Oecologia (1999) 119:140148

Springer-Verlag 1999

hlenberg Konrad Fiedler Jan Beck Eva Mu

Mud-puddling behavior in tropical butteries: in search of proteins or minerals?

Received: 5 October 1998 / Accepted: 7 December 1998

Abstract We experimentally investigated the attraction of adult butteries to moist soil and dirt places (a behavior termed `mud-puddling') in two species-rich tropical communities on the island of Borneo. At a rain forest site, 227 individuals (46 species) were attracted to the baits, compared to 534 individuals (54 species) at a farmland site. With one single exception, all attracted butteries were males. Of various salt and amino acid solutions, only sodium was accepted, but overall, albumin solutions turned out to be the most attractive puddling resource. Buttery families diered consistently in their resource preferences. Representatives of the families Papilionidae and Pieridae more often visited NaCl solutions, but still accepted albumin, whereas representatives of the Nymphalidae, Hesperiidae and, in particular, Lycaenidae preferred the protein resource. In experiments using decoys prepared from pinned buttery specimens, representatives of the Papilionidae and Pieridae were more strongly attracted to baits provided with decoys made from conspicuous, medium-sized yellow Eurema species (Pieridae), whereas dummies made from small, cryptically colored lycaenids (Prosotas and Caleta species) were ineective. Decoys did not inuence the attraction of lycaenid butteries towards baits. Hence, visual cues play an important role in locating puddling resources for papilionids and pierids, while for lycaenid butteries searching for nitrogen sources, olfactory cues emitted by decaying organic matter are more likely to be important. The strong attraction of male butteries to nitrogen-rich resources suggests that, as in the case of sodium, these nutrients may increase reproductive success. Key words Tropical butteries Protein food Sodium Reproductive biology Visual cues
J. Beck E. Mu hlenberg K. Fiedler (8) Lehrstuhl Tiero kologie I, Universita t Bayreuth, D-95440 Bayreuth, Germany e-mail: konrad.edler@uni-bayreuth.de

Introduction
Adult Lepidoptera (butteries and moths) of many species frequently visit moist ground, perspiration, tears, excrements, or animal carcasses to suck water and dissolved nutrients, a behavior conventionally termed `mud-puddling' (e.g., Arms et al. 1974; Adler 1982; Adler and Pearson 1982). Although this behavior is known from temperate-zone as well as tropical habitats, it is far more common in tropical regions. Large numbers of individuals may congregate at puddles, often neatly arrayed in groups of conspecics or of butteries of similar external appearance (e.g. Larsen 1991). Mudpuddling is thought to serve as a means of acquiring essential adult resources, particularly sodium. In most lepidopterans, the males pass complex spermatophores to their mates (Drummond 1984) which are used to transfer, besides sperm, various types of nuptial gifts. These gifts may consist of minerals such as sodium (Pivnick and McNeil 1987; Smedley and Eisner 1995, 1996) or calcium phosphate (Lai-Fook 1991), they may contain toxic secondary plant metabolites (Schneider 1992, 1993; Eisner and Meinwald 1995), or they contribute nutrients such as amino acids (Arms et al. 1974; Boggs and Gilbert 1979). Due to the low sodium content of land plants, many terrestrial herbivores (including humans; e.g., Haas and Harrison 1977) are expected to crave sodium. Among the Lepidoptera with their herbivorous caterpillar stages, mineral reserves assembled during the larval phase may often be limiting, and there should be strong selection for strategies to replenish mineral stocks. Toxic plant metabolites which serve as pheromone precursors and nuptial gifts by male butteries are collected through a special, sexually selected behavioral repertoire (`pharmacophagy': e.g. Schneider 1992, 1993). Mudpuddling might be seen as the analogous case with regard to minerals. This idea is supported by the fact that most individuals engaged in puddling behavior are males (e.g., Adler 1982).

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Occasionally, however, female butteries visit mud puddles (e.g., in montane North America: Boggs and Jackson 1991; Sculley and Boggs 1996). In the latter two studies, the propensity for puddling behavior diered consistently between sexes and varied with age. Hence, the mating system of a given species, the competitive abilities and current nutrient status of the individuals involved, and actual environmental conditions all aect the choice of resources available at puddling sites. Puddling behavior has probably evolved from the drinking of water (Adler 1982), and in dry habitats the need for water may be a prime reason for visiting puddles (e.g., Larsen 1991). In the Californian checkerspot buttery Euphydryas editha bayensis, puddling only occurred in years of extreme drought (Launer et al. 1996). In the dry steppe biomes of Turkey, huge numbers of blue butteries (Lycaenidae, mostly Polyommatini) regularly aggregate at puddles (Hesselbarth et al. 1995; K. Fiedler, unpublished observations), whereas the same, or very closely related, species visit such resources much less often in the cooler and more humid climate of central Europe (K. Fiedler, unpublished observations). Nevertheless, even under arid conditions, the clear majority of butteries attracted to puddles are males. In perhumid tropical rainforests, where puddling is common, the need for water as a primary motivation for puddling may safely be neglected except in El Nin o years. In addition, many puddling butteries and moths eject surplus water (Ba nziger 1973; Adler 1982), conrming that substances dissolved in water, rather than water itself, are the principal resource accumulated through puddling. To our knowledge, all experimental data supporting the hypothesis that searching for sodium is the prime reason for mud-puddling in the Lepidoptera come from temperate regions where mud-puddling is less pronounced than in the tropics. Furthermore, only in few studies and with a small number of species have buettype experiments been conducted oering simultaneously sodium and a variety of other, potentially puddling-related substances (e.g., Arms et al. 1974; Adler and Pearson 1982). In none of these studies was a protein included as a possible puddling stimulus, although Ba nziger (1973, 1988), for example, has hinted at the potential role of proteins in the ecology of lachryphagous moths. Hence, current conceptions of the evolutionary ecology of mud-puddling in butteries might be seriously biased with regard to geographic region, 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 potential as puddling stimuli in two Oriental tropical buttery communities under natural eld conditions. We also present data on how butteries may locate suitable puddling sites. In particular, we address the question whether buttery decoys at a puddling site increase the attractiveness of such resources, which are certainly rare and patchily distributed in a rainforest environment.

Materials and methods

Field methods Field work was done in 2 consecutive years, October to November 1996 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 protected rainforest with a rich and rather well studied buttery fauna (Ha user et al. 1997). The study site was situated near Poring Hot Springs at the southeastern margin of the park (634 N, 11633 E, elevation 500 m a.s.l.). While inside the park the vegetation is dominated by mixed hill dipterocarp forest (Kitayama 1992), outside the park limits, agricultural areas cultivated by local farmers and wasteland vegetation predominate. Two dierent sites were chosen for their relatively high abundance of puddling butteries. One was situated on the banks of the Mamut River in a slightly disturbed, but still tall closed dipterocarp forest; the other site was along a small river outside the park, surrounded by cultivated farmland. The rst set of experiments, conducted at the Mamut River in 1996, was designed to determine which substances are most attractive to various species in the focal buttery community. Nine dierent solutions and deionized water as a control were presented to the butteries in petri dishes (diameter 9 cm) lined with white lter paper. The following substances (all dissolved in deionized water, 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 albumin, Roth, Germany in 1997). If necessary, due to strong evaporation on hot sunny days, test solutions were replenished to minimize variation in the nutrient concentrations oered. The ten petri dishes (nine resources plus water) were arranged immediately adjacent to each other in a square on the sand of a sunlit riverbank during peak hours of buttery activity, from 0900 to around 1600 hours. Relative positions of the dishes were changed regularly. Four replicate sets were oered simultaneously, spaced well from each other (minimum distance between two sets 10 m). The behavior of butteries was observed with binoculars (10 42) from a distance of about 7 m to minimize disturbance. Butteries were considered sucking at the bait if they were actually seen drinking (easy to see in large Papilionidae) or if they sat in a petri dish for at least 1 min. In total, the bait sets were observed for 154 h on 35 days. Large-bodied butteries (families Papilionidae, Pieridae, Nymphalidae) accepting a bait were caught (36 of 47 individuals), identied (after Corbet and Pendlebury 1992), marked (if released, to avoid pseudoreplicates) and released. Representatives of taxonomically dicult groups like blues (Lycaenidae) and skippers (Hesperiidae) were caught (114 out of 180 individuals), marked and released, or killed and later identied (Seki et al. 1991). Butteries that could not be determined by sight and resisted capture could always be identied to subfamily level at least. The very low recapture rate of marked animals (7 out of 71) indicated that pseudoreplication among the unmarked butteries was negligible. Determination of sex was possible in 153 out of 227 specimens (either using the preserved vouchers, or on the basis of conspicuous sex dimorphism in wing pattern). The second set of experiments, mainly aimed at assessing the eect of buttery dummies on the attractiveness of resources for other free-ying butteries, was conducted in 1997 at a natural puddling site along a small creek in cultivated landscape. For these experiments, only albumin and NaCl (the two substances which had turned out to be most attractive in 1996) were oered, at the same concentrations as in the previous year. Resource solutions just covered the bottom of each petri dish and were replenished if needed (on 4 of 14 observation days, when eld work exceeded

142 4 h). No lter paper was used, in order to minimize visual attraction of butteries to the resources. Dummies were prepared from specimens of buttery species which were most commonly observed at puddles in the study region. As a representative of the larger, conspicuously colored butteries, we used male specimens of Eurema sari and Eurema simulatrix (Pieridae). At many natural puddling sites, members of the small-bodied Lycaenidae were most abundant. Two dierent sets of lycaenid dummies were tested. Male Prosotas spp. (P. pia and P. nora) served as representatives for the majority of more cryptically colored lycaenids, whereas the black-and-white-marked Caleta elna and C. manovus were used as examples of more conspicuous lycaenids. As dummies, we took eld-caught butteries (only fresh, intact specimens) which were killed, mounted on insect pins with the wings dorsally folded, and dried. Care was taken that the pinned dummies closely resembled the natural posture of puddling individuals. For experiments, either a single dummy or a group of ve was placed at a petri dish as decoy. Petridishes with no decoy served as controls. Hence, six combinations of puddling substances (albumin, NaCl) and decoys (none, one, ve) formed one test set (distance between neighboring petri dishes 30 cm). Every 30 min, the relative positions of the dishes and dummies were changed to minimize positional bias. Two sets of six test dishes were oered simultaneously, spaced 3 m apart from each other. One set contained Eurema dummies over the entire duration of the experiment. In the other set, we tested lycaenid dummies (Prosotas in the rst half of the experiment, Caleta in the second). Between subsequent days, the positions of the two sets were also exchanged to rule out position eects. From a distance of about 5 m, all butteries which settled down at the resources were recorded. Only visits at which the butteries remained at the resources for more than 30 s and drinking behavior could be positively conrmed (uncoiling of the proboscis, observations through binoculars) were included. Total observation time amounted to 53 h distributed over 14 days. As in the rst experiment, insects were identied by sight whenever possible (using Otsuka 1988). Vouchers of taxonomically dicult groups (102 of 534 individuals altogether, especially Lycaenidae) were caught and preserved for later identication. Statistical analysis From our eld experiments, we obtained data on the acceptance of the various resources with or without decoys. For the 1996 experiments, these frequency data were tabulated in a two-way contingency table (with buttery family and resource type as factors) and subjected to standard v2 statistics (Sachs 1992). Observed frequencies of acceptance of a resource versus water were tested against the null hypothesis of equal distribution. Evaluation was restricted to the more commonly used resources [NaCl and albumin; also Ca2(PO4)3 and urea for Lycaenidae]. To control for spurious signicance results arising from multiple comparisons, we applied the sequential Bonferroni approach (Rice 1989) within each set of hypotheses to be tested. Signicant results passing this criterion are marked with an asterisk. The 1997 data set had a more complex structure, since four factors were involved (buttery family, resource type, decoy type, and decoy number). This data set was evaluated using the log-linear analysis modul of StatSoft (1995). First, a log-linear model was constructed using those eects that signicantly contributed to variation as measured by signicant partial v2 values, and the t of this `optimized' model to the observed data was checked. Second, each of the included interaction eects was separately tested, by removing it from the model and analyzing the dierence in the goodness of t between the simplied and optimized model (measured as dierence of the v2 values) for signicance. Finally, the signicant eects were localized by standard v2 statistics in the reduced cross-tabulated two-way frequency tables. We performed all analyses primarily on the level of buttery families (i.e., Papilionidae, Pieridae, Nymphalidae, Lycaenidae and Hesperiidae). There are three reasons for this. First, most individual species in this tropical buttery community were so scarce that during our experimental period only small sample sizes were attained per species. Second, many of the more abundant lycaenid species (e.g. in the genus Prosotas) are impossible to identify to species level without capturing (and thus disturbing) the animals. Third, butteries from various species in the same family have often been observed to aggregate at puddles (Larsen 1991). Therefore it seems safe to assume for a rst approach that members of the same buttery family, if they visit mud puddles at all, have common resource requirements, while species-specic dierences are less pronounced. Where our samples were large enough, we tested the validity of this assumption on lower taxonomic levels (genera, species).

Results
Puddling preferences in a forest buttery community A total of 227 observations of drinking butteries representing at least 46 dierent species (see Appendix) were made at the baits in 1996 in a forest habitat (Table 1). Among 153 specimens where sex could be determined, all were males except 1 single female Charaxes bernardus (Nymphalidae). The ve buttery families were represented at the baits in greatly dierent numbers of individuals. The bulk of observations (76.2%) came from blues (Lycaenidae, mostly tribe Polyommatini), followed by Nymphalidae (13.2%), Papilionidae (6.6%), Hesperiidae (3.2%), and Pieridae (0.4%). Albumin and NaCl had by far the highest rates of acceptance. CaCl2 was never accepted, and the averse behavior of the butteries occasionally contacting such

Table 1 Acceptance of all tested puddling substances across buttery families (data from Mamut River, Borneo, 1996) All butteries Albumin NaCl Ca2(PO4)3 Glycine Lysine MgCl2 KCl Urea CaCl2 Water Total 107 72 14 12 7 3 3 9 0 4 227 Lycaenidae 73 66 11 5 4 2 1 8 0 3 173 Nymphalidae 17 2 1 2 3 1 3 1 0 0 30 Papilionidae 7 3 2 3 0 0 0 0 0 0 15 Pieridae 1 0 0 0 0 0 0 0 0 1 2 Hesperiidae 6 1 0 0 0 0 0 0 0 0 7

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baits indicated that this salt solution even had repellent properties. Lycaenidae clearly preferred albumin and NaCl over 2 pure water (v2 1 = 64.5, v1 = 57.5; both P < 0.0001*), while phosphate and urea were not accepted signicantly more often than water (v2 1 = 4.57, P = 0.038 for phosphate; v2 1 = 2.27, P = 0.132 for urea; both not signicant after sequential Bonferroni correction for ve tests within Lycaenidae). All other substances, including the amino acids, were only rarely visited. If all lycaenid species (>23) are pooled, there was no signicant difference in attractiveness between albumin and NaCl (v2 1 = 0.35, P = 0.55). For the few species or genera which were represented by larger numbers of individuals, 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 preferred over pure water (v2 1 = 17.0, P < 0.0001*) as well as over NaCl (v2 1 = 11.8, P < 0.001*; sequential Bonferroni correction for two tests within Nymphalidae). The 30 puddling visits were distributed over at least 12 species. Vindula dejone (ve visits) was the commonest nymphalid species and only occurred at albumin baits. In the Papilionidae, the only statistically signicant preference was again observed for albumin over water (v2 1 = 7.0, P < 0.01), and Papilio memnon, the commonest species (ve visits), exclusively utilized albumin. Otherwise, so few individuals appeared at the baits that no detailed analyses could be performed. Under tropical, warm and humid conditions, the albumin solution rapidly started decaying and then emitted a strong smell. An apparent preference for albumin might also emerge if such smells serve as cues to locate resources rich in sodium (e.g., animal carcasses). We

tested whether a smelly albumin solution (0.16 g/ml H2O) combined with 0.1 M NaCl would attract more butteries than albumin alone. Four replicates of a set of two petri dishes each (one with, one without NaCl added) were exposed at the same test site. No signicant dierence was found in the acceptance of pure albumin (18 visits) and the NaCl-enriched albumin solution (12 visits; v2 1 = 1.2, P = 0.273). The results of our experiments along Mamut River in 1996 strongly indicated that, besides sodium, proteins like albumin are an important adult resource sought by tropical butteries engaged in puddling behavior. For no family did any of the mineral resources or amino acids attract more individual butteries than the albumin bait, and only lycaenids showed up in roughly equal numbers at protein and sodium sources. Attraction of butteries to sodium and albumin During the 1997 experiments in a deforested tropical habitat, 534 butteries representing 54 species (see Appendix) accepted the experimental puddling resources (Table 2). Of 102 voucher specimens all were males. Representatives of the Lycaenidae again accounted for the majority of observations (75.5%), followed by Papilionidae and Pieridae (8.1% each), Nymphalidae (5.2%) and Hesperiidae (3.2%). Abundance relations deviated from the experimental series the year before (v2 4 = 18.82, P < 0.0009), but this was largely due to a prominent dierence in one family, the Pieridae (43 vs 2 specimens observed). Overall, considerably more butteries (10.08 individuals/h) were seen at the non-forest study site in 1997 than in 1996 within the forest (1.47 individuals/h).

Table 2 Acceptance of articial puddling resources (NaCl or albumin solutions) in experiments with dierent numbers (0, 1, 5) of Eurema (Pieridae), Prosotas and Caleta (both Lycaenidae) decoys NaCl 0 Eurema dummies Pieridae Papilionidae Lycaenidae Nymphalidae Hesperiidae Prosotas dummies Pieridae Papilionidae Lycaenidae Nymphalidae Hesperiidae Caleta dummies Pieridae Papilionidae Lycaenidae Nymphalidae Hesperiidae Column totals 0 0 7 2 0 1 0 4 1 1 1 0 0 0 0 17 1 2 0 6 0 1 0 0 3 0 0 0 0 4 0 0 16 5 22 25 4 2 0 0 1 4 0 0 1 0 3 2 1 65 Albumin 0 0 0 62 3 4 1 1 22 1 1 0 1 41 1 1 139 1 3 2 57 3 6 0 0 31 2 0 0 1 45 3 1 154 5 12 8 58 4 1 0 0 16 2 0 0 4 36 2 0 143 39 35 194 14 12 2 2 80 6 2 2 6 129 8 3 534 Row totals

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Log-linear analysis revealed that frequencies of butteries accepting the baits diered signicantly between families, resource types, decoy types, and decoy numbers (Table 3). In addition, only two-way interactions between buttery family and the design variables (resource, decoy type, and decoy number) were signicant, while all other two-way and all three-way interactions were not. The log-linear model including only the signicant two-way interactions and the three-way interaction between the design variables closely tted the observed frequencies (v2 48 = 48.8, P = 0.441), and comparisons of models omitting one of the above twoway interactions with this optimized log-linear model revealed that all these interactions were statistically signicant (family resource: v2 4 = 81.9, P < 0.0001; family decoy type: v2 = 16.223, P < 0.05; fami8 = 44.7, P < 0.0001). ly decoy number: v2 8 A closer analysis of the cross-tabulation of the three signicant two-way interactions (Table 4) shows that in the 1997 data set, lycaenids, nymphalids and hesperiids clearly preferred albumin over NaCl, while about twice as many papilionids and pierids were attracted to NaCl resources as compared to albumin (v2-tests for homogeneity between resources: Lycaenidae: v2 1 = 275.2, P < 0.000001*; Nymphalidae: v2 1 = 7.00, P = 0.0082*; Hesperiidae: v2 1 = 7.12, P = 0.0076*; Papilionidae: 2 v2 1 = 1.88, P = 0.17; Pieridae: v1 = 2.81, P = 0.093; sequential Bonferroni correction for ve tests).

Table 3 Partial associations between the variables buttery family (Papilionidae, Pieridae, Nymphalidae, Lycaenidae, and Hesperiidae), resource type (albumin or NaCl solution), decoy type (dummies made of pinned Eurema, Prosotas, or Caleta butteries), and decoy number (none, one, ve) in the 1997 data set Eect df v2 717.2 210.4 109.6 7.38 83.5 17.9 46.4 4.36 4.24 6.94 9.99 5.02 23.85 2.74 P <0.00001 <0.00001 <0.00001 0.025 <0.00001 0.022 <0.00001 0.113 0.120 0.139 0.266 0.755 0.09 0.603

Family 4 Resource 1 Decoy type 2 Decoy number 2 Family resource 4 Family decoy type 8 Family decoy number 8 Resource decoy type 2 Resource decoy number 2 Decoy type decoy number 4 Family resource decoy type 8 Family resource decoy number 8 Family decoy type decoy number 16 Resource decoy type decoy number 4

P < 0.000001*), with the larger group of ve decoys being most attractive (Table 4). In contrast, the number of decoys did not inuence visitation frequencies in any of the other three families (Lycaenidae: v2 2 = 2.36, P = 0.308; Nymphalidae: v2 = 1.14, P =0.565; Hes2 = 3.65, P = 0.162). periidae: v2 2

Discussion
Eects of decoys on puddling butteries Eurema decoys strongly increased the attractiveness of resources to papilionids (v2 2 = 45.3, P < 0.000001*) as well as pierids (v2 2 = 63.8, P < 0.000001*; sequential Bonferroni correction for ve tests) over resources equipped with lycaenid dummies or without decoy at all (Table 4). For the remaining three families, the presence of decoys had no noticeable eect on the acceptance of articial puddling resources (Lycaenidae: v2 P = 0.575; Nymphalidae: v2 2 = 0.50, 2 = 1.11, P = 0.779; Hesperiidae: v2 2 = 3.65, P = 0.162). Similarly, decoy number had a strong eect on papilionids 2 (v2 2 = 58.7, P < 0.000001*) and pierids (v2 = 44.9, In most studies on puddling behavior of butteries and moths, the need for sodium has been emphasized as the main causal factor (Arms et al. 1974; Smedley and Eisner 1995, 1996). The results presented above show clearly that in the two tropical buttery communities under investigation, proteins like albumin were also in demand. Although we have no chemical data on potential sodium `contamination' of the albumin test chemicals, even the presence of substantial amounts of sodium in the resources could not explain the consistent dierences which we observed between buttery families. The butteries accepted none of the other minerals or nitrogen sources (urea or two amino acids) with the possible exception of phosphate (cf. Lai-Fook 1991),

Table 4 Cross-tabulation of the three signicant two-way interactions as revealed by log-linear modelling of the data presented in Table 2

Papilionidae Buttery family resource NaCl Albumin Family decoy type Eurema All lycaenids No decoy Family decoy number None One Five 26 17 35 6 2 2 3 38

Pieridae 27 16 39 1 3 3 5 35

Nymphalidae 7 21 9 11 8 8 8 12

Lycaenidae 35 385 125 142 136 136 146 121

Hesperiidae 3 14 8 2 7 7 8 2

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which was occasionally taken up by lycaenids. Hence, at our study sites, mud-puddling served the twofold function of providing sodium as well as protein resources, and these nutrients were almost exclusively gathered by males. We observed signicant dierences between buttery families in their response to puddling sources. Nymphalids and hesperiids were primarily attracted to albumin, whereas papilionids and pierids visited sodium resources more frequently, though representatives of the latter two families by no means ignored protein baits. These observations agree well with earlier ndings. The most detailed experimental studies on sodium as a prime stimulus for puddling behavior in butteries were conducted with one temperate-zone papilionid (Arms et al. 1974) and one pierid (Adler and Pearson 1982). On the other hand, many tropical nymphalids and hesperiids are particularly attracted to carrion or feces (e.g., carnivore excrement, bird droppings: Larsen 1991; Austin and Riley 1995; Sourakov and Emmel 1995). Lycaenids were the most abundant visitors to our articial puddling sites. Albumin was an important resource throughout, but while in the 1996 experiments, roughly equal numbers of lycaenids were attracted to albumin and NaCl, the albumin baits were strongly preferred in the 1997 trials. We have no unequivocal explanation for this discrepancy. The two buttery communities diered considerably in species composition (So rensen index of similarity: 0.50; see Appendix). Within the Lycaenidae, representatives of the Nacaduba section (sensu Hirowatari 1992: genus Nacaduba) and 1983: Lycaenopsis section (sensu Eliot and Kawazoe genera Actyolepis, Celastrina, Megisba, Monodontides, Udara) were only, or more strongly, represented at the Mamut River site, and butteries in the Lycaenopsis section were the only lycaenids which visited NaCl more often than albumin (13 vs 5 cases). However, in both years, the same lycaenid species made up the majority of the visitors at puddling sites (i.e., Caleta, Prosotas, Ionolyce, Anthene spp.). These same lycaenid species visited sodium and albumin in roughly equal frequencies in 1996, but clearly preferred albumin in 1997. Albumin sources used for baiting diered between the years (chicken egg albumin resulting in a rather colorless solution in 1996, bovine serum albumin in 1997 giving a dark solution). So, either characteristics of the baits (dierences in chemical or visual properties of the two albumin types) or environmental factors (e.g., resource availability; see also Launer et al. 1996) rather than species composition were more likely causes for the between-year variation. Dierent species within the same taxonomic entity (such as family or genus) may have dierent resource requirements with regard to mud-puddling, e.g., depending on the mating system of each species (Karlsson 1995, 1996; Sculley and Boggs 1996). Our experimental data as well as eld observations, however, indicate that in the Bornean buttery communities studied here, puddling preferences are quite consistent within taxo-

nomic family boundaries. All common lycaenid genera (Caleta, Prosotas, Ionolyce, Anthene) showed the same strong attraction to albumin as the lycaenids as a whole. Often ve to ten species of these genera co-occur at the same natural puddles. Similarly, the albumin preference of nymphalids was mirrored at lower taxonomic levels (Charaxes, Polyura, Vindula, and Phalanta almost exclusively appeared at albumin baits), as was the slight sodium preference of papilionids (Graphium and Pathysa preferred NaCl). As one counter example, birdwing butteries (Troides brookiana) often occurred at natural puddling sites along the Mamut River, but never visited the baits, whereas other papilionids were attracted. Hence, our analyses at the family level as a rst approach are consistent with observations on lower taxonomic levels as well as eld experience and thus likely reveal real patterns, but more detailed studies at the species level would be worthwhile. Sodium gathered by male butteries and moths during mud-puddling is transferred, sometimes in large amounts, to their female mates through the spermatophore, and this nuptial gift increases reproductive success (e.g., Adler and Pearson 1982; Pivnick and McNeil 1987; Smedley and Eisner 1996). Similarly, proteins obtained by male butteries could well increase their reproductive success. The striking predominance of males at protein sources strongly suggests that as with sodium collecting this behavior has evolved through sexual selection. The studies of Karlsson (1994, 1995, 1996) and Karlsson and Wickman (1989) on a variety of temperate-zone buttery species suggest that nitrogen allocation is a critical factor in their reproductive biology (Boggs 1981), and that among long-lived and/or polyandrous species in particular, there should be strong selection for mechanisms to improve nitrogen budgets. According to Ba nziger (1973, 1990), only certain teardrinking adult moths have so far been shown to possess protease activity in their midgut, but the capacity to digest proteins might be more widespread in lepidopterans which routinely utilize a protein-rich adult diet. However, even if butteries attracted to protein baits would turn out to lack any protease, they could still benet from these nitrogen resources. Under humid tropical conditions in particular, any protein source (such as carrion or excrement) will be quickly decomposed by bacteria and fungi, making available a variety of smaller nitrogen-rich molecules like amino acids or oligopeptides. Amino acids from adult food are incorporated by various butteries (Wheeler 1996). Male North American Papilio glaucus imbibe amino acids by chance when at mud puddles and subsequently transmit them through the spermatophore to their mates (Arms et al. 1974). Neotropical Heliconius derives amino acids from pollen, and pollen-feeding is essential to increase longevity and reproduction in these outstandingly long lived butteries (Gilbert 1972; Dunlap-Pianka et al. 1977). Flower nectar can be enriched by amino acids dissolved from pollen that fell into the ower (Erhardt and Baker 1990), increasing

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the availability of amino acids for nectar-feeding butteries. However, evidence for the role of amino acids in the nutrition of nectar-feeding butteries is discordant. While some species preferentially drink nectars enriched with amino acids (Pieris rapae: Alm et al. 1989), others do not (Battus philenor, Ornithoptera priamus: Erhardt 1991, 1992). In various species, no positive eect of nectar amino acids on longevity or fecundity was found (e.g., Hill 1989; Hill and Pierce 1989), whereas in others, amino acids were benecial (Murphy et al. 1983). Interestingly, neither in earlier experiments (Arms et al. 1974) nor in our experiments in Borneo were amino acid solutions (glycine, lysine) attractive to butteries. With regard to the use and importance of adult resources, lepidopterans occupy a continuum between two extremes (Dunlap-Pianka et al. 1977; Boggs 1981, 1986; Karlsson 1994; Wheeler 1996). While some species (in particular many moths) utilize exclusively (or at least predominantly) resources stored throughout larval development for somatic maintenance and reproduction, others supplement their nitrogen budget by adult feeding or through multiple mating. The rst strategy typically coincides with a short adult life span and a short, but intense period of reproduction, while the second strategy selects for prolonged life and more even, life-long egg production. An extreme example for the latter strategy are the pollen-feeding Heliconius species (Gilbert 1972; Karlsson 1994). From our observations that protein sources are strongly attractive to a variety of tropical butteries, in particular in the families Lycaenidae, Nymphalidae, and Hesperiidae, one might deduce that these species are on average more long-lived than temperate-zone butteries, and multiple matings may be more common. Unfortunately, data on longevity and mating frequency under natural conditions are too scant for most tropical butteries to empirically examine this idea. In addition, protein-feeding could be more widespread among temperate-zone butteries than has so far been documented. In the experimental studies on papilionids and pierids, for example, no protein resources were tested (Arms et al. 1974; Adler and Pearson 1982). However, in laboratory experiments with one European lycaenid buttery (Polyommatus icarus), albumin was largely rejected, while sodium was strongly attractive (M. von Kor, U. Hammon and K. Fiedler, unpublished observations). Furthermore, attraction to potentially proteinrich adult resources is much more conspicuous among tropical butteries (at carrion, dung, bird droppings), and in moths, the specialized habits of tear-drinking or blood-sucking are virtually restricted to the tropics (Ba nziger 1973, 1988, and references therein). Hence, a higher need for replenishment of protein reserves might be generally more pronounced in tropical regions. Unfortunately, no chemical data are yet available on the substances which are really present at natural puddles, and the presence and concentrations of sodium have not been established. Although sodium may often be a rare resource in outwashed tropical soils (e.g., Ross and

Dykes 1996), nitrogen is limiting in many ecological settings, and both resources should commonly co-occur at carrion or animal excrement. Irrespective of the chemical nature of the puddling stimulus (protein or sodium), butteries require mechanisms to locate such resources which are usually rare and patchily distributed. We have shown that the often brightly colored butteries (at least as seen by the human observer; cf. Endler 1990) from the families Pieridae and Papilionidae used the presence and number of other conspicuous butteries as cues to locate puddling sites, but did not respond to lycaenid dummies. The mostly cryptic Lycaenidae (at least when in natural resting position), in contrast, did not respond to any of the decoys oered. These ndings suggest that visual orientation is of paramount importance for papilionids and pierids, which may use a generalized search image when looking for puddling sites. Decoys were particularly eective if oered in groups, and small lycaenids obviously did not t into the search image of Pieridae and Papilionidae species present at the study sites. Such searching behavior would explain the common observation of papilionids and pierids congregated at puddles. Congregations will further enhance the attractiveness of a site to newly arriving butteries. Visual orientation for locating puddling sites should be especially advantageous when searching for resources which are barely detectable by chemoreceptors from a distance (such as sodium). The lycaenids and nymphalids, in contrast, did not respond to the visual stimuli oered in our experiments and appeared to rely mainly on olfactory cues which emanate from decaying protein resources. Together, the two contrasting ways of locating puddling resources plus the dierences between buttery families in resource preferences explain well the proximate mechanisms for buttery grouping at puddling sites. However, other causal factors may also be involved. For example, in large aggregations at mud puddles, the predation risk for each individual buttery might be reduced (Hamilton 1971). Our experimental study of puddling behavior in two diverse tropical buttery communities shows that, besides sodium, nitrogen sources are important. The signicance of proteins in puddling behavior has so far been overlooked, probably due to a geographical as well as taxonomic bias of previous studies on this sexually selected behavior. For selected species, what eects the uptake of nitrogen from puddles may have on tnessrelated variables such as lifespan, fecundity, and attractiveness to mates need to be tested. Furthermore, it will be interesting to see whether tropical mud-puddling buttery communities consistently dier from those so far studied in temperate zones. As already formulated by Boggs and Jackson (1991), mud-puddling by butteries is certainly not a simple matter.
Acknowledgements We thank the directors of Sabah Parks, Datuk Lamri Ali and Francis Liew, for granting permission to work at Mt. Kinabalu Park, and the overall very helpful sta (especially

147 Justinah Francis) for their kind support and hospitality. We also appreciate critical comments on the manuscript from Carol Boggs, Hans Ba nziger, and an anonymous reviewer. Thanks are further due to Christian Schulze for manifold suggestions, to K. Eduard Linsenmair for logistic support, and to Silke Doerk for help during the eld work in 1996. Supported by grants from the Deutsche Forschungsgemeinschaft. Appendix (contd.) 1996 Hypolycaena amasa Hypolycaena erylus Ionolyce helicon Jamides celeno Megisba malaya Monodontides musina Nacaduba berenice Nacaduba beroe Nacaduba subperusia Petrelaea dana Prosotas aluta Prosotas bhutea Prosotas dubiosa Prosotas gracilis Prosotas nora Prosotas pia Udara dilecta Udara dilectissima Una usta Hesperiidae Odontoptilum pygela Pithauria marsena Potanthus omaha Seseria anis Species total Diversity (Williams' a 95% condence interval) 1997 X X X X

X X X X X X X X X X X X X X X X X X 46 17.4 3.7

Appendix Buttery species observed at the articial puddling resources in the two study periods 1996 Papilionidae Graphium agamemnon Graphium bathycles Graphium doson Graphium eurypylus Graphium evemon Graphium procles Graphium sarpedon Lamproptera meges Papilio helenus Papilio memnon Pathysa agates Pathysa antiphates Pathysa delessertii Pieridae Appias panda Cepora iudith Cepora pactolicus Eurema cf. ada Eurema andersonii Eurema sari Eurema simulatrix Eurema sp. indet. Hebomoia glaucippe Ixias undatus Prioneris cornelia Nymphalidae Athyma asura Athyma larymna Athyma sp. indet. Euripus nyctelius Charaxes bernardus Chersonesia intermedia Chersonesia sp. indet. Cirrhochroa sp. indet. Cyrestis maenalis Cyrestis nivea Moduza procris Neptis clinia Paduca fasciata Phalanta alcippe Polyura athamas Rhinopalpa polynice Vindula dejone Vindula erota Lycaenidae Actyolepis puspa Anthene emolus Anthene lycaenina Caleta elna Caleta manovus Catopyrops ancyra Celastrina lavendularis Cheritra freja Discolampa ethion 1997 X X X X X X X X X X X X X X X X X X X X X X X

X X X X X X X X X X 54 15.0 2.3

X X X X

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