All references to figures and tables are from Yeager, M. E. and A.R. Hughes. 2025. Functional trait analysis reveals the hidden stability of multitrophic communities. Ecology. In press.

Study system
This study was conducted along the southern shore of Rhode Island across six coastal ponds: Green Hill (GH) Pond, Ninigret Pond (NP), Point Judith (PJ) Pond, Potter Pond (PP), Quonochontaug Pond (QP), and Winnapaug Pond (WP; Appendix S1: Fig. S1 of Yeager and Hughes, 2025). Fish communities in these ponds mostly consist of marine species connected to the ocean via a breachway that reassemble each year via larval dispersal from the ocean (Satchwill and Sisson 1991a, 1991b, Sisson and Satchwill 1991). The fish communities found throughout these ponds encompass a range of trophic levels (2 - 4.5) and feeding modes (Appendix S6: Table S1, Fig S1). Here, we examined 13 fish functional traits which can be categorized into three functional roles: (1) energy acquisition, (2) locomotion, and (3) nutrient recycling (Villéger et al. 2017). Fish communities are a good model system to ask these types of questions due to a strong foundation in the functional trait literature, offering a thorough understanding of the functional roles fish provide and functionally meaningful traits to measure (Dumay et al. 2004, Mason et al. 2007, Villéger et al. 2010, 2017, Albouy et al. 2011, Stuart-Smith et al. 2013, Mouillot et al. 2013, Yeager et al. 2017, McLean et al. 2019).

Fish community collections
From June to October 2018, we sampled six coastal pond fish communities monthly via 150-foot beach seine in conjunction with the Rhode Island Department of Environmental Management (RIDEM) fish and macroinvertebrate survey. This survey has been ongoing since 2010, with fish and macroinvertebrates identified to the lowest taxonomic level, enumerated and a subset measured to infer population size structure. Fish collections in 2018 for this study were collected via the same methods as the RIDEM survey. The fish in these communities span trophic level ranges from to 2.1 to 4.5, consisting of detritivores, invertivores, and piscivores. We targeted 38 species that account for 99.4% of total abundance across the survey. For each species, we aimed to collect 20 individuals evenly distributed across their size range, informed from past survey data. Upon collection, fish were either transferred into seawater containers for excretion incubations or euthanized immediately via a seawater-clove oil (Eugenol extract, Syzygium aromaticum) mixture (IACUC protocol #: 18-0622R). Once euthanized, fish were held on ice before returning to the lab to conduct morphometric analysis. We collected a total of 708 fish across 27 species and 23 families. We analyzed a subset of 200 fish for nutrient recycling traits, resulting in an average of 18.63 + 2.6 fish per species for energy acquisition and locomotion traits and an average of 7.48 + 0.91 fish per family for nutrient recycling traits.

Nutrient recycling traits
To quantify nutrient recycling traits, we conducted excretion incubations, targeting 27 fish families (N = 1-3 species per family), which account for 99.7% of total abundance across the survey. For each family, we targeted 10 individuals evenly distributed across their size range. Directly after fish were removed from the seine net, individuals were placed into separate 3-liter (L) sterile plastic bags of seawater directly taken from that site before seine collection and allowed to incubate for 30 minutes (Appendix S2: Fig. S1f). During incubations, plastic bags were placed in a large cooler to ensure minimal stress. Directly after incubations, fish were transferred to a seawater-clove oil mixture for euthanasia. Two 60-millilieter (mL) 0.7-micrometer (μm) filtered water samples were taken from each plastic bag directly before and after each incubation trial, resulting in a pre- and post-incubation water sample for both N and P concentrations. Water samples were placed on ice and frozen immediately once returning from the field and kept in a -20 degrees Celsius (°C) freezer until processing.

We analyzed all water samples for concentrations of ammonium (NH4+) and phosphate (PO43-) using two spectra-photometric assays of phenolhypochlorite (Solórzano 1969) and molybdenum blue (Murphy and Riley 1962) methods as modified by Whiles et al. (2009). To quantify the N and P contribution for each fish, we took the difference of N and P concentrations from the pre- and post-incubation water samples (Appendix S2: Fig. S1g). Lastly, we took the ratio of change of N to P for each fish to calculate the N:P functional trait.

Morphological traits
To collect traits which inform energy acquisition and locomotion functional roles, we measured 15 morphometrics. For each fish, we took a series of five photos: (i) lateral full body, (ii) lateral head, (iii) lateral head with mouth protruded, (iv) ventral full body, and (v) anterior with mouth open, all with a ruler in shot for length standardization (Appendix S2: Fig. S1). Using ImageJ analysis (Schneider et al. 2012), we measured 15 morphometrics which were used to calculate five energy acquisition traits (oral gape surface, oral gape shape, oral gape position, protrusion, eye size) and five locomotion traits (eye position, body transverse surface, body transverse shape, pectoral fin position, caudal peduncle throttling) (Appendix S2: Table S1a-e; Albouy et al. 2011). The 10 continuous functional trait measurements are commonly used in morphological studies on fishes, have been connected to diet or movement (Sibbing and Nagelkerke 2000, Dumay et al. 2004, Mason et al. 2007, Villéger et al. 2010), and they are easily measured and broad enough to apply to any fish species.