Red-footed and masked boobies stable isotope data

Animals that co-occur in a region (sympatry) may share the same environment (syntopy), and niche differentiation is expected among closely related species competing for resources. The masked booby (Sula dactylatra) and smaller congeneric red-footed booby (Sula sula) share breeding grounds. In addition to the inter-specific size difference, females of both species are also larger than the respective males (reversed sexual size dimorphism). Although both boobies consume similar prey, sometimes in mixed-species flocks, each species and sex may specialize in terms of their diet or foraging habitats. We examined inter- and intra-specific differences in isotopic values  (δ13C and δ15N) in these pelagically feeding booby species during the incubation period at Clarion Island, Mexico, to quantify the degrees of inter- and intra-specific niche partitioning throughout the annual cycle. During incubation, both species preyed mainly on flyingfish and squid, but masked boobies had heavier food loads than red-footed boobies. There was no overlap in isotopic niches between masked and red-footed boobies during breeding (determined from whole blood), but there was a slight overlap during the non-breeding period (determined from body feathers). Female masked boobies had a higher trophic position than conspecific males during breeding; however, no such pattern was detected in red-footed boobies. These results provide evidence of inter- and intra-specific niche partitioning in these tropical seabird species, particularly during the breeding period and in the more dimorphic species. Our results suggest that these closely related species use different strategies to cope with the same tropical marine environment.

Methods
This study was conducted at Clarion Island, Revillagigedo Archipelago, Mexico (18°21’7.53” N, 114°43’18.61” W)

Individual masked and red-footed boobies were captured at their nest by hand or using a hand net from a distance of 1–2 m.

A few drops of blood (~ 0.15 mL) were collected from the brachial vein of individual birds using a 25 G needle and non-coated capillary tubes. The blood samples were placed on glass microscope slides and air-dried. Whole blood reflects the diet assimilated during the previous 3–4 weeks (Vander Zanden et al., 2015), and whole blood samples therefore provide information on the bird’s diet during the incubation and/or pre-laying period. Body feathers were collected from the ventral part of adult birds and stored in individual paper bags. Body feathers were considered optimal given that they are easy to collect and do not impair the flight ability of the sampled birds (Jaeger et al., 2009; Bighetti et al., 2022). In contrast to whole blood, body feathers integrate information about the diet during the period when the feather was formed (from weeks to months; Grecian et al., 2015, Petalas et al., 2024), which is usually during the non-breeding period for these boobies (Grace et al., 2020; Schreiber et al., 2020). As mentioned before, no signs of molting, such as missing primary feathers, worn feathers of feather growth were observed while manipulating the individuals, supporting that molting occurs outside the breeding season.

Dried whole blood samples (0.2–0.6 mg) were scraped from the slides and placed in tin cups in the laboratory. The feathers were immersed in a 2:1 chloroform and methanol solvent to remove surface oils and associated contaminants (Hobson et al., 2014). The samples did not undergo lipid extraction, and the low C:N (all < 4) mass ratios indicated that mathematical correction for high lipid content was not required (Post et al., 2007). The isotope values of all samples were analyzed at the Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany using a Flash elemental analyzer (Thermo Fisher Scientific, Bremen, Germany) connected in sequence via a ConFlo (Thermo Fisher Scientific, Bremen, Germany) to a stable isotope ratio mass spectrometer (Delta V; Thermo Fisher Scientific, Bremen, Germany). The instrument was flushed with chemically pure helium gas for measurements. Stable isotope ratios were expressed in delta notation indicating the deviation from international standards (in air nitrogen for nitrogen and V-PDB for carbon), according to the equation: δX=[(Rsample/Rstandard) −1], where X is 13C or 15N and R is the ratio 13C/12C or 15N/14N, respectively. Secondary isotopic reference materials were tyrosine (δ13C: -23.96 ± 0.02 ‰; δ15N: 4.36 ± 0.04 ‰) and leucine (δ13C: -30.15± 0.05‰; δ15N: 10.82 ± 0.08 ‰). The analytical precision of both δ13C and δ15N was of < 0.2 ‰.