Received 10 May 2002 Accepted 8 July 2002 Published online 11 September 2002 Sandpipers (Scolopacidae) switch from monoester to diester preen waxes during courtship and incubation, but why? Jeroen Reneerkens1*, Theunis Piersma1,2 and Jaap S. Sinninghe Damsté1 1 Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands Centre for Ecological and Evolutionary Studies, Zoological Laboratory, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands 2 Recently, a shift in preen wax composition, from lower molecular weight monoesters to higher molecular weight diesters, was described for individuals of a sandpiper species (red knot, Calidris canutus) that were about to leave for the tundra breeding grounds. The timing of the shift indicated that diester waxes served as a quality signal during mate choice. Here, this hypothesis is evaluated on the basis of a survey of preen wax composition in 19 sandpiper species. All of these species showed the same shift observed in the highArctic breeding red knots. As the shift also occurred in temperate breeding species, it is not speciŽc to tundra-breeding sandpipers. Both sexes produced the diester waxes during the incubation period until hatching, in addition to the short period of courtship, indicating that diesters’ functions extend beyond that of a sexually selected ‘make-up’. The few non-incubating birds examined (males of curlew sandpipers (C. ferruginea) and ruffs (Philomachus pugnax)) had the lowest likelihood of secreting diesters, indicating a functional role for diester preen waxes during incubation. We propose that diester preen waxes enhance olfactory crypticism at the nest. Keywords: uropygial gland; sandpipers; mate choice; mating system; chemical ecology 1. INTRODUCTION A complete and abrupt shift has recently been discovered in the chemical composition of secretions from the uropygial gland (preen gland) in high-Arctic breeding red knots (Calidris canutus) (Piersma et al. 1999). Although these secretions were previously considered invariable and taxon-speciŽc ( Jacob & Ziswiler 1982), a rare class of diesters (Sinninghe Damsté et al. 2000) completely replaces the usual mixture of monoesters at the start of courtship in this species. The uropygial gland secretions are preened into the plumage (hence the name ‘preen waxes’) and several functions have been proposed ( Jacob & Ziswiler 1982). Preen waxes may delay feather wear, keep feathers exible (Stettenheim 1972) and waterproof (Elder 1954; but see Fabricius 1959; Elowson 1984) and have anti-dermatophytic characteristics ( Jacob et al. 1997). This compositional shift indicated an additional function for diester preen waxes during the period of courtship and mating (Piersma et al. 1999). Diester preen waxes are more viscous than monoester mixtures and they may be difŽcult to preen into the plumage under the prevailing cold temperatures during the summer season in the high Arctic. The change to a preen wax mixture that brings about additional costs led Piersma et al. (1999) to propose that diester preen waxes may function as a sexually selected quality signal, perhaps by enhancing the appearance or reectance of the plumage. In this study, we explore the idea that diester preen waxes function as a sexual signal. We do so in a compara- * Author for correspondence (reneer@nioz.nl) . Proc. R. Soc. Lond. B (2002) 269, 2135–2139 DOI 10.1098/rspb.2002.2132 tive way by studying the chemical composition of preen gland secretions before, during and after the reproductive period in 19 closely related sandpiper species of the Charadriiform family Scolopacidae. 2. METHODS (a) Birds Sandpipers were caught at various stages of their annual cycle and preen wax samples were collected. All investigated species are migratory and use different areas for reproduction and wintering, often thousands of kilometres apart. Except for blacktailed godwits (Limosa limosa), redshanks (Tringa totanus), Asian dowitchers (Limnodromus semipalmatus) and some of the ruffs (Philomachus pugnax) that breed in temperate regions, all investigated sandpipers reproduce on the (sub-) Arctic tundra (table 1). They typically winter in (sub-) tropical or temperate coastal salt-water habitats (Piersma 1997). To reach the Arctic breeding areas between late May and early June, sandpipers make longdistance ights of thousands of kilometres, with one or two intermediate refuelling stops in wetland habitats (summarized in Piersma et al. 1996). (b) Sex and life-cycle stage The composition of preen wax secretions was studied in relation to sex and life-cycle stage. We determined the sex of the birds by examining sex-speciŽc plumage traits, size differences and/or sex-speciŽc behaviour (e.g. incubation in some of the species). The sexually monomorphic red knots were sexed using a standard and veriŽed molecular technique (Baker et al. 1999). Most sanderlings (C. alba), semipalmated (C. pusilla), Baird’s (C. bairdii) and white-rumped sandpipers (C. fuscicollis) were not sexed individually. Sexes of red phalaropes (Phalaropus fulicarius), Hudsonian godwits (L. haemastica) and bar-tailed 2135 Ó 2002 The Royal Society species adults common name (scientiŽc name) breeding range spring migration godwits black-tailed godwit (Limosa limosa) Hudsonian godwit (Limosa haemastica) bar-tailed godwit (Limosa lapponica) temperate low Arctic high Arctic 1/10 0/3 0.5/34 shanks redshank (Tringa totanus) temperate 0/7 turnstones ruddy turnstone (Arenaria interpres) high Arctic 0/40 phalaropes red phalarope (Phalaropus fulicarius) low and high Arctic dowitchers Asian dowitcher (Limnodromus semipalmatus) short-billed dowitcher (Limnodromus griseus) temperate low Arctic red knot (Calidris canutus) sanderling (Calidris alba) semipalmated sandpiper (Calidris pusilla) western sandpiper (Calidris mauri ) little stint (Calidris minuta) Temminck’s stint (Calidris temminckii ) white-rumped sandpiper (Calidris fuscicollis) Baird’s sandpiper (Calidris bairdii ) dunlin (Calidris alpina) curlew sandpiper (Calidris ferruginea) ruff (Philomachus pugnax) high Arctic high Arctic low and high Arctic high Arctic high Arctic low and high Arctic high Arctic high Arctic low and high Arctic high Arctic temperate–high Arctic sandpipers all species percentage prebreeding incubation 11/11 1/1 chick guarding 0/9 0.5/2 0/4 15/15 0.5/2 0/19 24/26 2/3 14.5/15 4/4 juveniles’ breeding grounds 0/6 0/3 0/4 0/3 0/33 0/18 0.5/1 0/9 0/8 0.5/1 0.5/19 0/10 0/2 0/13 5/7 11.5/20 32.5/33 11/11 2/2 3/4 16.5/17 11/11 6/6 0.5/1 0/2 0/14 0/20 0/8 0/5 0/9 65/81 80 177.5/185 96 4/13 13 1/155 0 0/59 0 11/12 0/9 10/92 13/308 4 0/3 0/1 1.5/2 3/3 0/2 0.5/14 0/26 45/48 1.5/2 0.5/65 0.5/20 0/6 0/4 0/2 winter 2/2 1/1 10.5/12 autumn migration 0/16 0/2 0/7 0/8 0/63 0 Functional shifts in preen wax composition subfamily 2136 J. Reneerkens and others Proc. R. Soc. Lond. B (2002) Table 1. Frequencies of diester preen waxes that individuals of 19 sandpiper species secrete during spring migration, pre-breeding, incubation, chick guarding, autumn migration and during winter. (Individuals that secreted mixtures of monoesters and diesters were scored as 0.5.) Functional shifts in preen wax composition Limosa limosa Arenaria interpres J. Reneerkens and others 2137 Calidris ferruginea a) b) c) retention time Figure 1. Gas chromatograms of typical (a) monoester, (b) mono/diester, and (c) diester secretions of black-tailed godwit (Limosa limosa), ruddy turnstone (Arenaria interpres) and curlew sandpiper (Calidris ferruginea). godwits (L. lapponica) could not be compared, as a single individual or only one of the sexes was caught during courtship and incubation. Birds caught shortly after arrival on the Arctic breeding grounds were considered to be in the period of mate choice and courtship. High-Arctic breeding sandpipers start courtship displays within a few days of arrival (e.g. Reneerkens et al. 2002). Sometimes they arrive already paired-up, as observed in some of the curlew sandpipers (H. Schekkerman and I. Tulp, personal communication). Birds on the breeding grounds with fully developed brood-patches were considered to be incubating even if not caught on the nest. In redshanks and western sandpipers (C. mauri ), it was possible to relate the composition of preen-gland secretions to the number of days before hatching. Hatching dates were calculated from known laying dates, using the incubation lengths (24 days and 19 days, respectively) measured at the study sites (A. Niehaus and W. Tijssen, personal communication). (c) Sample processing By softly massaging the nipple of the preen gland, a tiny sample of preen wax can be obtained on a cotton bud. The waxes were dissolved in ethyl acetate. We then evaporated the solvent with a gentle ow of nitrogen gas and weighed the waxes (ranging from 0.1–4.3 mg). Subsequently, the waxes were redissolved in ethyl acetate to a concentration of 1 mg ml2 1 and injected into a gas chromatograph (GC). Details of the analytic procedures are described elsewhere (Dekker et al. 2000). Gas chromatograms of the wax mixtures are characteristic for either mono- and diesters (Piersma et al. 1999; Sinninghe Damsté et al. 2000) enabling easy classiŽcation of samples into three groups: (i) monoesters, (ii) diesters and (iii) a mixture of monoand diesters (Žgure 1). This classiŽcation was conŽrmed by GC and by GC followed by mass spectrometry analysis of hydrolysed waxes (cf. Dekker et al. 2000). We scored the fraction of diesters in the preen waxes as: 0, only monoesters; 0.5, mixture Proc. R. Soc. Lond. B (2002) of mono- and diesters; and 1, predominantly (more than 95%) diesters. 3. RESULTS During migration and in winter, all 19 investigated sandpiper species secreted mixtures of monoester preen waxes. As in red knots (Piersma et al. 1999), shifts from mono- to diester waxes only occurred at the start of courtship and mating (table 1; Žgure 2). Shortly before departure to the breeding grounds (usually late May), diester waxes were produced by a few individuals. By contrast, shortly after arrival on the breeding grounds, the majority of individuals (80%) secreted diesters (table 1). This indicates that the shift in preen wax composition occurs around arrival on the Arctic tundra. Almost all individuals (96%) produced diesters during incubation (table 1). Only a few incubating redshanks and western sandpipers secreted (some) monoester compounds Žve days or less before hatching. However, most adults with chicks secreted monoester preen waxes (table 1), indicating a sharp shift from diesters to monoesters at hatching. During autumn migration and winter preengland secretions never consisted of diesters (table 1). Recently edged juveniles (63 individuals of eight species) only secreted monoester waxes (table 1). Redshanks showed more overlap in the temporal pattern of mono- and diester secretion than other species (Žgure 2). This is caused by the temporal overlap of lifecycle stages (table 1). Where veriŽable, diester secretion during courtship and incubation occurred evenly in both sexes. However, curlew sandpipers and ruffs were different. In curlew sandpipers, only two of the 10 male curlew sandpipers secreted complete diester mixtures shortly after arrival on the breeding grounds. By contrast, eight of the 10 females from this period secreted diester waxes only 2138 J. Reneerkens and others Functional shifts in preen wax composition Calidris canutus Limosa lapponica Calidris ferruginea Calidris alba Arenaria interpres Calidris minuta Calidris bairdii Calidris fuscicollis Calidris mauri Phalaropus fulicarius Calidris temminckii Calidris alpina Calidris pusilla Limosa haemastica Limnodromus griseus Limnodromus semipalmatus Philomachus pugnax Limosa limosa Tringa totanus April May June July August Figure 2. Seasonal changes in chemical composition of preen waxes of adult birds in 19 sandpiper species. Species are ordered from top to bottom on the basis of median lattitude of their breeding range, with the northernmost breeding species Žrst. Squares, monoesters; triangles, mixture of mono- and diesters; Žlled circles, diesters. (Mann–Whitney U-test with tied ranks, U1,20 = 82.0, p = 0.008). Of the 49 male and 43 female ruffs caught during spring migration (table 1), none of the males and 14 of the females produced (mixtures with) diesters (Žgure 2). All captive female (and no male) ruffs sampled during the period of incubation secreted diester preen waxes ( J. Reneerkens and D. B. Lank, unpublished data). 4. DISCUSSION (a) How common are changes from mono- to diester preen waxes? Jacob & Poltz (1973) and Jacob (1978) characterized preen wax components of seven shorebird species, including three of the species that we investigated (redshank, red knot and dunlin) and found monoesters with some traces of diesters. It is not known when their samples were taken. In this study, we demonstrated that in all 19 species examined, during the short period of mate choice and incubation, wax composition shifted from mono- to diesters. As temperate breeding species start their reproductive activities earlier than high-Arctic breeding species, diesters are secreted earlier in temperate breeding species (blacktailed godwit, redshank and ruff; Žgure 2). (b) What function(s) do diester preen waxes serve? In all 19 sandpiper species, diester waxes are secreted during the relatively brief periods of courtship and incubation, indicating a common function shaped by the speciŽc demands during these life-cycle stages. Because adults (as well as juveniles) of Arctic breeding species excrete monoesters after hatching, Arctic conditions (e.g. low temperatures, strong winds and high ultraviolet radiation) are unlikely to have selectively favoured diester preen waxes as a way of plumage protection. As they also occur in redshanks, black-tailed godwits and Asian dowitProc. R. Soc. Lond. B (2002) chers, shifts to diesters are not restricted to Arctic breeders. The more distantly related oystercatcher (Haematopus ostralegus, Haematopodidae) breeds and overwinters in western Europe and also shifts to diester preen waxes during the breeding season ( J. Reneerkens, unpublished data). Ruffs and curlew sandpipers are, to our knowledge, the only two investigated sandpipers in which incubation is completely or largely restricted to females. Secretion of diester waxes is also restricted to female ruffs and occurs signiŽcantly more often in female than in male curlew sandpipers. In wild-type and domesticated mallards (Anas platyrhynchos), females, but not males, show similar qualitative shifts from mono- to diester preen waxes during courtship and incubation ( Jacob et al. 1979; Kolattukudy et al. 1987). In mallards, incubation is also restricted to females. The change to diester wax secretion in incubating individuals indicates that diesters are important for birds on the nest. Diester preen waxes have higher molecular weights than monoesters and consequently are less volatile. Thus, they may reduce the smell and enhance olfactory crypticism. If diesters make it more difŽcult for mammalian predators, such as Arctic foxes (Alopex lagopus), to smell out the bird on the nest, a shift from mono- to diester preen waxes during incubation would have a large selective advantage. Piersma et al. (1999) proposed that diesters enhance sexually selected quality signals that make the plumage brighter or shinier, enabling visual discrimination of Žt mates during mate choice. The present study does not falsify this hypothesis, but indicates that it is not the whole story because diester preen waxes are also secreted during incubation, that is, after fertilization. Small differences in the smell or in the visibility of plumage due to different wax compositions may not be detectable by the human senses (e.g. Viitala et al. 1995). However, if different preen wax compositions can be distinguished by conspeciŽcs, Functional shifts in preen wax composition they could potentially play a part during mate choice before becoming fully functional during incubation. We thank our NIOZ colleagues Anne Dekinga, Maurine Dietz, Graciela Escudero, Petra de Goeij, Anita Koolhaas, Luisa Mendes and especially Bernard Spaans, for their help with bird catching and preen wax sampling on locations worldwide. Leo Bruinzeel, Nicola Baccetti, Welmoed Ekster, Frank Engelen, Adrian Farmer, Katharine Graham, Niko Groen, Tómas Gunnarsson, Joe Jehl Jr, Joop Jukema, Jan van der Kamp, Tatyana Ê ke Kirikova, Joanna Klima, Oscar Langevoord, Dov Lank, A Lindström, Ron Mes, Guy Morrison, Amanda Niehaus, Leon Peters, ringing group ‘Calidris’, Castricum ringing group, Daniel Ruthrauff, Hans Schekkerman, Mikhail Soloviev, Adriano Talamelli, Wim Tijssen, Ingrid Tulp, Douwe van der Zee and several anonymous contributors also provided preen wax samples. J.R. thanks the Canadian Forces for their support at the military station at Alert and the Canadian Wildlife Service for Žnancial and logistic support of work in 1999, and Jack Stephens and Kurt Burnham of the Peregrine Fund for hospitality in Thule, Greenland. John WingŽeld is acknowledged for enabling J.R. to participate in Želdwork in Thule. Muriël van den Anker and Soledad van Eijk helped in the laboratory. We thank Pavel S. Tomkovich, Ingrid Tulp, Hans Schekkerman and Dov Lank for early feedback on the results, and Phil Battley, Sue Moore and an anonymous referee for improving an earlier draft. The work is Žnancially supported through the Netherlands Organization for ScientiŽc Research (NWO) by ALW grant 810.34.003 and a PIONIER-grant to T.P. This is NIOZ publication 3700. REFERENCES Baker, A. J., Piersma, T. & Greenslade, A. D. 1999 Molecular versus phenotypic sexing in red knots. Condor 101, 887–893. Dekker, M. H. A., Piersma, T. & Sinninghe Damsté, J. S. 2000 Molecular analysis of intact preen waxes of Calidris canutus (Aves: Scolopacidae) by gas chromatography/mass spectrometry. Lipids 35, 533–541. Elder, W. H. 1954 The oil gland of birds. Wilson Bull. 66, 6–31. Elowson, A. M. 1984 Spread-wing postures and the water repellency of feathers: a test of Rijke’s hypothesis. Auk 101, 371–383. Fabricius, E. 1959 What makes plumage waterproof ? Wildfowl Trust Rep. 10, 105–113. Proc. R. Soc. Lond. B (2002) J. 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