over 80%, it was determined that the most important food components of the P. punctatus diet were Gracilaria spp. (15.24%), Hypnea musciformis (8.58%), Jania mexicana (5.79%), Sphacelaria spp. (4%), and Dictyota flabellata (3.7%). NIOM had the highest IRI value with 45.92% (Table 1; Fig. 1). For females, 47 stomachs contained food, of which the most important components were NIOM (43.34%), Gracilaria (15.03%), Hypnea musciformis (10.39%), Jania mexicana (5.97%), and Dictyota flabellata (3.90%). These components made up over 80% of the index of relative importance (IRI) (Fig. 2). For males, 39 stomachs had food; the most important components were NIOM (50.31%), Gracilaria (15.51%), Hypnea musciformis (5.70%), Jania mexicana (5.19%) and Sphacelaria spp. (4.75%) (Fig. 2). The female to male proportion was significantly different from 1:1; there were 1F:0.8M (x2= p>0.05). For small sizes, there were 17 stomachs with food; the most important components of the diet were NIOM (49.90%), Gracilaria (19.33%), Hypnea musciformis (7.35%), Jania

mexicana (4.08%), Sphacelaria spp. (4. 51%) and Dictyota flabellata (3.71%) (Fig. 3). For medium sizes there were 59 stomachs with food; 80% of the diet was made up by NIOM MONI (46.85%), Gracilaria (14.14%), Hypnea musciformis (8.45%), Jania mexicana (5.70%), Sphacelaria spp. (3.78%) and Dictyota flabellata (3.73%) (Fig. 3). For large individuals, 10 stomachs contained food. In this category, the most important components were NIOM (35.49%), Gracilaria (18.74%), Hypnea musciformis (10%), Jania mexicana (6.98%), Sphacelaria spp. (5.05%) and Dictyota flabellata (4.17%) (Fig. 3). For the seasonal trophic analysis, sampled months were divided into warm and cold seasons, based on in situ temperature records, according to Lavn and Marinone (2003). The months of November-December 2004 and February-April 2005 were considered cold months (22-25 C), and the months of June to October 2005 were considered warm months (26-31 C). For the cold season, 47 stomachs with food were analyzed. During this period P. punctatus fed on NIOM (46.68%), Gracilaria (17.39%), Hypnea muscirormis (7.85%), Jania mexicana (6.86%), Dictyota crenulata (3.5%), Dictyota flabellata (3.15%) and Sphacelaria spp. (2.58%) (Fig. 4). For the warm period, there were 39 stomachs with food; NIOM (44.43%), Gracilaria (13.28%), Hypnea musciformis (9%), Jania mexicana (4.89%), Dictyota crenulata (1.30%), Dictyota flabellata (4.06%) and Sphacelaria spp. (5.44%) were the most important trophic components (Fig. 4). When the Levins index was calculated, a low diet width (Bi = 0.13) was observed in the general trophic spectrum, as well as when analyzing by sex, size or season (Table 2). Due to P. punctatus preference for consuming a reduced number of algae species (Gracilaria

spp., Hypnea musciformis, Jania mexicana, Sphacelaria spp. and Dictyota flabellata), this species can then be characterized as an herbivore with a specialist trophic behaviour. The analysis of similarities of diet composition by gender (ANOSIM, PRIMER 6 v. 6.1.6) showed no differences by sex (R = 0.01, p = 33.41), size (R = 0.14, p = 31), or season (R = 0.01, p = 32.1). The maximum weight recorded was 1564 g and the minimum was 136 g. The maximum length was 43 cm and the minimum was 18 cm. The weight-length (W-L) relationship was estimated for 86 organisms. A value of r2 = 0.955 was obtained, the a value was 0.040 and the standard error 0.040. The b value was 2.803 with a standard error of 0.101. The results obtained indicate that P. punctatus is a species with allometric growth. DISCUSSION Stomach content analysis of fish is important to understand the different trophic interactions in the marine ecosystem (Jackson et al., 2000). Within the trophic interactions in the environment, herbivory is one of the most influential, because this interaction constitutes the base of trophic chains, and therefore conditions the flow of matter and energy through communities (Crawley, 1983; Horn, 1989). Despite this, there are few studies worldwide that focus on the study of food habits of herbivorous species (Horn, 1992), and there are even fewer studies of herbivorous fishes in the Gulf of California. According to the results obtained, it was confirmed that the yellowtail surgeonfish P. punctatus is a strictly herbivorous fish, which feeds on 22 algal species grouped in three divisions: Phaeophyta, Chlorophyta and Rhodophyta. These correspond to 34.4% of the floral diversity reported for the Cabo Pulmo Los Frailes area. The yellowtail surgeonfish

fed on a third of the algae present in the area. According to Anaya-Reyna & RiosmenaRodrguez (1996), Mateo-Cid et al. (2000) and Riosmena-Rodrguez et al. (2005), in this reef there are 61 algae species during the year. The yellowfin surgeonfish diet was made up mainly of red algae (Gracilaria spp., Hypnea musciformis, and Jania mexicana). This could be due to the fact that these red algae have floridean starch, sugars, glycosides and sucrose as storage compounds (Bold & Wynne 1978), which can probably be hydrolyzed by the enzymes present in teleosts. Additionally to extracellular components, these algae are made up of a great variety of sugars, some of which have alpha bonds, and can be hydrolyzed. Regarding this, Montgomery and Gerking (1980) point out that this combination of characteristics can mean that red algae carbohydrates are more susceptible to digestion than green and brown algae carbohydrates, and could eventually be used directly as a source of energy (Cceres & Ojeda, 2000). It is mentioned in the literature that there is a negative effect of brown algae consumption by herbivorous fish due to the presence of secondary metabolites such as pachydictyol-A, dictyol-B, dictyol-B acetate, dictyol-H, dictyopterernes A and B (Ragan & Jenzen, 1977; Steinberg, 1989; Sale, 1991). These compounds have been demonstrated to be a dissuasive to the action of herbivorous fish (Paul & Hay, 1986; Targett et al., 1986). The leaves of some of these algae are very tough, particularly in Laminaria (e.g. Lessonia), and act as mechanic inhibitors to the trophic action of small-sized herbivores (Lobel & Ogden, 1981; Lobel, 1981). Contrary to what has been reported, in the yellowtail surgeonfish P. punctatus diet we found a low biomass but high frequency of occurrence of brown algae such as Sphacelaria

spp., Dictyota flabellata, D. crenulata and Padina concrescens during the entire year of sampling (all these species have at least one secondary metabolite). These results also contrast with what was found by Montgomery et al. (1980a), who analyzed stomach contents of seven yellowtail surgeonfish individuals in Punta Colorada, Baja California Sur, Mexico, and mentioned that these fish avoided including brown algae in their diet. The brown algae consumption in the present study could be due to the large size of analyzed specimens. It is considered that large-sized fish could endure chemical deterrents, and also need to consume large quantities of food to complement their diet. The agastric characteristic and size of yellowtail surgeonfish could provide this fish with a greater tolerance to secondary metabolite toxins (dicterpenes A and B, terpenes pachydictyol-A, dictyol-B, dictyol-B acetate, dictyol-H, dictyopterernes A and B) of brown algae than other gastric grazers of smaller size (Sale 1991; Prez-Espaa, 1994), since the proportion of toxins to biomass in the yellowtail surgeonfish is lower than that of its smaller-sized competitors. It is worth mentioning that in all analyzed digestive tracts a great quantity of sand was found, which in total weighed 1,553 g, and could help with algae digestion, as has been determined for other herbivorous species (Lobel, 1981). P. punctatus does not have a differentiated stomach or an end sac with endosymbionts that could help break cellular walls. The quantity of non-identified organic matter (NIOM) was very high in all stomachs, from which we can infer that the digestive processes of the yellowtail surgeonfish are carried out constantly, since NIOM is food in a highly digested state.

The trophic spectrum similarity analysis by sex (males and females), size (small, medium and large), and season (warm and cold), showed no significant differences for yellowtail surgeonfish. One possible explanation is that the yellowtail surgeonfish forms schools of dozens to several hundred individuals (Montgomery et al., 1980a; Thomson et al., 2000), which are made up of both sexes and different sizes, which graze in the same locations, with no segregation in their feeding habits. This strategy allows them to feed in areas where other territorial herbivorous species (Stegastes rectifraenum, S. acapulcoensis, S. flavilatus) defend their feeding zone from other conspecifics and other herbivorous species in a vigorous manner (Moreno-Snchez, 2009). Despite the fact that the yellowtail surgeonfish diet included a large number of algal species (22), according to the Levins index it had a specialist trophic behaviour; this can be explained by their marked preference for a reduced number of particular algal species, since the diet was dominated by three items (Glacilaria spp., Hypnea musciformis and Jania mexicana). This result contrasts with Montgomery et al. (1980a), who mention that the yellowtail surgeonfish is a non-selective generalist grazer, who takes small filaments of multispecific algae. It is worth mentioning that in that study only seven organisms were analyzed, which could explain in part this difference, since in this study a larger number of specimens were analyzed during an annual cycle. Results are therefore based on more complete data, which generated a very detailed characterization of the trophic conduct of this species. We also have to take into account that a specialist species is characterized by feeding on very specific resources independently of their abundance in the feeding area (Gerking,

1994). Regarding this, Ortz-Aguirre (2010) mention that the red algae biomass made up 34% of the total, below brown algae (63%) in the rocky reef of Punta Arenas, B.C.S., and mention that the species on which P. punctatus fed (Gracilaria spp., Hypnea musciformis and Jania mexicana) do not constitute more than 50 gm2. It can therefore be assumed that this species searches for and selects algae. P. punctatus coinhabits with other herbivorous species that form dense schools, such as Acanthurus triostegus. According to Abitia-Crdenas et al. (2011), this species fed in the same area, mainly on the green alga Ulva linza, which can make up over 50% of its diet, as well as on Gelidiella spp. (15%) and Polysiphonia simplex (9%). This feeding segregation can therefore allow the co-existence of both species. This same behaviour has been reported for other surgeonfish species such as A. lineatus and Ctenochae tusstriatus, who due to different dentition, mandible shape and digestive tracts, can use different algae types in a same area (Montgomery et al., 1989). This could probably also happen between P. puntatus and A. triostegus. There are few studies that focus on the weight-length relationship of reef fishes in the Gulf of California (Balart et al., 2006; Trujillo-Milln, 2009). The allometric growth of P. punctatus does not agree with what was found by Trujillo-Milln (2009), who reports for this same species an isometric growth. That result could be due to differences in the sizes and weights recorded (mostly juveniles). We conclude that the yellowtail surgeonfish P. punctatus uses a wide variety of algae (red, brown and green), but feeds preferentially on Gracilaria spp., Hypnea musciformis and Jania mexicana, due to the nutritive and energetic value of these algae. There are no

changes in diet by season, sex or size since P. punctatus individuals feed by forming dense schools, which allows them to graze any reef area without regard to the presence of territorial herbivorous species. AKNOWLEDGMENTS The authors thank the Instituto Politcnico nacional (IPN) for support received through COFAA and EDI. XGMS thanks the National Science and Technology Council (CONACyT) for economic support through the retention program. Table 1. General diet of yellowtail surgeonfish P. punctatus in the Los Frailes reef, BCS, Mexico. Figure 1. Study area where the yellowtail surgeonfish P. punctatus specimens were caught. Figure 2. Trophic spectrum of the yellowtail surgeonfish P. punctatus. Figure 3. Diet of male and female P. punctatus. Figure 4. Trophic spectrum of P. punctatus by size interval, shown as relative importance index percentages. Figure 5. Seasonal index of relative importance of P. punctatus. Table 2. General, by size, by sex and by season diet width of P. punctatus according to the Levins index.