J. Phycol. 48, 956–965 (2012) Ó 2012 Phycological Society of America DOI: 10.1111/j.1529-8817.2012.01173.x FATTY ACID SIGNATURES DIFFERENTIATE MARINE MACROPHYTES AT ORDINAL AND FAMILY RANKS1 Aaron W. E. Galloway2 Friday Harbor Laboratories, School of Aquatic and Fishery Sciences, University of Washington, 620 University Rd., Friday Harbor, WA, 98250, USA Kevin H. Britton-Simmons, David O. Duggins Friday Harbor Laboratories, University of Washington, 620 University Rd., Friday Harbor, WA, 98250, USA Paul W. Gabrielson University of North Carolina Herbarium, CB# 3280, Coker Hall, Chapel Hill, NC, 27599-3280, USA and Michael T. Brett Civil and Environmental Engineering, University of Washington, Box 352700, Seattle, WA, 98195-2700, USA Primary productivity by plants and algae is the Abbreviations: ALA, a-linolenic acid (18:3x3); fundamental source of energy in virtually all food ARA, arachidonic acid (20:4x6); DHA, docosa- webs. Furthermore, photosynthetic organisms are hexaenoic acid (22:6x3); EFA, essential fatty the sole source for x-3 and x-6 essential fatty acids acids; EPA, eicosapentaenoic acid (20:5x3); FA, (EFA) to upper trophic levels. Because animals can- fatty acids; FAME, fatty acid methyl esters; GC, not synthesize EFA, these molecules may be useful gas chromatograph; GCMS, gas chromatography- as trophic markers for tracking sources of primary mass spectrometry; LIN, linoleic acid (18:2x6); production through food webs if different primary MSI, multiple stable isotopes; PCA, principal producer groups have different EFA signatures. We components analysis; PERMANOVA, permuta- tested the hypothesis that different marine macro- tional multivariate analysis of variance; PERM- phyte groups have distinct fatty acid (FA) signatures DISP, permutational test of multivariate by conducting a phylogenetic survey of 40 marine dispersions; SDA, stearidonic acid (18:4x3); SI, macrophytes (seaweeds and seagrasses) representing stable isotope; SJA, San Juan Archipelago 36 families, 21 orders, and four phyla in the San Juan Archipelago, WA, USA. We used multivariate statistics to show that FA composition differed sig- nificantly (P < 0.001) among phyla, orders, and fam- Photosynthetic organisms are the source of virtu- ilies using 44 FA and a subset of seven EFA ally all energy in food webs. Upper trophic level (P < 0.001). A second analysis of published EFA consumers are constrained by this production (e.g., data of 123 additional macrophytes confirmed that Power 1992), but for many systems the relative this pattern was robust on a global scale (P < 0.001). importance of different sources of production to This phylogenetic differentiation of macrophyte consumer communities is debated and poorly taxa shows a clear relationship between macrophyte resolved (e.g., see Pace et al. 2004, Brett et al. phylogeny and FA content and strongly suggests that 2009). In nearshore marine and aquatic environ- FA signature analyses can offer a viable approach to ments, sources of primary production may be clarifying fundamental questions about the contribu- autochthonous [e.g., macrophytes (macroalgae and tion of different basal resources to food webs. seagrasses), single-celled phytoplankton (diatoms, Moreover, these results imply that taxa with com- dinoflagellates)], or allochthonous (e.g., terrestrial mercially valuable EFA signatures will likely share plants). Assessing the relative importance of these such characteristics with other closely related taxa distinct basal resources in marine food webs has that have not yet been evaluated for FA content. been a complex problem because direct observation or gut content analysis is not possible for many pri- Key index words: algal systematics; essential fatty mary consumers. The use of stable isotopes (SI; acids; fatty acids; food web biomarkers; marine Duggins et al. 1989, Kaehler et al. 2000, Page et al. algae; marine macrophytes; primary production; 2008) and fatty acids (FA; Budge et al. 2008, seagrasses Richoux and Froneman 2008, Copeman et al. 2009) as biomarkers in this regard has shown promise but 1Received 22 November 2011. Accepted 15 March 2012. these approaches assume that all important food 2Author for correspondence: email [email protected]. sources are identified, adequately characterized, and 956 MARINEMACROPHYTEFATTYACIDS 957 represented in mixing models. Moreover, FA and SI each other (Stengel et al. 2011), which are known can only be used when all relevant primary produc- to exhibit an astounding array of FA (Harwood and tion sources have distinct signatures, which is often Guschina 2009), even among closely related taxa. not the case with SI. Herein, we investigated the Lang et al. (2011) recently demonstrated a signifi- phylogenetic differentiation of FA content in the cant phylogenetic signal in the FA composition of four major macrophyte phyla found worldwide in cultured microalgae, and several studies have nearshore marine habitats, to evaluate the potential reported on the FA content of marine macrophytes of FA signature analysis for clarifying fundamental in different parts of the world (Khotimchenko 1998, questions about energy sources in food webs. Graeve et al. 2002, Khotimchenko et al. 2002, FA are necessary constituents of the tissues of all Hanson et al. 2010, Kumari et al. 2010). However, living organisms. Because FA have distinct chemical whether or not marine macrophytes segregate taxo- structures, it is possible to routinely identify up to nomically with respect to their FA composition has 70 FA within a given organism (Iverson 2009). The not been demonstrated explicitly for a diverse identities and quantities of FA in a given sample assemblage of taxa. constitute the FA ‘signature’. Of particular interest We conducted a broad survey of the FA content are the essential FA (EFA), generally defined as x-3 of 40 NE Pacific marine macrophyte taxa represent- and x-6 FA families, which animals are unable to ing 36 families, in 21 orders, across four phyla synthesize (Bell and Tocher 2009), and as such are (Table 1; seagrasses, Anthophyta; brown algae, Och- potentially conservative molecular biomarkers. In rophyta; green algae, Chlorophyta; red algae, Rho- addition, EFA are especially useful in a food web dophyta) in the San Juan Archipelago (SJA), NE context because they are important for physiological Pacific, to evaluate the taxonomic resolution of mac- processes (Sargent et al. 1999, Muller-Navarra rophytes as basal resources in a food web context. 2008), including survival, growth, and reproduction We compared this analysis with an evaluation of in a wide range of aquatic species (Brett and Muller- published macrophyte FA data (1994–2010) from Navarra 1997), but are only synthesized in biologi- an additional 123 independently collected taxa from cally relevant amounts by plants and algae (Glady- 36 families, in 21 orders (Table S1 in supplementary shev et al. 2009). Previous research on the role of material) across the same four phyla in all major algae as a supply of EFA has focused on the contri- oceans of the world. Specifically, we asked: (i) Does bution of EFA by phytoplankton to aquatic food macrophyte FA composition differ among phyloge- webs (Kainz et al. 2004, Ravet et al. 2010). netic categories of phylum, order, or family level The role of nearshore macrophytes as a subsidy using 44 FA in the SJA dataset? (ii) Is the same taxo- (e.g., Polis et al. 1997) source of EFA for marine nomic resolution achieved with the SJA dataset food webs is unknown. This is particularly surprising using only a subset of seven EFA? (iii) Are locally due to the high productivity (Mann 1973, Duarte observed patterns consistent with published global and Cebrian 1996) and known importance of ben- macrophyte EFA data? thic algae for nearshore invertebrate assemblages (e.g., Dunton and Schell 1987). As little as 10% of this production is believed to be directly consumed METHODS by herbivores as standing stock (Mann 1988). The NE Pacific macrophytes. vast majority of this energy is exported as a spatial Selection of taxa: Our goal was to compare FA signatures subsidy to subtidal (Duggins et al. 1989), intertidal from the four major marine macrophyte phyla present world- (Rodriguez 2003), pelagic (Kaehler et al. 2006), and wide in nearshore waters. We selected species to maximize taxonomic diversity by creating a list of 80 macrophyte species terrestrial (Polis and Hurd 1995) food webs. The that we expected to find during a May sampling period (see patterns of FA composition of marine macrophytes below) in the SJA. We removed species from the list that were may be used to further explain the role of macro- difficult to identify due to lack of reliable morphological ⁄ ana- phytes as a source of EFA to higher trophic levels tomical traits (Gabrielson et al. 2006). The list was filtered to and to increase the resolution of marine food web maximize the number of orders. Once an order was repre- models if these patterns are conserved in consum- sented by one taxon, we selected multiple taxa within that ers. The transfer of EFA synthesized by plants and order: (i) if each species was from a different family, (ii) if the different species could be found in different habitats (e.g., algae to food webs should be of fundamental inter- intertidal vs. subtidal) and (iii) in the case of Laminariales est to resource managers, as these primary produc- (kelps), we included eight species because of their biomass ers are the ultimate source of x-3 and x-6 EFA in dominance in the drift (Britton-Simmons et al. 2009) in the higher trophic level consumers, which are harvested SJA. Recent molecular work has shown that the green algal for human consumption. species commonly referred to as Ulva lactuca in the NE Pacific Northeast (NE) Pacific nearshore marine macro- and other temperate marine waters in the northern and southern hemisphere has been misidentified (O’Kelly et al. phyte communities contain a very diverse mix of 2010). We therefore extracted DNA from two specimens we species (>640 taxa) representing four phyla (Gabri- had identified as U.