Jan Beck á Eva MuÈ hlenberg á Konrad Fiedler

Mud-puddling behavior in tropical butter¯ies:in search of proteins or minerals?

Received: 5 October 1998 /Accepted: 7 December 1998

Abstract We experimentally investigated the attractionof adult butter¯ies to moist soil and dirt places (a be-havior termed `mud-puddling') in two species-richtropical communities on the island of Borneo. At a rainforest site, 227 individuals (46 species) were attracted tothe baits, compared to 534 individuals (54 species) at afarmland site. With one single exception, all attractedbutter¯ies were males. Of various salt and amino acidsolutions, only sodium was accepted, but overall, albu-min solutions turned out to be the most attractivepuddling resource. Butter¯y families di�ered consis-tently in their resource preferences. Representatives ofthe families Papilionidae and Pieridae more often visitedNaCl solutions, but still accepted albumin, whereasrepresentatives of the Nymphalidae, Hesperiidae and, inparticular, Lycaenidae preferred the protein resource. Inexperiments using decoys prepared from pinned butter-¯y specimens, representatives of the Papilionidae andPieridae were more strongly attracted to baits providedwith decoys made from conspicuous, medium-sizedyellow Eurema species (Pieridae), whereas dummiesmade from small, cryptically colored lycaenids (Prosotasand Caleta species) were ine�ective. Decoys did not in-¯uence the attraction of lycaenid butter¯ies towardsbaits. Hence, visual cues play an important role in lo-cating puddling resources for papilionids and pierids,while for lycaenid butter¯ies searching for nitrogensources, olfactory cues emitted by decaying organicmatter are more likely to be important. The strong at-traction of male butter¯ies to nitrogen-rich resourcessuggests that, as in the case of sodium, these nutrientsmay increase reproductive success.

Key words Tropical butter¯ies á Protein food á Sodium áReproductive biology á Visual cues

Introduction

Adult Lepidoptera (butter¯ies and moths) of manyspecies frequently visit moist ground, perspiration, tears,excrements, or animal carcasses to suck water and dis-solved nutrients, a behavior conventionally termed`mud-puddling' (e.g., Arms et al. 1974; Adler 1982;Adler and Pearson 1982). Although this behavior isknown from temperate-zone as well as tropical habitats,it is far more common in tropical regions. Large num-bers of individuals may congregate at puddles, oftenneatly arrayed in groups of conspeci®cs or of butter¯iesof similar external appearance (e.g. Larsen 1991). Mud-puddling is thought to serve as a means of acquiringessential adult resources, particularly sodium.

In most lepidopterans, the males pass complexspermatophores to their mates (Drummond 1984) whichare used to transfer, besides sperm, various types ofnuptial gifts. These gifts may consist of minerals such assodium (Pivnick and McNeil 1987; Smedley and Eisner1995, 1996) or calcium phosphate (Lai-Fook 1991), theymay contain toxic secondary plant metabolites (Schnei-der 1992, 1993; Eisner and Meinwald 1995), or theycontribute nutrients such as amino acids (Arms et al.1974; Boggs and Gilbert 1979).

Due to the low sodium content of land plants, manyterrestrial herbivores (including humans; e.g., Haas andHarrison 1977) are expected to crave sodium. Amongthe Lepidoptera with their herbivorous caterpillar stag-es, mineral reserves assembled during the larval phasemay often be limiting, and there should be strong se-lection for strategies to replenish mineral stocks. Toxicplant metabolites which serve as pheromone precursorsand nuptial gifts by male butter¯ies are collectedthrough a special, sexually selected behavioral repertoire(`pharmacophagy': e.g. Schneider 1992, 1993). Mud-puddling might be seen as the analogous case with re-gard to minerals. This idea is supported by the fact thatmost individuals engaged in puddling behavior are males(e.g., Adler 1982).

Oecologia (1999) 119:140±148 Ó Springer-Verlag 1999

J. Beck á E. MuÈ hlenberg á K. Fiedler (&)Lehrstuhl TieroÈ kologie I, UniversitaÈ t Bayreuth,D-95440 Bayreuth, Germanye-mail: konrad.®edler@uni-bayreuth.de

Occasionally, however, female butter¯ies visit mudpuddles (e.g., in montane North America: Boggs andJackson 1991; Sculley and Boggs 1996). In the latter twostudies, the propensity for puddling behavior di�eredconsistently between sexes and varied with age. Hence,the mating system of a given species, the competitiveabilities and current nutrient status of the individualsinvolved, and actual environmental conditions all a�ectthe choice of resources available at puddling sites.

Puddling behavior has probably evolved from thedrinking of water (Adler 1982), and in dry habitats theneed for water may be a prime reason for visiting pud-dles (e.g., Larsen 1991). In the Californian checkerspotbutter¯y Euphydryas editha bayensis, puddling only oc-curred in years of extreme drought (Launer et al. 1996).In the dry steppe biomes of Turkey, huge numbers ofblue butter¯ies (Lycaenidae, mostly Polyommatini)regularly aggregate at puddles (Hesselbarth et al. 1995;K. Fiedler, unpublished observations), whereas thesame, or very closely related, species visit such resourcesmuch less often in the cooler and more humid climate ofcentral Europe (K. Fiedler, unpublished observations).Nevertheless, even under arid conditions, the clear ma-jority of butter¯ies attracted to puddles are males. Inperhumid tropical rainforests, where puddling is com-mon, the need for water as a primary motivation forpuddling may safely be neglected except in El NinÄ oyears. In addition, many puddling butter¯ies and mothseject surplus water (BaÈ nziger 1973; Adler 1982), con-®rming that substances dissolved in water, rather thanwater itself, are the principal resource accumulatedthrough puddling.

To our knowledge, all experimental data supportingthe hypothesis that searching for sodium is the primereason for mud-puddling in the Lepidoptera come fromtemperate regions where mud-puddling is less pro-nounced than in the tropics. Furthermore, only in fewstudies and with a small number of species have bu�et-type experiments been conducted o�ering simulta-neously sodium and a variety of other, potentiallypuddling-related substances (e.g., Arms et al. 1974;Adler and Pearson 1982). In none of these studies was aprotein included as a possible puddling stimulus, al-though BaÈ nziger (1973, 1988), for example, has hinted atthe potential role of proteins in the ecology of lach-ryphagous moths. Hence, current conceptions of theevolutionary ecology of mud-puddling in butter¯iesmight be seriously biased with regard to geographic re-gion, taxonomic coverage, or resources examined.

In the present study we tested a set of ®ve minerals,two amino acids, urea, and a protein for their potentialas puddling stimuli in two Oriental tropical butter¯ycommunities under natural ®eld conditions. We alsopresent data on how butter¯ies may locate suitablepuddling sites. In particular, we address the questionwhether butter¯y decoys at a puddling site increase theattractiveness of such resources, which are certainly rareand patchily distributed in a rainforest environment.

Materials and methods

Field methods

Field work was done in 2 consecutive years, October to November1996 by J.B. and August to September 1997 by E.M., in Mt.Kinabalu National Park (Malaysia, Sabah) in northeast Borneo.With 730 km2, the park covers a relatively large area of protectedrainforest with a rich and rather well studied butter¯y fauna(HaÈ user et al. 1997). The study site was situated near Poring HotSprings at the southeastern margin of the park (6°34¢ N, 116°33¢ E,elevation 500 m a.s.l.). While inside the park the vegetation isdominated by mixed hill dipterocarp forest (Kitayama 1992), out-side the park limits, agricultural areas cultivated by local farmersand wasteland vegetation predominate. Two di�erent sites werechosen for their relatively high abundance of puddling butter¯ies.One was situated on the banks of the Mamut River in a slightlydisturbed, but still tall closed dipterocarp forest; the other site wasalong a small river outside the park, surrounded by cultivatedfarmland.

The ®rst set of experiments, conducted at the Mamut River in1996, was designed to determine which substances are most at-tractive to various species in the focal butter¯y community. Ninedi�erent solutions and deionized water as a control were presentedto the butter¯ies in petri dishes (diameter 9 cm) lined with white®lter paper. The following substances (all dissolved in deionizedwater, at 0.1 M concentration; chemicals from Merck or Roth,Germany, purity >99.5%) were used: NaCl, KCl, MgCl, CaCl2,Ca3(PO4)2, urea [CO(NH2)2], glycine (NH2CH2COOH), and lysine[NH2(CH2)4CH(NH2)COOH]. A saturated solution of albumin(approx. 0.16 g/ml H2O) served as a standardized protein source(chicken egg albumin, Roth, Germany in 1996, bovine serum al-bumin, Roth, Germany in 1997). If necessary, due to strongevaporation on hot sunny days, test solutions were replenished tominimize variation in the nutrient concentrations o�ered.

The ten petri dishes (nine resources plus water) were arrangedimmediately adjacent to each other in a square on the sand of asunlit riverbank during peak hours of butter¯y activity, from 0900to around 1600 hours. Relative positions of the dishes were chan-ged regularly. Four replicate sets were o�ered simultaneously,spaced well from each other (minimum distance between two sets10 m). The behavior of butter¯ies was observed with binoculars(10 ´ 42) from a distance of about 7 m to minimize disturbance.Butter¯ies were considered sucking at the bait if they were actuallyseen drinking (easy to see in large Papilionidae) or if they sat in apetri dish for at least 1 min. In total, the bait sets were observed for154 h on 35 days.

Large-bodied butter¯ies (families Papilionidae, Pieridae,Nymphalidae) accepting a bait were caught (36 of 47 individu-als), identi®ed (after Corbet and Pendlebury 1992), marked (ifreleased, to avoid pseudoreplicates) and released. Representativesof taxonomically di�cult groups like blues (Lycaenidae) andskippers (Hesperiidae) were caught (114 out of 180 individuals),marked and released, or killed and later identi®ed (Seki et al.1991). Butter¯ies that could not be determined by sight andresisted capture could always be identi®ed to subfamily level atleast. The very low recapture rate of marked animals (7 out of71) indicated that pseudoreplication among the unmarked but-ter¯ies was negligible. Determination of sex was possible in 153out of 227 specimens (either using the preserved vouchers, or onthe basis of conspicuous sex dimorphism in wing pattern).

The second set of experiments, mainly aimed at assessing thee�ect of butter¯y dummies on the attractiveness of resources forother free-¯ying butter¯ies, was conducted in 1997 at a naturalpuddling site along a small creek in cultivated landscape. For theseexperiments, only albumin and NaCl (the two substances whichhad turned out to be most attractive in 1996) were o�ered, at thesame concentrations as in the previous year. Resource solutionsjust covered the bottom of each petri dish and were replenished ifneeded (on 4 of 14 observation days, when ®eld work exceeded

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4 h). No ®lter paper was used, in order to minimize visual attrac-tion of butter¯ies to the resources.

Dummies were prepared from specimens of butter¯y specieswhich were most commonly observed at puddles in the study re-gion. As a representative of the larger, conspicuously coloredbutter¯ies, we used male specimens of Eurema sari and Euremasimulatrix (Pieridae). At many natural puddling sites, members ofthe small-bodied Lycaenidae were most abundant. Two di�erentsets of lycaenid dummies were tested. Male Prosotas spp. (P. piaand P. nora) served as representatives for the majority of morecryptically colored lycaenids, whereas the black-and-white-markedCaleta elna and C. manovus were used as examples of more con-spicuous lycaenids. As dummies, we took ®eld-caught butter¯ies(only fresh, intact specimens) which were killed, mounted on insectpins with the wings dorsally folded, and dried. Care was taken thatthe pinned dummies closely resembled the natural posture ofpuddling individuals.

For experiments, either a single dummy or a group of ®ve wasplaced at a petri dish as decoy. Petridishes with no decoy served ascontrols. Hence, six combinations of puddling substances (albu-min, NaCl) and decoys (none, one, ®ve) formed one test set (dis-tance between neighboring petri dishes 30 cm). Every 30 min, therelative positions of the dishes and dummies were changed tominimize positional bias. Two sets of six test dishes were o�eredsimultaneously, spaced 3 m apart from each other. One set con-tained Eurema dummies over the entire duration of the experiment.In the other set, we tested lycaenid dummies (Prosotas in the ®rsthalf of the experiment, Caleta in the second). Between subsequentdays, the positions of the two sets were also exchanged to rule outposition e�ects. From a distance of about 5 m, all butter¯ies whichsettled down at the resources were recorded. Only visits at whichthe butter¯ies remained at the resources for more than 30 s anddrinking behavior could be positively con®rmed (uncoiling of theproboscis, observations through binoculars) were included. Totalobservation time amounted to 53 h distributed over 14 days.

As in the ®rst experiment, insects were identi®ed by sightwhenever possible (using Otsuka 1988). Vouchers of taxonomicallydi�cult groups (102 of 534 individuals altogether, especiallyLycaenidae) were caught and preserved for later identi®cation.

Statistical analysis

From our ®eld experiments, we obtained data on the acceptance ofthe various resources with or without decoys. For the 1996 exper-iments, these frequency data were tabulated in a two-way contin-gency table (with butter¯y family and resource type as factors) andsubjected to standard v2 statistics (Sachs 1992). Observed fre-quencies of acceptance of a resource versus water were testedagainst the null hypothesis of equal distribution. Evaluation wasrestricted to the more commonly used resources [NaCl and albu-min; also Ca2(PO4)3 and urea for Lycaenidae]. To control forspurious signi®cance results arising from multiple comparisons, weapplied the sequential Bonferroni approach (Rice 1989) within each

set of hypotheses to be tested. Signi®cant results passing this cri-terion are marked with an asterisk.

The 1997 data set had a more complex structure, since fourfactors were involved (butter¯y family, resource type, decoy type,and decoy number). This data set was evaluated using the log-linearanalysis modul of StatSoft (1995). First, a log-linear model wasconstructed using those e�ects that signi®cantly contributed tovariation as measured by signi®cant partial v2 values, and the ®t ofthis `optimized' model to the observed data was checked. Second,each of the included interaction e�ects was separately tested, byremoving it from the model and analyzing the di�erence in thegoodness of ®t between the simpli®ed and optimized model (mea-sured as di�erence of the v2 values) for signi®cance. Finally, thesigni®cant e�ects were localized by standard v2 statistics in thereduced cross-tabulated two-way frequency tables.

We performed all analyses primarily on the level of butter¯yfamilies (i.e., Papilionidae, Pieridae, Nymphalidae, Lycaenidae andHesperiidae). There are three reasons for this. First, most individualspecies in this tropical butter¯y community were so scarce thatduring our experimental period only small sample sizes were attainedper species. Second, many of the more abundant lycaenid species(e.g. in the genus Prosotas) are impossible to identify to species levelwithout capturing (and thus disturbing) the animals. Third, butter-¯ies from various species in the same family have often been ob-served to aggregate at puddles (Larsen 1991). Therefore it seems safeto assume for a ®rst approach that members of the same butter¯yfamily, if they visit mud puddles at all, have common resource re-quirements, while species-speci®c di�erences are less pronounced.Where our samples were large enough, we tested the validity of thisassumption on lower taxonomic levels (genera, species).

Results

Puddling preferences in a forest butter¯y community

A total of 227 observations of drinking butter¯ies rep-resenting at least 46 di�erent species (see Appendix)were made at the baits in 1996 in a forest habitat(Table 1). Among 153 specimens where sex could bedetermined, all were males except 1 single female Char-axes bernardus (Nymphalidae). The ®ve butter¯y fami-lies were represented at the baits in greatly di�erentnumbers of individuals. The bulk of observations(76.2%) came from blues (Lycaenidae, mostly tribePolyommatini), followed by Nymphalidae (13.2%),Papilionidae (6.6%), Hesperiidae (3.2%), and Pieridae(0.4%). Albumin and NaCl had by far the highest ratesof acceptance. CaCl2 was never accepted, and the aversebehavior of the butter¯ies occasionally contacting such

Table 1 Acceptance of all tested puddling substances across butter¯y families (data from Mamut River, Borneo, 1996)

All butter¯ies Lycaenidae Nymphalidae Papilionidae Pieridae Hesperiidae

Albumin 107 73 17 7 1 6NaCl 72 66 2 3 0 1Ca2(PO4)3 14 11 1 2 0 0Glycine 12 5 2 3 0 0Lysine 7 4 3 0 0 0MgCl2 3 2 1 0 0 0KCl 3 1 3 0 0 0Urea 9 8 1 0 0 0CaCl2 0 0 0 0 0 0Water 4 3 0 0 1 0Total 227 173 30 15 2 7

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baits indicated that this salt solution even had repellentproperties.

Lycaenidae clearly preferred albumin and NaCl overpure water (v21 = 64.5, v21 = 57.5; both P < 0.0001*),while phosphate and urea were not accepted signi®cantlymore often than water (v21 = 4.57, P = 0.038 forphosphate; v21 = 2.27, P = 0.132 for urea; both notsigni®cant after sequential Bonferroni correction for ®vetests within Lycaenidae). All other substances, includingthe amino acids, were only rarely visited. If all lycaenidspecies (>23) are pooled, there was no signi®cant dif-ference in attractiveness between albumin and NaCl(v21 = 0.35, P = 0.55). For the few species or generawhich were represented by larger numbers of individu-als, this pattern persisted at lower taxonomic levels(NaCl/albumin; Ionolyce helicon: 7/6; Prosotas spp.: 5/6;Caleta spp.: 29/33).

Among the Nymphalidae, albumin was preferredover pure water (v21 = 17.0, P < 0.0001*) as well asover NaCl (v21 = 11.8, P < 0.001*; sequential Bonfer-roni correction for two tests within Nymphalidae). The30 puddling visits were distributed over at least 12 spe-cies. Vindula dejone (®ve visits) was the commonestnymphalid species and only occurred at albumin baits.In the Papilionidae, the only statistically signi®cantpreference was again observed for albumin over water(v21 = 7.0, P < 0.01), and Papilio memnon, the com-monest species (®ve visits), exclusively utilized albumin.Otherwise, so few individuals appeared at the baits thatno detailed analyses could be performed.

Under tropical, warm and humid conditions, the al-bumin solution rapidly started decaying and then emit-ted a strong smell. An apparent preference for albuminmight also emerge if such smells serve as cues to locateresources rich in sodium (e.g., animal carcasses). We

tested whether a smelly albumin solution (0.16 g/mlH2O) combined with 0.1 M NaCl would attract morebutter¯ies than albumin alone. Four replicates of a set oftwo petri dishes each (one with, one without NaCladded) were exposed at the same test site. No signi®cantdi�erence was found in the acceptance of pure albumin(18 visits) and the NaCl-enriched albumin solution (12visits; v21 = 1.2, P = 0.273).

The results of our experiments along Mamut River in1996 strongly indicated that, besides sodium, proteinslike albumin are an important adult resource sought bytropical butter¯ies engaged in puddling behavior. For nofamily did any of the mineral resources or amino acidsattract more individual butter¯ies than the albumin bait,and only lycaenids showed up in roughly equal numbersat protein and sodium sources.

Attraction of butter¯ies to sodium and albumin

During the 1997 experiments in a deforested tropicalhabitat, 534 butter¯ies representing 54 species (see Ap-pendix) accepted the experimental puddling resources(Table 2). Of 102 voucher specimens all were males.Representatives of the Lycaenidae again accounted forthe majority of observations (75.5%), followed byPapilionidae and Pieridae (8.1% each), Nymphalidae(5.2%) and Hesperiidae (3.2%). Abundance relationsdeviated from the experimental series the year before(v24 = 18.82, P < 0.0009), but this was largely due to aprominent di�erence in one family, the Pieridae (43 vs 2specimens observed). Overall, considerably more but-ter¯ies (10.08 individuals/h) were seen at the non-foreststudy site in 1997 than in 1996 within the forest (1.47individuals/h).

Table 2 Acceptance of arti®cial puddling resources (NaCl or albumin solutions) in experiments with di�erent numbers (0, 1, 5) of Eurema(Pieridae), Prosotas and Caleta (both Lycaenidae) decoys

NaCl Albumin Row totals

0 1 5 0 1 5

Eurema dummiesPieridae 0 2 22 0 3 12 39Papilionidae 0 0 25 0 2 8 35Lycaenidae 7 6 4 62 57 58 194Nymphalidae 2 0 2 3 3 4 14Hesperiidae 0 1 0 4 6 1 12

Prosotas dummiesPieridae 1 0 0 1 0 0 2Papilionidae 0 0 1 1 0 0 2Lycaenidae 4 3 4 22 31 16 80Nymphalidae 1 0 0 1 2 2 6Hesperiidae 1 0 0 1 0 0 2

Caleta dummiesPieridae 1 0 1 0 0 0 2Papilionidae 0 0 0 1 1 4 6Lycaenidae 0 4 3 41 45 36 129Nymphalidae 0 0 2 1 3 2 8Hesperiidae 0 0 1 1 1 0 3

Column totals 17 16 65 139 154 143 534

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Log-linear analysis revealed that frequencies of but-ter¯ies accepting the baits di�ered signi®cantly betweenfamilies, resource types, decoy types, and decoy numbers(Table 3). In addition, only two-way interactions be-tween butter¯y family and the design variables (re-source, decoy type, and decoy number) were signi®cant,while all other two-way and all three-way interactionswere not. The log-linear model including only the sig-ni®cant two-way interactions and the three-way inter-action between the design variables closely ®tted theobserved frequencies (v248 = 48.8, P = 0.441), andcomparisons of models omitting one of the above two-way interactions with this optimized log-linear modelrevealed that all these interactions were statisticallysigni®cant (family ´ resource: v24 = 81.9, P < 0.0001;family ´ decoy type: v28 = 16.223, P < 0.05; fami-ly ´ decoy number: v28 = 44.7, P < 0.0001).

A closer analysis of the cross-tabulation of the threesigni®cant two-way interactions (Table 4) shows that inthe 1997 data set, lycaenids, nymphalids and hesperiidsclearly preferred albumin over NaCl, while about twice asmany papilionids and pierids were attracted to NaCl re-sources as compared to albumin (v2-tests for homoge-neity between resources: Lycaenidae: v21 = 275.2,P < 0.000001*; Nymphalidae: v21 = 7.00, P = 0.0082*;Hesperiidae: v21 = 7.12, P = 0.0076*; Papilionidae:v21 = 1.88, P = 0.17; Pieridae: v21 = 2.81, P = 0.093;sequential Bonferroni correction for ®ve tests).

E�ects of decoys on puddling butter¯ies

Eurema decoys strongly increased the attractiveness ofresources to papilionids (v22 = 45.3, P < 0.000001*) aswell as pierids (v22 = 63.8, P < 0.000001*; sequentialBonferroni correction for ®ve tests) over resourcesequipped with lycaenid dummies or without decoy at all(Table 4). For the remaining three families, the pres-ence of decoys had no noticeable e�ect on the accep-tance of arti®cial puddling resources (Lycaenidae:v22 = 1.11, P = 0.575; Nymphalidae: v22 = 0.50,P = 0.779; Hesperiidae: v22 = 3.65, P = 0.162). Simi-larly, decoy number had a strong e�ect on papilionids(v22 = 58.7, P < 0.000001*) and pierids (v22 = 44.9,

P < 0.000001*), with the larger group of ®ve decoysbeing most attractive (Table 4). In contrast, the numberof decoys did not in¯uence visitation frequencies in anyof the other three families (Lycaenidae: v22 = 2.36,P = 0.308; Nymphalidae: v22 = 1.14, P=0.565; Hes-periidae: v22 = 3.65, P = 0.162).

Discussion

In most studies on puddling behavior of butter¯ies andmoths, the need for sodium has been emphasized as themain causal factor (Arms et al. 1974; Smedley and Eis-ner 1995, 1996). The results presented above showclearly that in the two tropical butter¯y communitiesunder investigation, proteins like albumin were also indemand. Although we have no chemical data onpotential sodium `contamination' of the albumin testchemicals, even the presence of substantial amounts ofsodium in the resources could not explain the consistentdi�erences which we observed between butter¯y fami-lies. The butter¯ies accepted none of the other mineralsor nitrogen sources (urea or two amino acids) with thepossible exception of phosphate (cf. Lai-Fook 1991),

Table 3 Partial associations between the variables butter¯y family(Papilionidae, Pieridae, Nymphalidae, Lycaenidae, and Hesper-iidae), resource type (albumin or NaCl solution), decoy type(dummies made of pinned Eurema, Prosotas, or Caleta butter¯ies),and decoy number (none, one, ®ve) in the 1997 data set

E�ect df v2 P

Family 4 717.2 <0.00001Resource 1 210.4 <0.00001Decoy type 2 109.6 <0.00001Decoy number 2 7.38 0.025Family ´ resource 4 83.5 <0.00001Family ´ decoy type 8 17.9 0.022Family ´ decoy number 8 46.4 <0.00001Resource ´ decoy type 2 4.36 0.113Resource ´ decoy number 2 4.24 0.120Decoy type ´ decoy number 4 6.94 0.139Family ´ resource ´ decoy type 8 9.99 0.266Family ´ resource ´ decoy number 8 5.02 0.755Family ´ decoy type ´ decoy number 16 23.85 0.09Resource ´ decoy type ´ decoy number 4 2.74 0.603

Table 4 Cross-tabulation ofthe three signi®cant two-wayinteractions as revealed bylog-linear modelling of the datapresented in Table 2

Papilionidae Pieridae Nymphalidae Lycaenidae Hesperiidae

Butter¯y family ´ resourceNaCl 26 27 7 35 3Albumin 17 16 21 385 14

Family ´ decoy typeEurema 35 39 9 125 8All lycaenids 6 1 11 142 2No decoy 2 3 8 136 7

Family ´ decoy numberNone 2 3 8 136 7One 3 5 8 146 8Five 38 35 12 121 2

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which was occasionally taken up by lycaenids. Hence, atour study sites, mud-puddling served the twofold func-tion of providing sodium as well as protein resources,and these nutrients were almost exclusively gathered bymales.

We observed signi®cant di�erences between butter¯yfamilies in their response to puddling sources.Nymphalids and hesperiids were primarily attracted toalbumin, whereas papilionids and pierids visited sodiumresources more frequently, though representatives of thelatter two families by no means ignored protein baits.These observations agree well with earlier ®ndings. Themost detailed experimental studies on sodium as a primestimulus for puddling behavior in butter¯ies were con-ducted with one temperate-zone papilionid (Arms et al.1974) and one pierid (Adler and Pearson 1982). On theother hand, many tropical nymphalids and hesperiidsare particularly attracted to carrion or feces (e.g., car-nivore excrement, bird droppings: Larsen 1991; Austinand Riley 1995; Sourakov and Emmel 1995).

Lycaenids were the most abundant visitors to ourarti®cial puddling sites. Albumin was an important re-source throughout, but while in the 1996 experiments,roughly equal numbers of lycaenids were attracted toalbumin and NaCl, the albumin baits were stronglypreferred in the 1997 trials. We have no unequivocalexplanation for this discrepancy. The two butter¯ycommunities di�ered considerably in species composi-tion (SoÈ rensen index of similarity: 0.50; see Appendix).Within the Lycaenidae, representatives of the Nacadubasection (sensu Hirowatari 1992: genus Nacaduba) andLycaenopsis section (sensu Eliot and Kawazoe 1983:genera Actyolepis, Celastrina, Megisba, Monodontides,Udara) were only, or more strongly, represented at theMamut River site, and butter¯ies in the Lycaenopsissection were the only lycaenids which visited NaCl moreoften than albumin (13 vs 5 cases). However, in bothyears, the same lycaenid species made up the majorityof the visitors at puddling sites (i.e., Caleta, Prosotas,Ionolyce, Anthene spp.). These same lycaenid speciesvisited sodium and albumin in roughly equal frequenciesin 1996, but clearly preferred albumin in 1997. Albuminsources used for baiting di�ered between the years(chicken egg albumin resulting in a rather colorless so-lution in 1996, bovine serum albumin in 1997 giving adark solution). So, either characteristics of the baits(di�erences in chemical or visual properties of the twoalbumin types) or environmental factors (e.g., resourceavailability; see also Launer et al. 1996) rather thanspecies composition were more likely causes for the be-tween-year variation.

Di�erent species within the same taxonomic entity(such as family or genus) may have di�erent resourcerequirements with regard to mud-puddling, e.g., de-pending on the mating system of each species (Karlsson1995, 1996; Sculley and Boggs 1996). Our experimentaldata as well as ®eld observations, however, indicate thatin the Bornean butter¯y communities studied here,puddling preferences are quite consistent within taxo-

nomic family boundaries. All common lycaenid genera(Caleta, Prosotas, Ionolyce, Anthene) showed the samestrong attraction to albumin as the lycaenids as a whole.Often ®ve to ten species of these genera co-occur at thesame natural puddles. Similarly, the albumin preferenceof nymphalids was mirrored at lower taxonomic levels(Charaxes, Polyura, Vindula, and Phalanta almost ex-clusively appeared at albumin baits), as was the slightsodium preference of papilionids (Graphium and Pathysapreferred NaCl). As one counter example, birdwingbutter¯ies (Troides brookiana) often occurred at naturalpuddling sites along the Mamut River, but never visitedthe baits, whereas other papilionids were attracted.Hence, our analyses at the family level as a ®rst ap-proach are consistent with observations on lower taxo-nomic levels as well as ®eld experience and thus likelyreveal real patterns, but more detailed studies at thespecies level would be worthwhile.

Sodium gathered by male butter¯ies and moths dur-ing mud-puddling is transferred, sometimes in largeamounts, to their female mates through the sperm-atophore, and this nuptial gift increases reproductivesuccess (e.g., Adler and Pearson 1982; Pivnick andMcNeil 1987; Smedley and Eisner 1996). Similarly,proteins obtained by male butter¯ies could well increasetheir reproductive success. The striking predominance ofmales at protein sources strongly suggests that ± as withsodium collecting ± this behavior has evolved throughsexual selection. The studies of Karlsson (1994, 1995,1996) and Karlsson and Wickman (1989) on a variety oftemperate-zone butter¯y species suggest that nitrogenallocation is a critical factor in their reproductive biol-ogy (Boggs 1981), and that among long-lived and/orpolyandrous species in particular, there should be strongselection for mechanisms to improve nitrogen budgets.

According to BaÈ nziger (1973, 1990), only certain tear-drinking adult moths have so far been shown to possessprotease activity in their midgut, but the capacity todigest proteins might be more widespread in lepidop-terans which routinely utilize a protein-rich adult diet.However, even if butter¯ies attracted to protein baitswould turn out to lack any protease, they could stillbene®t from these nitrogen resources. Under humidtropical conditions in particular, any protein source(such as carrion or excrement) will be quickly decom-posed by bacteria and fungi, making available a varietyof smaller nitrogen-rich molecules like amino acids oroligopeptides.

Amino acids from adult food are incorporated byvarious butter¯ies (Wheeler 1996). Male North AmericanPapilio glaucus imbibe amino acids by chance when atmud puddles and subsequently transmit them through thespermatophore to their mates (Arms et al. 1974). Neo-tropical Heliconius derives amino acids from pollen, andpollen-feeding is essential to increase longevity and re-production in these outstandingly long lived butter¯ies(Gilbert 1972; Dunlap-Pianka et al. 1977). Flower nectarcan be enriched by amino acids dissolved from pollen thatfell into the ¯ower (Erhardt and Baker 1990), increasing

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the availability of amino acids for nectar-feeding butter-¯ies. However, evidence for the role of amino acids in thenutrition of nectar-feeding butter¯ies is discordant.Whilesome species preferentially drink nectars enriched withamino acids (Pieris rapae: Alm et al. 1989), others do not(Battus philenor, Ornithoptera priamus: Erhardt 1991,1992). In various species, no positive e�ect of nectaramino acids on longevity or fecunditywas found (e.g.,Hill1989; Hill and Pierce 1989), whereas in others, aminoacids were bene®cial (Murphy et al. 1983). Interestingly,neither in earlier experiments (Arms et al. 1974) nor in ourexperiments inBorneowere amino acid solutions (glycine,lysine) attractive to butter¯ies.

With regard to the use and importance of adult re-sources, lepidopterans occupy a continuum between twoextremes (Dunlap-Pianka et al. 1977; Boggs 1981, 1986;Karlsson 1994; Wheeler 1996). While some species (inparticular many moths) utilize exclusively (or at leastpredominantly) resources stored throughout larval de-velopment for somatic maintenance and reproduction,others supplement their nitrogen budget by adult feedingor through multiple mating. The ®rst strategy typicallycoincides with a short adult life span and a short, butintense period of reproduction, while the second strategyselects for prolonged life and more even, life-long eggproduction. An extreme example for the latter strategyare the pollen-feeding Heliconius species (Gilbert 1972;Karlsson 1994).

From our observations that protein sources arestrongly attractive to a variety of tropical butter¯ies, inparticular in the families Lycaenidae, Nymphalidae, andHesperiidae, one might deduce that these species are onaverage more long-lived than temperate-zone butter¯ies,and multiple matings may be more common. Unfortu-nately, data on longevity and mating frequency undernatural conditions are too scant for most tropical but-ter¯ies to empirically examine this idea. In addition,protein-feeding could be more widespread among tem-perate-zone butter¯ies than has so far been documented.In the experimental studies on papilionids and pierids,for example, no protein resources were tested (Armset al. 1974; Adler and Pearson 1982). However, inlaboratory experiments with one European lycaenidbutter¯y (Polyommatus icarus), albumin was largelyrejected, while sodium was strongly attractive (M. vonKor�, U. Hammon and K. Fiedler, unpublished obser-vations). Furthermore, attraction to potentially protein-rich adult resources is much more conspicuous amongtropical butter¯ies (at carrion, dung, bird droppings),and in moths, the specialized habits of tear-drinking orblood-sucking are virtually restricted to the tropics(BaÈ nziger 1973, 1988, and references therein). Hence, ahigher need for replenishment of protein reserves mightbe generally more pronounced in tropical regions. Un-fortunately, no chemical data are yet available on thesubstances which are really present at natural puddles,and the presence and concentrations of sodium have notbeen established. Although sodium may often be a rareresource in outwashed tropical soils (e.g., Ross and

Dykes 1996), nitrogen is limiting in many ecologicalsettings, and both resources should commonly co-occurat carrion or animal excrement.

Irrespective of the chemical nature of the puddlingstimulus (protein or sodium), butter¯ies require mecha-nisms to locate such resources which are usually rare andpatchily distributed. We have shown that the oftenbrightly colored butter¯ies (at least as seen by the hu-man observer; cf. Endler 1990) from the families Pier-idae and Papilionidae used the presence and number ofother conspicuous butter¯ies as cues to locate puddlingsites, but did not respond to lycaenid dummies. Themostly cryptic Lycaenidae (at least when in naturalresting position), in contrast, did not respond to any ofthe decoys o�ered. These ®ndings suggest that visualorientation is of paramount importance for papilionidsand pierids, which may use a generalized search imagewhen looking for puddling sites. Decoys were particu-larly e�ective if o�ered in groups, and small lycaenidsobviously did not ®t into the search image of Pieridaeand Papilionidae species present at the study sites. Suchsearching behavior would explain the common obser-vation of papilionids and pierids congregated at puddles.Congregations will further enhance the attractiveness ofa site to newly arriving butter¯ies. Visual orientation forlocating puddling sites should be especially advanta-geous when searching for resources which are barelydetectable by chemoreceptors from a distance (such assodium). The lycaenids and nymphalids, in contrast, didnot respond to the visual stimuli o�ered in our experi-ments and appeared to rely mainly on olfactory cueswhich emanate from decaying protein resources.

Together, the two contrasting ways of locating pud-dling resources plus the di�erences between butter¯yfamilies in resource preferences explain well the proxi-mate mechanisms for butter¯y grouping at puddlingsites. However, other causal factors may also be in-volved. For example, in large aggregations at mudpuddles, the predation risk for each individual butter¯ymight be reduced (Hamilton 1971).

Our experimental study of puddling behavior in twodiverse tropical butter¯y communities shows that, be-sides sodium, nitrogen sources are important. The sig-ni®cance of proteins in puddling behavior has so farbeen overlooked, probably due to a geographical as wellas taxonomic bias of previous studies on this sexuallyselected behavior. For selected species, what e�ects theuptake of nitrogen from puddles may have on ®tness-related variables such as lifespan, fecundity, and at-tractiveness to mates need to be tested. Furthermore, itwill be interesting to see whether tropical mud-puddlingbutter¯y communities consistently di�er from those sofar studied in temperate zones. As already formulated byBoggs and Jackson (1991), mud-puddling by butter¯iesis certainly not a simple matter.

Acknowledgements We thank the directors of Sabah Parks, DatukLamri Ali and Francis Liew, for granting permission to work atMt. Kinabalu Park, and the overall very helpful sta� (especially

146

Justinah Francis) for their kind support and hospitality. We alsoappreciate critical comments on the manuscript from Carol Boggs,Hans BaÈ nziger, and an anonymous reviewer. Thanks are furtherdue to Christian Schulze for manifold suggestions, to K. EduardLinsenmair for logistic support, and to Silke Doerk for help duringthe ®eld work in 1996. Supported by grants from the DeutscheForschungsgemeinschaft.

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Hypolycaena amasa XHypolycaena erylus XIonolyce helicon X XJamides celeno XMegisba malaya XMonodontides musina XNacaduba berenice XNacaduba beroe XNacaduba subperusia XPetrelaea dana XProsotas aluta XProsotas bhutea X XProsotas dubiosa X XProsotas gracilis X XProsotas nora X XProsotas pia X XUdara dilecta X XUdara dilectissima XUna usta X

HesperiidaeOdontoptilum pygela X XPithauria marsena X XPotanthus omaha XSeseria a�nis X

Species total 46 54Diversity(Williams' a � 95%con®dence interval)

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