Ecology and mechanisms of parasitism of Iridomyrmex by the thermophilic ant, Melophorus anderseni

Ecology and mechanisms of parasitism of Iridomyrmex by the thermophilic ant, Melophorus anderseni | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ecology and mechanisms of parasitism of Iridomyrmex by the thermophilic ant, Melophorus anderseni Ben Hoffmann, Sara Hu, Adrian A. Smith, Andrew V. Suarez, Terry McGlynn This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7361241/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Melophorus anderseni is a thermophilic ant species that nests near colonies of Iridomyrmex on which it conducts brood raids. We examined their colony founding behavior and foraging ecology to evaluate possible mechanisms that may facilitate interspecific nest raiding. Specifically, wee 1) examined colony establishment and survival, 2) patterns of worker activity (walking speed, foraging and raiding behavior), and 3) compared cuticular hydrocarbon profiles of M. anderseni and I. reburrus workers. Following nuptial flights, M. anderseni queens established colonies in clusters around I. reburrus colonies, with an average distance to the nearest Iridomyrmex nest of 133.1(± 8.9) cm (range 25 to 650 cm). The average number of M. anderseni queens per Iridomyrmex nest entrance was 8.4 (± 1.9), and the average distance to the nearest M. anderseni neighbor was 44.9 (± 5.3) cm (range 10 to 233 cm). Queen survival was lower in clusters that were farther from the nearest I. reburrus nest or to other M. anderseni queens. Iridomyrmex reburrus activity was negatively related to soil temperature, with no activity occurring above 50°C. In contrast, M. anderseni activity was positively related to temperature with peak activity occurring just above 50°C. Melophorus anderseni worker activity commenced when soil temperatures exceeded 37.4°C, and activity increased until mid-day. Activity ceased in the afternoon at temperatures lower than that for activity commencement, on average at 36.7°C. Activity of M. anderseni at I. reburrus nests followed that of activity at M. anderseni nests. Workers of M. anderseni had an average foraging duration of 63 seconds and a mean foraging distance of 249 cm. Of the 49 foragers followed, 42 (86%) entered an I. reburrus nest, and 32 (76.2%) of those entered the closest nest entrance. Of the 1,444 observations of M. anderseni exiting an I. reburrus nest, they stole an item in 122 (8.4%) instances, with 117 (95.9%) of these items being brood. Workers of M. anderseni had faster running speeds (mean 9.72 cm/s, peak 18 cm/s) than I. reburrus workers (7.05 cm/s). On average, the speed of M. anderseni exiting an I. reburrus nest was more than twice the speed of I. reburrus. Extracts of I. reburrus and M. anderseni worker cuticular hydrocarbon profiles were qualitatively identical in that no compounds were unique to either species. Saturated hydrocarbons with chain lengths between 25 and 29 carbons comprised the majority of hydrocarbons found on the cuticle of both species. The relative abundances of compounds within the profiles of both species were also similar. Two other sympatric Iridomyrmex and Melophorus species shared very few compounds with I. reburrus and M. anderseni . Our results support that M. anderseni specializes on brood raiding from I. reburrus nests. Both behavior (running speed) and chemical mimicry are likely used in combination to facilitate this specialized foraging. Additional research is still needed to determine the source (e.g. genetic versus environmental) of M. anderseni’s hydrocarbon profiles, and if geographic variation in host use and chemical mimicry exists. chemical mimicry cuticular hydrocarbons Iridomyrmex reburrus nest raiding parasite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The ability to distinguish familiar individuals from strangers is essential for the evolution and maintenance of animal societies. Behavioral discrimination allows individuals to direct costly beneficial behaviors towards group members, who are often relatives, and exclude or defend against non-group members. Consequently, well-developed recognition systems are common in social and colonial organisms (Sherman et al. 1997 , Starks 2004 , Suarez et al. 2020 ). Ants have finely tuned recognition systems that are critical to social organization, division of labor, and territoriality (Stuart and Herbers 2000 , Sturgis and Gordon 2012 , Larsen et al. 2016 ). The ability to detect and exclude non-nestmates is essential for colony defense from competitors, predators and parasites. Colony and species-specific cuticular hydrocarbon profiles are used for nestmate recognition (Bonavita-Cougourdan et al. 1987 , Lahav et al. 1999 , Lucas et al. 2005 ), and components of these profiles can be employed by ant and non-ant social parasites to cloak their presence from hosts (Franks et al. 1990 , Lenoir et al. 1997 , Akino et al. 1999 , Lambardi et al. 2007 , Hojo et al. 2014 ). These cues can be biosynthesized directly by the social parasites and/or acquired from their hosts through physical contact (Lenoir et al. 2001 ). Many parasitic species have evolved strategies to take advantage of the resources and labor within colonies of eusocial insects (Lenoir et al. 2001 , D’Ettorre et al. 2002). Social parasites come from a wide variety of taxa and employ diverse mechanisms to circumvent recognition by host colonies (Buschinger 1986, D’Ettorre and Heinze 2001 , Als et al. 2004 , Buschinger 2009 , Rettenmeyer et al. 2010, Parker 2016 , Hölldobler and Kwapich 2022 ). These mechanisms include chemical mimicry of nestmate recognition cues (Lenoir et al. 2001 ), other forms of chemical signaling (e.g. not related to nest mate recognition; Lenoir et al. 2001 ), and morphological or behavioral mimicry (e.g. stridulation; Geiselhardt et al. 2007 , Barbero et al. 2009 , Di Giulio et al. 2015). Likewise, the duration that parasitic species associate with their host varies greatly. Many social parasites take up residence inside ant colonies for at least part of their life cycle, while others specialize on cleptobiosis (Breed et al. 2012 ). Workers of cleptobiotic species steal resources such as food, brood, nesting materials, or other items of value, either from members of the same species or a different species (Breed et al. 1990 , 2012 ). Raids are often accompanied by aggressive interactions between host and raiding workers, but in some species, the “thieves” utilize repulsive chemicals (Blum et al. 1980 ) or exhibit specialized behaviors that may serve to reduce the chance of getting caught (McGlynn et al. 2015 ). Melophorus anderseni is a thermophilic ant species that nests near colonies of Iridomyrmex sanguineus and I. reburrus on which it conducts brood raids (Agosti 1997 ). Agosti ( 1997 ) noted that M. anderseni workers were able to make their way through a guarded entrance, into brood chambers and escape with brood. However, it is unclear what mechanism allows these raids to be successful, especially because Iridomyrmex workers are highly aggressive. One reason could be attributed to the behavior of M. anderseni foragers straddling motionless Iridomyrmex host workers. Agosti ( 1997 ) suggested that M. anderseni workers hugged and rubbed the straddled workers to acquire the I. sanguineus smell (cuticular hydrocarbons). Alternatively, thermophilic species like Melophorus often have adaptations to foraging at high temperatures including longer legs and increased running speeds (Sommer and Wehner 2012 , Hurlbert et al. 2008 ). Therefore, M. anderseni may be able to avoid conflict while raiding by either selectively foraging at particularly hot times of the day when Iridomyrmex workers are less active, or by being able to simply outrun them. In this study, we examined 1) colony establishment and survival, 2) activity (walking speed, foraging and raiding behavior), and 3) cuticular hydrocarbon profiles of M. anderseni workers to study its ecology and evaluate possible mechanisms that may facilitate interspecific nest raiding behavior of I. reburrus . MATERIALS AND METHODS Study sites and organisms Most work was conducted on the grounds of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) laboratories in Darwin (12 ° 24’42” S, 130 ° 55’17” E), Northern Territory, Australia, which is the type location for M. anderseni (Agosti 1997 ). Data were also collected from Maxwell Creek on Melville Island, approximately 100 km north of Darwin (11 ° 27’27” S, 130 ° 35’08” E) and in Humpty Doo approximately 20 km south of Darwin (12 34’27” S, 131 06’05” E). The region has a tropical monsoonal climate, with a wet season (November-March) with an average rainfall of approximately 1600mm and relatively high temperatures and humidity (35C, > 80% respectively), and a dry season (April-October) with almost no rain and relatively low temperatures and humidity (31C, 30%) (Taylor & Tulloch 1985 ). Melophorus show a seasonal and daily pattern of being most active during peak temperatures when Iridomyrmex are the least active (Andersen 1983, Hoffmann 1998 , Muser et al. 2005 , Schultheiss et al. 2012 ). Both I. sanguineus and I. reburrus construct polydomous nests interconnected by walkways that can be many tens of metres long. To date, nothing has been detailed about the nests of M. anderseni other than they have “entrances at the outskirts of the [ Iridomyrmex ] nest, with much narrower entrances, so that… the [ Iridomyrmex ] could not enter” (Agosti 1997 ). Colony establishment and survival At CSIRO, prior to the first known M. anderseni nuptial flight, all nests of I. sanguineus and I. reburrus were marked and mapped on the days prior to 9 February 2014. At approximately mid-day on 9 February 2014, an M. anderseni nuptial flight occurred, and as many M. anderseni queens as possible were located and their nuptial nest entrances marked. The location of these nuptial nests was easily identified by the small pile of soil freshly excavated by the queens. On the same afternoon, M. anderseni queens were also found at Humpty Doo beside three I. reburrus nests, and the nest entrances of both species were marked. A second and final nuptial flight occurred on 22 February 2014, but M. anderseni queens were only found at CSIRO. In the days after both flights, distances were measured between each M. anderseni queen (101 queens) to the nearest Iridomyrmex nest entrance, as well as the distance between each M. anderseni queen to the closest M. anderseni queen. The nuptial M. anderseni nests were monitored for activity daily for 6 weeks to determine when workers first emerged, and again between late June and early July 2014 to quantify longer-term colony survival. Activity was confirmed either by direct observations of workers, or the presence of this species’ distinctive soil mound beside a marker. Additionally, two clusters of mature M. anderseni colonies around I. reburrus nests were located and mapped mid-February at CSIRO approximately 200 meters from where the nuptial queens landed. Activity All activity data were collected at CSIRO. Two clusters of mature M. anderseni colonies around I. reburrus nests, located approximately 200 meters from where the new queens were establishing colonies, were used to collect activity, foraging, and speed data between 31 March 2014 and 1 May 2014. Observations were made between 9:00 AM to 5:00 PM for nine days. Conditions during this period varied in cloud cover and rain. Cloud cover became light, and rain became infrequent by early May. If rain was medium to heavy and persisted for more than 20 minutes, data collection for the day stopped. Partial-day data were not included in the analyses. Both species were not always active at the same time, thus data was not always collected for both species over all nine days. To determine the number of days that colonies were active, ten colonies were monitored for 30 days, between 23 May 2014 and 21 June 2014. Colonies were monitored for activity between 11:00 AM and 2:00 PM daily, when T S reached a minimum of 37.4 ° C. A colony was categorized as active when the nest entrance was unplugged, and a worker appeared. M. anderseni colony activity was measured by counting the number of workers seen going in or out of their own nest within a five-minute period every hour. Items brought back to the nest were noted. I. reburrus activity was quantified by counting the number of workers passing a point on a nearby trail for five minutes every hour. M. anderseni activity at I. reburrus nest entrances was quantified for 20 minutes every hour after M. anderseni activity for the associated cluster of colonies had initiated for the day. Raiding was quantified by counting the number of M. anderseni going in or out of the I. reburrus nest entrance. General behavioral observations were made, and the number and type of items taken from the I. reburrus nest were noted. Soil (T S ) and ambient (T A ) temperature data were recorded using a Fluke ® thermometer with thermocouple (Fluke Corporation, Everett, WA) immediately before measuring activity. T S was recorded beside the M. anderseni nest entrance, I. reburrus trail, and at the I. reburrus nest entrance depending on the activity being measured. For T S the thermocouple sensor tip was covered with a small amount of dirt to prevent erroneous readings caused by solar radiation. T A was recorded 1 m above the mound, nest or point on the trail, with the thermocouple sensor tip placed in the shade of a 20 cm 2 piece of cardboard covered with foil held 30 cm higher. Foraging behavior of M. anderseni Foraging was defined as commencing when a worker left a nest and did not immediately return, and ending when it either entered an I. reburrus nest or returned to its own nest. Data were collected on three days in late March and early April by following 49 haphazardly chosen workers from nine colonies. We quantified foraging distance (defined as the absolute farthest distance from their nest, not the total distance travelled), foraging duration, any items returned to the nest, and also noted any seemingly relevant behavioral observations. When an I. reburrus nest was entered by a worker of M. anderseni , we determined whether it was the closest I. reburrus nest entrance. Speed The running speeds of M. anderseni and I. reburrus workers were measured on three days in June 2014 at two I. reburrus nest entrances and four nearby I. reburrus foraging trails. Videos set at 29.97 frames per second were recorded using an iPhone 5 or an Olympus TG-830 set on a tripod. Individuals were tracked for a minimum distance of 5 cm and maximum of 30 cm. 95 M. anderseni workers entering and exiting the I. reburrus nest (including those with and without items) were recorded and compared with 63 I. reburrus workers entering and exiting their own nest, and those on foraging trails. Videos were analyzed using the distance tracking feature of Kinovea 0.8.15 (Kinovea.org) to determine nest entering and exit speeds and running speeds. For M. anderseni , nest exit speed was split between workers with and without a stolen item. Running speed for I. reburrus was the speed of workers on the foraging trails, but because M. anderseni did not forage on trails and was not practicable to film away from nest entrances, running speed was calculated from the combination of data for all its activities. Chemical Mimicry To determine if chemical mimicry was a possible mechanism allowing interspecific nest raiding behavior in this system, we examined worker cuticular hydrocarbon profiles of M. anderseni , I. reburrus and other sympatric Melophorus and Iridomyrmex species that do not display parasitism. Specifically, we sampled workers from the following number of colonies at 3 sites: I. reburrus (n = 2 colonies) and M. anderseni (n = 11) from CSIRO, I. reburrus (n = 2) and M. anderseni (n = 2) from Melville Island, I. reburrus (n = 2), I. anceps (n = 2), and I. pallidus (n = 3) from Darwin, and Melophorus sp 1. (n = 1) and Melophorus sp. 11 (n = 1) from Gunlom Falls in Kakadu National Park. We tried to collect other sympatric Melophorus specimens from Darwin, but we were unable to find any. Samples were collected live, frozen, and stored at -20C in glass vials filled with drierite desiccant until they were shipped to the University of Illinois, Urbana for analysis. Hydrocarbon extraction was conducted by placing the ants in 300µl of hexane for 5 minutes. The resultant extract was filtered through a glass wool plug in a glass pipette and concentrated down to 10µl, 1µl of which was injected into an Agilent 7890 gas chromatograph (Agilent Technologies, Santa Clara, CA), connected to an Agilent 5977 mass selective detector. The GC injection port and the transfer line were set to 260 ºC. The column temperature was held at 60 ºC for 2 min, increased to 220ºC at 40 ºC/min, and then to 315 ºC at 4 ºC/min and held for 5min. Helium was the carrier gas at 1 ml/min, and samples were injected in splitless mode with a purge time of 0.75 min. Electron impact ionization mass spectra were obtained using 70 eV ionizing voltage, with a source temperature of 230 ºC. Major compounds were identified from a combination of their mass spectra, looking specifically for molecular ions or enhanced ions due to fragmentation on either side of methyl branch points, and by comparison of their retention indices to those of straight-chain hydrocarbons. Analysis For all analyses of colony establishment and survival, the data of queens from the two nuptial flights were combined. Statistical tests were conducted using R software version 3.1.0. Linear, polynomial (2nd degree), and logarithmic regressions were used to determine the relationships between M. anderseni and I. reburrus activity with soil temperature. The models were ranked for suitability using Akaike’s Information Criterion (AIC), which favors both model fit and model simplicity (Burnham & Anderson 2002 ). Foraging duration and distance data of the M. anderseni workers, and speed of the M. anderseni workers and I. reburrus workers were compared using Mann-Whitney U-tests. For analysis of chemical profiles, relative compound abundances of each worker were calculated relative to the cumulative total abundance of all compounds within the extract. Non-metric multi-dimensional scaling was used to analyze the similarity of profiles (Primer 6, PRIMER-E Ltd., Ivybridge, UK), with Chord (normalized Euclidean) distances used to calculate the distance matrices. Analysis of Similarity (ANOSIM) was used to test the statistical separation in ordination space of the profiles of M. anderseni vs I. reburrus workers and M. anderseni vs all other Iridomyrmex and Melophorus workers other than I. reburrus . RESULTS Colony establishment and survival One hundred and one M. anderseni queens were located making nuptial nests, 62 at CSIRO and 11 at Humpty Doo on the first flight date, and another 28 at CSIRO on the second. The queens were highly clustered, establishing at CSIRO on the first flight around only five nest entrances of one I. reburrus colony and one nest entrance of a second colony. At Humpty Doo they established around 3 nest entrances of one colony, and on the second flight at CSIRO they established around five nest entrances of a different colony. Considered together, the mean (± SE) distance to the nearest Iridomyrmex nest was 133.1 ± 8.9 cm, ranging from 25 to 650 cm. Mean queens per Iridomyrmex nest entrance was 8.4 ± 1.9. Mean distance to the nearest M. anderseni neighbor was 44.9 ± 5.3 cm, ranging from 10 to 233 cm. The first workers were observed emerging from incipient colonies after 56 days on 6 April 2014. Only 78 queens produced workers, and after five months, only 27 (35%) of the nests had signs of activity, being 14 of 36 (38.8%), 11 of 23 (47%), 2 of 7 (28.6%), and 0 of 13 (0%) for the four clusters respectively. The two mature colony clusters had a mean of 1 ± 0.5 and 11.3 ± 2.2 M. anderseni colonies per I. reburrus nest entrance and the M. anderseni nests had a mean distance of 341.6 ± 51 cm and 154.8 ± 32.4 cm from the nearest I. reburrus nest entrance respectively (range 27–461 cm). M. anderseni in clusters that were more widely separated (greater distance to nearest I. reburrus nest and to nearest M. anderseni neighbor) had a lower proportion of estimated queens surviving. The colonies in the cluster with a total estimated 0% surviving queens, commenced with an average distance of 220.4 ± 36 cm from the nearest I. reburrus nest entrance and were on average 94.1 ± 26.2 cm apart from the nearest neighboring M. anderseni neighbor, whereas colonies in the cluster with a total estimated 47% of queens surviving had on average 118.4 ± 11.6 cm from the nearest I. reburrus nest entrance and an average of 38.2 ± 8.4 cm apart from the nearest M. anderseni neighbor. Similarly, in the clusters established prior to the survey, the cluster with an average of 11.33 ± 2.21 colonies per I. reburrus nest entrance had colonies that were closer (154.8 ± 51 cm) to the nearest I. reburrus nest entrance compared to the cluster with only one (± 0.52) colony per I. reburrus nest entrance (341.6 ± 32.4 cm from the nearest I. reburrus nest entrance). Although nearest neighbor distances for the established colonies were not measured, the colonies in the second cluster were a meter apart at minimum, which is greater than the estimated nearest neighbor distances observed in the first cluster. Activity Soil temperatures at M. anderseni nests and I. reburrus nests were similar, peaking at approximately midday (Fig. 1 ). I. reburrus activity had a negative relationship with soil temperature (R 2 = 0.52, P < 0.0001), with no activity occurring above 50°C, whereas M. anderseni activity was positively related to temperature (R 2 = 0.35, P < 0.0001) with peak activity occurring just above 50°C (Fig. 2 ). M. anderseni worker activity commenced when soil temperatures exceeded 37.4°C (X = 39.2°C). Up to 11 am, the number of M. anderseni workers exiting their nests exceeded the number of workers entering (Fig. 3 ). Activity increased until mid-day, after which it declined (Fig. 3 ). Activity at peak temperatures (12pm and 1pm) accounted for more than half of all its activity. Activity ceased in the afternoon at temperatures lower than that for activity commencement, on average at 36.7°C (low of 33.2°C) (Fig. 2 ). Activity of M. anderseni at I. reburrus nests followed that of activity at M. anderseni nests, rising rapidly following activity initiation and peaking at midday (Fig. 4 ). More M. anderseni workers entered I. reburrus nests than were exiting up to the last three hours of activity. The mean (± SE) number of days each M. anderseni colony was active in the 30-day period was 9.9 ± 2.7 days, ranging from one to 22 days. Foraging behavior Mean M. anderseni foraging duration was 63 ± 9 s and mean foraging distance was 249 ± 19 cm. Of the 49 foragers followed, 42 (86%) entered an I. reburrus nest, and 32 (76.2%) of those entered the closest nest entrance. There was no difference (Mann-Whitney U-test: U = 126, Z = 0.6, P = 0.56) in foraging duration between workers that did or didn’t enter an I. reburrus nest (63 vs 61s respectively), but workers that entered an I. reburrus nest travelled more than three times farther than those that did not (281 vs 93 cm respectively; Mann-Whitney U-test: U = 11, Z = 3.9, P = 0.0001; Table 1 ). Of the workers that entered an I. reburrus nest, there was no difference in foraging duration between workers that entered the nearest or a farther I. reburrus nest entrance (62 vs 67 s respectively; Mann-Whitney U-test: U = 115, Z = 1.3, P = 0.19), despite workers entering a farther nest entrance travelling 62% further (Mann-Whitney U-test: U = 61, Z = 2.9, P = 0.004). Of the 1,444 observations of M. anderseni being observed exiting I. reburrus nests, in 122 (8.4%) they were observed stealing at item, with 117 (95.9%) of these items being brood, and the other five items being seeds or other invertebrates (beetle, caterpillar, spider). Four out of the five non-brood items were abandoned either immediately or soon after exiting the I. reburrus nest entrance. Table 1 Mean (± SE) foraging duration and distance of M. anderseni workers that did or did not enter an Iridomyrmex nest, and if it was the nearest Iridomyrmex nest entrance to the forager’s nest. Entered nest Did not enter nest Entered nearest entrance Entered farther entrance Duration (s) 63 ± 10.8 61.4 ± 13.6 61.7 ± 13.7 67.3 ± 13.6 Distance (cm) 281.4 ± 20 93.1 ± 9.5 245.3 ± 19 396.9 ± 41.3 Speed Mean M. anderseni running speed (9.72 ± 0.35 cm/s) was faster (P = 0.0005) than I. reburrus running speed (7.05 ± 0.46; Table 2 ). The speed of M. anderseni exiting an I. reburrus nest without a stolen item was more than twofold the speed of I. reburrus. An M. anderseni exiting a nest with a stolen item was also faster than one without a stolen item (12.7 ± 0.5 cm/s and 9.8 ± 0.3 cm/s respectively; Mann-Whitney U-test: U = 161, Z = 4.2, P < 0.0001). In the few instances when M. anderseni were recorded running along the I. reburrus foraging trails their speeds were as high as 18 cm/s. Table 2 Mean (± SE) M. anderseni and I. reburrus speed (cm/s) while foraging, and entering or exiting an I. reburrus nest, as well as metrics of respective Mann-Whitney U-tests. M. anderseni I. reburrus U Z P Foraging 9.72 ± 0.35 7.05 ± 0.46 479 -3.48 0.0005 Entering 6.98 ± 0.48 5.50 ± 0.41 235 -1.98 0.0479 Exiting 9.82 ± 0.31 4.54 ± 0.35 8 -6.74 < 0.0001 Chemical mimicry Extracts of I. reburrus and M. anderseni worker cuticular hydrocarbon profiles were qualitatively identical in that no compounds were unique to either species in all locations (Fig. 5 ). Saturated hydrocarbons with chain lengths between 25 and 29 carbons comprised most hydrocarbons found on the cuticle of both species (Table 3 ). The relative abundances of compounds within the profiles of both species were also similar, such that the NMDS analysis displayed strong clustering of their profiles (Fig. 6 ; ANOSIM: Global R = 0.014, P = 0.412). The two other sympatric Iridomyrmex and Melophorus species shared very few compounds with I. reburrus and M. anderseni (Figs. 5 , 6 , Table 3 ), and their profiles separated greatly in ordination space from those of M. anderseni (ANOSIM: Global R = 0.929, P = 0.001). Table 3 Compound identifications corresponding to number labels of Fig. 1 . Relative compound abundances given in average (minimum, maximum). Kovat’s retention index (RI) and diagnostic ion listed for each compound, when available. Sample sizes: 6 I. reburrus , 13 M. anderseni , 1 I. anceps , 1 M. sp1. # Compound RI Diagnostic ions I. reburrus M. anderseni I. anceps M. sp.1 1 Pentacosane 25 352 3.14 (1.77, 4.92) 1.9 (0, 4.29) 0.74 12.58 2 13-;11-Methylpentacosane 25.32 351 (M+-15), 197/197; 169/225 2.96 (1.23, 5.41) 3.12 (1.35, 5.98) 0.29 0 3 Hexacosane 26 366 0.79 (0.52, 1.14) 0.7 (0, 1.79) 0.37 0.44 4 14-; 12-Methylhexacosane 26.31 365 (M+-15), 211/197; 183/225 1.46 (0.42, 2.78) 0.07 (0, 0.45) 0 0 5 Heptacosane 27 380 11.87 (4.84, 20.21) 10.84 (2.8, 33.47) 1.14 1.45 6 13-; 11-Methylheptacosane 27.31 379 (M+-15), 197/225; 169/253 22.22 (18.96, 25.26) 24.57 (14.28, 29.25) 0 0 7 9-; 7-Methylheptacosane 27.39 379 (M+-15), 141/281; 113/309 2.84 (2.14, 4.38) 3.95 (2.05, 22.14) 0 0 8 3-Methylheptacosane 27.72 379 (M+-15), 57/365 6.36 (4.18, 8.69) 6.7 (4.24, 12.63) 0.38 0 9 5, 15-Dimethylheptacosane 27.79 393 (M+-15), 351/85, 197/239 4.29 (1.97, 6.54) 3.84 (2.52, 5.68) 0.39 0 10 Octacosane 28 394 0.52 (0, 1.28) 0.29 (0, 1.65) 0 0 11 3, 11-Dimethylheptacosane 28.03 393 (M+-15), 379/57, 253/183 3.58 (2.35, 5.12) 3.94 (1.31, 6.04) 0.71 0 12 14, 16-Dimethyloctacosane 28.3 407 (M+-15), 239/211, 197/253 2.18 (1.55, 3.15) 2.97 (0, 5.1) 0 0 13 Nonacosane 29 408 2.97 (0.74, 8.3) 3.44 (0.88, 12.76) 4.23 1.18 14 15-; 13-Methylnonacosane 29.29 407 (M+-15), 225; 197/253; 5.14 (3.16, 8.03) 6.92 (5.45, 9.49) 3.21 0 15 9-Methylnonacosane 29.34 407 (M+-15), 141/309 4 (3.56, 4.26) 3.92 (3.06, 5.09) 0.53 0 16 7-Methylnonacosane 29.39 407 (M+-15), 127/323 2.72 (1.86, 3.71) 2.99 (1.92, 3.98) 0 0 17 x, y-Dimethylnonacosane 29.66 6.55 (1.99, 8.93) 6.33 (1.04, 8.93) 5.83 0 DISCUSSION Colony establishment and survival The high number of M. anderseni queens (8.4 ± 1.9 queens) per I. reburrus nest entrance, and their close proximity to I. reburrus nest entrances (133.1 ± 8.9 cm), support a pattern of queens attempting to establish their colonies in the vicinity of I. reburrus nests. Although a direct association was not measured in this study (i.e. we did not map the locations of other ant species), the specialized feeding habits of M. anderseni make a strong case for association. Nest associations are also seen in other ant species with specialized feeding requirements like Myrmecocystus mexicanus for dead Pogonomyrmex occidentalis workers in north America, resulting in M. mexicanus having nest distributions associated with those of P. occidentalis (Cole et al. 2001 ). Notably, no conspecific fights were observed between M. anderseni workers despite their high colony densities. A lack of aggression has also been reported between workers of M. aeneovirens (Hoffmann 1998 ) although aggression has been found in M. bagoti (Muser et al. 2005 ). This suggests that competition for resources between neighboring M. anderseni colonies is low due to the presence of a high value and predictable food source. Other Melophorus species appear to have a more widely dispersed pattern of colony distribution compared to that of M. anderseni . M. bagoti has been reported to have a density of 3–13 nests per hectare (Muser et al. 2005 ; Schultheiss & Nooten 2013 ), and an average of ~ 20 meter between colonies (Muser et al. 2005 ). M. aeneovirens has been surveyed with an even lower density; five colonies in one hectare and 42.8 m to the nearest neighbor (Hoffmann 1998 ). Another unnamed Melophorus species had a range between nearest neighboring nests of 10.7 to 51 m (Schultheiss et al. 2012 ). However, despite the higher colony density of M. anderseni in localized areas near Iridomyrmex nests soon after nuptial flights are conducted, it is possible that later in the year following the extirpation of many incipient colonies, the average colony density over a large area (i,e, hectares) may not be so dissimilar to other Melophorus. Regardless, that M. anderseni colonies had greater apparent survival when spaced closer together than those farther apart indicates that such clustering is important for survival, presumably from attack by I. reburrus . Activity The thermophilic activity patterns of M. anderseni were congruent with those of other Melophorus . Aboveground activity did not occur below temperature at ant height above the soil being 37.4°C, which is close to the lowest temperature activity was recorded for the sympatric M. aeneovirens (35.2°C) (Hoffmann 1998 ), and lower than that of M. bagoti from the central deserts (> 40°C) (Christian & Morton 1992 ). Also consistent with all other Melophorus species studied (Hoffmann 1998 , Muser et al. 2005 , Schultheiss et al. 2012 ) was that activity is seemingly actively reduced and ceased prematurely despite soil temperature remaining above the initiation threshold. Notably, M. anderseni colonies were not active every day (range of 1–22 out of 30 days) despite suitable above-ground activity temperature being met and exceeded. The reasoning for this cannot be a seasonal influence because of the short timeframe of the study. Instead, we suspect that because of the high water, protein, and lipid content that would be present in stolen brood, foraging is not required every day for colony nutrition, and because of the dangerous nature of brood stealing, not foraging when a colony’s nutritional needs are met eliminates unnecessary risk of worker mortality. Foraging Behavior M. anderseni specializes in foraging inside I. reburrus nests and almost exclusively steals brood. Notably, its foraging success rate (8.4%) is a lower than that recorded of the other Melophorus species: M. aeneovirens (range of 12-23.6%; Hoffmann 1998 ), M. bagoti (~ 23%; Muser et al. 2005 ) and Melophorus sp (~ 20%; Schultheiss et al. 2012 ). This low rate is likely due to the high risk involved in stealing brood. How and why there was no difference in foraging duration between workers that entered the nearest or a farther I. reburrus nest entrance (the 62% difference in distance being statistically significant) remains unclear. Speed High running speed is associated with ants that forage in hot environments (Hurlbert et al. 2008 ; Zollikofer 1994 ). This is apparent in their morphology, with thermophilic ants having longer legs compared to closely related non-thermophilic species (Sommer and Wehner 2012 ). During the analysis of the video footage used to measure speed, M. anderseni appeared to “float” over I. reburrus workers when nest entrances were surrounded by these workers. This observation may not be an exaggeration given what is known about ant locomotion. Zollikofer ( 1994 ) found that at high running speeds, stride exceeds the maximum span of legs, revealing there is loss of contact with the ground. Besides speed itself being an advantage to avoid aggressive encounters with Iridomyrmex workers, in crowded areas inside the I. reburrus nests, these brief aerial moments would allow for minimizing contact with I. reburrus workers. Running speed used as an evasive tactic is found in another parasitic ant, Pseudomyrmex nigropilosus which walks 2.6 times faster than the three species it robs (Amador-Vargas 2012 ). Additionally, P. nigropilosus uses speed in combination with erratic direction changes, as well as unpredictable stopping and speeding up to further aid robbing behavior. Although not systematically measured in our study, this type of unpredictable movement is apparent in M. anderseni , particularly when attempting to enter I. reburrus nest entrances surrounded by I. reburrus workers. This could potentially explain why speed was slower for M. anderseni entering (6.98 ± 0.48 cm/s) than exiting (9.82 ± 0.31 cm/s) an I. reburrus nest. M. anderseni running speed (maximum recorded being 18 cm/s) is slower compared to other Melophorus and ecologically equivalent species on other continents. For example, M. bagoti runs at approximately 20 cm/s (Wystrach et al. 2014 ), Ocymyrmex barbiger up to 38 cm/s (Marsh 1985 ), and Cataglyphis bombycina , can run up to 85.5 cm/s (Pfeffer et al. 2019 ). Higher running speeds are associated with higher surface temperatures (Marsh 1985 ; Hurlbert et al. 2008 , Zollikofer 1994 ) and both M. bagoti and Cataglyphis spp have higher surface temperatures associated with their maximum activity levels in desert environments than what M. anderseni experiences in its tropical environment. Chemical Mimicry The worker cuticular hydrocarbon profiles of M. anderseni match those of I. reburrus , and profiles of both species are distinct from congeneric Melophorus and Iridomyrmex species, suggesting chemical mimicry is a likely tactic used by M. anderseni to gain access to Iridomyrmex nests. Chemical mimicry is a widespread strategy of both cleptoparasites who enter and exploit insect nests as well as social parasites who reside in host nests (Lenior et al. 2001; Bagnères & Lorenzi 2010 ). Temporary entering and exploiting nest resources is also known to be associated with other chemical strategies: chemical insignificance, when social parasites lack chemical recognition cues (e.g., Uboni et al. 2012 ) and incomplete chemical mimicry associated with a non-specific host association (e.g. Quezada-Euán et al 2013 ). Neither of these scenarios seem to be used by M. anderseni , and the degree to which M. anderseni replicates the chemical profile of I. reburrus is similar to social parasites that are obligate nest inhabitants (Lenior et al. 2001). Together, the chemical data corroborate the behavioral data suggesting that M. anderseni is an obligate and exclusive forager on I. reburrus nests, as their chemical profiles would likely only provide camouflage with I. reburrus . Whether or not M. anderseni generates or environmentally acquires a chemical profile matching I. reburrus is not known. However, hypotheses such as those made by Agosti ( 1997 ) that M. anderseni might gain chemical profiles of hosts through interactions with adult workers, remain a distinct possibility. Beyond the worker profiles analyzed in this study, we also anecdotally sampled M. anderseni queens from some of our study sites. While those samples were not replicated in a way appropriate for presenting in this study, we did observe that both foundress and established queens have an observable hydrocarbon profile with some overlap in compounds reported for workers in this study. A follow up study that investigates the expression of M. anderseni queen profiles would be fruitful for determining the relative contributions of genetic versus environmental cues in this mimicry. Declarations The authors declare that they have no financial or non-financial interests that are directly or indirectly related to this work. ACKNOWLEDGEMENTS We acknowledge Sara Hu for conducting this work as part of her Master’s thesis (Hu 2017). SH and TM were supported by the International Research Experiences for Students program of the National Science Foundation (OISE-1261015), and the Explorer’s Fund of the National Geographic Society. References Agosti D (1997) Two new engimatic Melophorus species (Hymenoptera: Formicidae) from Australia. J NY Entomol Soc 105:161-169 Akino T, Knapp JJ, Thomas JA, Elmes, GW (1999) Chemical mimicry and host specificity in the butterfly Maculinea rebeli , a social parasite of Myrmica ant colonies. Proc R Soc Lond B Biol Sci 266:1419-1426 Als TD, Vila R, Kandul NP, Nash DR, et al. (2004) The evolution of alternative parasitic life histories in large blue butterflies. 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J Insect Physiol 58:1259-1264 Wystrach A, Philippides A, Aurejac A, Cheng K, Graham P (2014) Visual scanning behaviours and their role in the navigation of the Australian desert ant Melophorus bagoti . J Comp Physiol A Neuroethol Sens Neural Behav Physiol 200:615-626 Zollikofer C (1994) Stepping patterns in ants. – 2. Influence of body morphology. J Exp Biol 192:107-118 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revisions Needed 19 Oct, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 14 Aug, 2025 First submitted to journal 12 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7361241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502337625,"identity":"da22b7ad-85c6-436d-885d-c00c3ba15109","order_by":0,"name":"Ben Hoffmann","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4010-4723","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":true,"prefix":"","firstName":"Ben","middleName":"","lastName":"Hoffmann","suffix":""},{"id":502337626,"identity":"f5e5c8e6-c155-424b-9932-13d0f1925add","order_by":1,"name":"Sara Hu","email":"","orcid":"","institution":"California State University Dominguez Hills","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Hu","suffix":""},{"id":502337627,"identity":"bba512d2-b08b-474f-a65a-cc9a761b5def","order_by":2,"name":"Adrian A. 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Times on the hour indicate temperatures recorded at \u003cem\u003eM. anderseni\u003c/em\u003e nests. Times 25 minutes after the hour indicate temperatures recorded at \u003cem\u003eI. reburrus\u003c/em\u003e nests, just before measuring \u003cem\u003eM. anderseni\u003c/em\u003e activity at the \u003cem\u003eI. reburrus\u003c/em\u003e nests.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/37f94d0410b19dabc970e11d.jpg"},{"id":89990551,"identity":"6158dc8c-f8e1-42d6-9573-10436d3c89e0","added_by":"auto","created_at":"2025-08-27 07:14:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58861,"visible":true,"origin":"","legend":"\u003cp\u003eRelationships between \u003cem\u003eI. reburrus \u003c/em\u003eactivity (circles) and \u003cem\u003eM. anderseni \u003c/em\u003eactivity (triangles) with soil temperature. Solid logarithmic regression line for \u003cem\u003eI. reburrus\u003c/em\u003e (y = 4888.5e\u003csup\u003e-0.099x\u003c/sup\u003e, r\u003csup\u003e2\u003c/sup\u003e=0.31, p=1.4e\u003csup\u003e-07\u003c/sup\u003e). \u0026nbsp;Dashed logarithmic regression line for \u003cem\u003eM. anderseni\u003c/em\u003e (y = 9E\u003csup\u003e-17\u003c/sup\u003ee\u003csup\u003e0.8264x\u003c/sup\u003e, r\u003csup\u003e2\u003c/sup\u003e=0.04, p=5.411e\u003csup\u003e-05\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/ea768896c562fc19c1ab1be5.jpg"},{"id":89990555,"identity":"0f33690e-4cc0-4d52-9d28-5172c0f942c6","added_by":"auto","created_at":"2025-08-27 07:14:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMelophorus\u003c/em\u003e \u003cem\u003eanderseni\u003c/em\u003e activity at their own nests. A) Total activity. B) Activity separated by In, Out, or Out/in.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/cde649bff1610fea813ed442.jpg"},{"id":89988785,"identity":"6770abdc-6816-442b-863f-196361725c8c","added_by":"auto","created_at":"2025-08-27 07:06:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62382,"visible":true,"origin":"","legend":"\u003cp\u003eMean \u003cem\u003eM. anderseni\u003c/em\u003e activity at \u003cem\u003eI. reburrus\u003c/em\u003e nests.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/4c455f9811fbb947076c4d45.jpg"},{"id":89990554,"identity":"e02eea84-87e7-439b-9b49-c17fc7b6151b","added_by":"auto","created_at":"2025-08-27 07:14:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108730,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative chromatograms with major compounds of the cuticular hydrocarbon profiles of \u003cem\u003eI. reburrus\u003c/em\u003e and \u003cem\u003eM. anderseni\u003c/em\u003elabeled and identified in Table 1. Those compounds when present in profiles of \u003cem\u003eI. anceps\u003c/em\u003e and \u003cem\u003eM.\u003c/em\u003e sp1 are labeled when present.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/70d762a521be65e473746139.jpg"},{"id":89988780,"identity":"3f372b65-ac9f-4563-84e4-b30284009331","added_by":"auto","created_at":"2025-08-27 07:06:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63670,"visible":true,"origin":"","legend":"\u003cp\u003eNMDS ordination of differences within and between various \u003cem\u003eMelophorus\u003c/em\u003eand \u003cem\u003eIridomyrmex\u003c/em\u003e species. Sample sizes: 6 \u003cem\u003eI. reburrus\u003c/em\u003e, 13 \u003cem\u003eM. anderseni\u003c/em\u003e, 1 all other species. 2D stress value = 0.01 which indicates an excellent two-dimensional representation of data variation.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/4a41729bd78972789d9438a9.jpg"},{"id":89992462,"identity":"f5ee88a5-e215-4ea1-abb1-5f54b72930ad","added_by":"auto","created_at":"2025-08-27 07:38:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1314865,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7361241/v1/7712f808-2b26-464f-8798-3106c4e92a3f.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEcology and mechanisms of parasitism of Iridomyrmex by the thermophilic ant, Melophorus anderseni\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe ability to distinguish familiar individuals from strangers is essential for the evolution and maintenance of animal societies. Behavioral discrimination allows individuals to direct costly beneficial behaviors towards group members, who are often relatives, and exclude or defend against non-group members. Consequently, well-developed recognition systems are common in social and colonial organisms (Sherman et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Starks \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Suarez et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ants have finely tuned recognition systems that are critical to social organization, division of labor, and territoriality (Stuart and Herbers \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Sturgis and Gordon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Larsen et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The ability to detect and exclude non-nestmates is essential for colony defense from competitors, predators and parasites. Colony and species-specific cuticular hydrocarbon profiles are used for nestmate recognition (Bonavita-Cougourdan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Lahav et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Lucas et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and components of these profiles can be employed by ant and non-ant social parasites to cloak their presence from hosts (Franks et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Lenoir et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Akino et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Lambardi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Hojo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These cues can be biosynthesized directly by the social parasites and/or acquired from their hosts through physical contact (Lenoir et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMany parasitic species have evolved strategies to take advantage of the resources and labor within colonies of eusocial insects (Lenoir et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, D\u0026rsquo;Ettorre et al. 2002). Social parasites come from a wide variety of taxa and employ diverse mechanisms to circumvent recognition by host colonies (Buschinger 1986, D\u0026rsquo;Ettorre and Heinze \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Als et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Buschinger \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Rettenmeyer et al. 2010, Parker \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, H\u0026ouml;lldobler and Kwapich \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These mechanisms include chemical mimicry of nestmate recognition cues (Lenoir et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), other forms of chemical signaling (e.g. not related to nest mate recognition; Lenoir et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and morphological or behavioral mimicry (e.g. stridulation; Geiselhardt et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Barbero et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Di Giulio et al. 2015). Likewise, the duration that parasitic species associate with their host varies greatly. Many social parasites take up residence inside ant colonies for at least part of their life cycle, while others specialize on cleptobiosis (Breed et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Workers of cleptobiotic species steal resources such as food, brood, nesting materials, or other items of value, either from members of the same species or a different species (Breed et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Raids are often accompanied by aggressive interactions between host and raiding workers, but in some species, the \u0026ldquo;thieves\u0026rdquo; utilize repulsive chemicals (Blum et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) or exhibit specialized behaviors that may serve to reduce the chance of getting caught (McGlynn et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eMelophorus anderseni\u003c/em\u003e is a thermophilic ant species that nests near colonies of \u003cem\u003eIridomyrmex sanguineus\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e on which it conducts brood raids (Agosti \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Agosti (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) noted that \u003cem\u003eM. anderseni\u003c/em\u003e workers were able to make their way through a guarded entrance, into brood chambers and escape with brood. However, it is unclear what mechanism allows these raids to be successful, especially because \u003cem\u003eIridomyrmex\u003c/em\u003e workers are highly aggressive. One reason could be attributed to the behavior of \u003cem\u003eM. anderseni\u003c/em\u003e foragers straddling motionless \u003cem\u003eIridomyrmex\u003c/em\u003e host workers. Agosti (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) suggested that \u003cem\u003eM. anderseni\u003c/em\u003e workers hugged and rubbed the straddled workers to acquire the \u003cem\u003eI. sanguineus\u003c/em\u003e smell (cuticular hydrocarbons). Alternatively, thermophilic species like \u003cem\u003eMelophorus\u003c/em\u003e often have adaptations to foraging at high temperatures including longer legs and increased running speeds (Sommer and Wehner \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Hurlbert et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, \u003cem\u003eM. anderseni\u003c/em\u003e may be able to avoid conflict while raiding by either selectively foraging at particularly hot times of the day when \u003cem\u003eIridomyrmex\u003c/em\u003e workers are less active, or by being able to simply outrun them.\u003c/p\u003e\u003cp\u003eIn this study, we examined 1) colony establishment and survival, 2) activity (walking speed, foraging and raiding behavior), and 3) cuticular hydrocarbon profiles of \u003cem\u003eM. anderseni\u003c/em\u003e workers to study its ecology and evaluate possible mechanisms that may facilitate interspecific nest raiding behavior of \u003cem\u003eI. reburrus\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy sites and organisms\u003c/h2\u003e\u003cp\u003eMost work was conducted on the grounds of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) laboratories in Darwin (12\u003cb\u003e\u0026deg;\u003c/b\u003e24\u0026rsquo;42\u0026rdquo; S, 130\u003cb\u003e\u0026deg;\u003c/b\u003e55\u0026rsquo;17\u0026rdquo; E), Northern Territory, Australia, which is the type location for \u003cem\u003eM. anderseni\u003c/em\u003e (Agosti \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Data were also collected from Maxwell Creek on Melville Island, approximately 100 km north of Darwin (11\u003cb\u003e\u0026deg;\u003c/b\u003e27\u0026rsquo;27\u0026rdquo; S, 130\u003cb\u003e\u0026deg;\u003c/b\u003e35\u0026rsquo;08\u0026rdquo; E) and in Humpty Doo approximately 20 km south of Darwin (12 34\u0026rsquo;27\u0026rdquo; S, 131 06\u0026rsquo;05\u0026rdquo; E). The region has a tropical monsoonal climate, with a wet season (November-March) with an average rainfall of approximately 1600mm and relatively high temperatures and humidity (35C, \u0026gt;\u0026thinsp;80% respectively), and a dry season (April-October) with almost no rain and relatively low temperatures and humidity (31C, 30%) (Taylor \u0026amp; Tulloch \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eMelophorus\u003c/em\u003e show a seasonal and daily pattern of being most active during peak temperatures when \u003cem\u003eIridomyrmex\u003c/em\u003e are the least active (Andersen 1983, Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Schultheiss et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Both \u003cem\u003eI. sanguineus\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e construct polydomous nests interconnected by walkways that can be many tens of metres long. To date, nothing has been detailed about the nests of \u003cem\u003eM. anderseni\u003c/em\u003e other than they have \u0026ldquo;entrances at the outskirts of the [\u003cem\u003eIridomyrmex\u003c/em\u003e] nest, with much narrower entrances, so that\u0026hellip; the [\u003cem\u003eIridomyrmex\u003c/em\u003e] could not enter\u0026rdquo; (Agosti \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eColony establishment and survival\u003c/h3\u003e\n\u003cp\u003eAt CSIRO, prior to the first known \u003cem\u003eM. anderseni\u003c/em\u003e nuptial flight, all nests of \u003cem\u003eI. sanguineus\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e were marked and mapped on the days prior to 9 February 2014. At approximately mid-day on 9 February 2014, an \u003cem\u003eM. anderseni\u003c/em\u003e nuptial flight occurred, and as many \u003cem\u003eM. anderseni\u003c/em\u003e queens as possible were located and their nuptial nest entrances marked. The location of these nuptial nests was easily identified by the small pile of soil freshly excavated by the queens. On the same afternoon, \u003cem\u003eM. anderseni\u003c/em\u003e queens were also found at Humpty Doo beside three \u003cem\u003eI. reburrus\u003c/em\u003e nests, and the nest entrances of both species were marked. A second and final nuptial flight occurred on 22 February 2014, but \u003cem\u003eM. anderseni\u003c/em\u003e queens were only found at CSIRO. In the days after both flights, distances were measured between each \u003cem\u003eM. anderseni\u003c/em\u003e queen (101 queens) to the nearest \u003cem\u003eIridomyrmex\u003c/em\u003e nest entrance, as well as the distance between each \u003cem\u003eM. anderseni\u003c/em\u003e queen to the closest \u003cem\u003eM. anderseni\u003c/em\u003e queen. The nuptial \u003cem\u003eM. anderseni\u003c/em\u003e nests were monitored for activity daily for 6 weeks to determine when workers first emerged, and again between late June and early July 2014 to quantify longer-term colony survival. Activity was confirmed either by direct observations of workers, or the presence of this species\u0026rsquo; distinctive soil mound beside a marker. Additionally, two clusters of mature \u003cem\u003eM. anderseni\u003c/em\u003e colonies around \u003cem\u003eI. reburrus\u003c/em\u003e nests were located and mapped mid-February at CSIRO approximately 200 meters from where the nuptial queens landed.\u003c/p\u003e\n\u003ch3\u003eActivity\u003c/h3\u003e\n\u003cp\u003eAll activity data were collected at CSIRO. Two clusters of mature \u003cem\u003eM. anderseni\u003c/em\u003e colonies around \u003cem\u003eI. reburrus\u003c/em\u003e nests, located approximately 200 meters from where the new queens were establishing colonies, were used to collect activity, foraging, and speed data between 31 March 2014 and 1 May 2014. Observations were made between 9:00 AM to 5:00 PM for nine days. Conditions during this period varied in cloud cover and rain. Cloud cover became light, and rain became infrequent by early May. If rain was medium to heavy and persisted for more than 20 minutes, data collection for the day stopped. Partial-day data were not included in the analyses. Both species were not always active at the same time, thus data was not always collected for both species over all nine days. To determine the number of days that colonies were active, ten colonies were monitored for 30 days, between 23 May 2014 and 21 June 2014. Colonies were monitored for activity between 11:00 AM and 2:00 PM daily, when T\u003csub\u003eS\u003c/sub\u003e reached a minimum of 37.4\u003cb\u003e\u0026deg;\u003c/b\u003eC.\u003c/p\u003e\u003cp\u003eA colony was categorized as active when the nest entrance was unplugged, and a worker appeared. \u003cem\u003eM. anderseni\u003c/em\u003e colony activity was measured by counting the number of workers seen going in or out of their own nest within a five-minute period every hour. Items brought back to the nest were noted. \u003cem\u003eI. reburrus\u003c/em\u003e activity was quantified by counting the number of workers passing a point on a nearby trail for five minutes every hour. \u003cem\u003eM. anderseni\u003c/em\u003e activity \u003cem\u003eat I. reburrus\u003c/em\u003e nest entrances was quantified for 20 minutes every hour after \u003cem\u003eM. anderseni\u003c/em\u003e activity for the associated cluster of colonies had initiated for the day. Raiding was quantified by counting the number of \u003cem\u003eM. anderseni\u003c/em\u003e going in or out of the \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance. General behavioral observations were made, and the number and type of items taken from the \u003cem\u003eI. reburrus\u003c/em\u003e nest were noted.\u003c/p\u003e\u003cp\u003eSoil (T\u003csub\u003eS\u003c/sub\u003e) and ambient (T\u003csub\u003eA\u003c/sub\u003e) temperature data were recorded using a Fluke\u003cb\u003e\u0026reg;\u003c/b\u003e thermometer with thermocouple (Fluke Corporation, Everett, WA) immediately before measuring activity. T\u003csub\u003eS\u003c/sub\u003e was recorded beside the \u003cem\u003eM. anderseni\u003c/em\u003e nest entrance, \u003cem\u003eI. reburrus\u003c/em\u003e trail, and at the \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance depending on the activity being measured. For T\u003csub\u003eS\u003c/sub\u003e the thermocouple sensor tip was covered with a small amount of dirt to prevent erroneous readings caused by solar radiation. T\u003csub\u003eA\u003c/sub\u003e was recorded 1 m above the mound, nest or point on the trail, with the thermocouple sensor tip placed in the shade of a 20 cm\u003csup\u003e2\u003c/sup\u003e piece of cardboard covered with foil held 30 cm higher.\u003c/p\u003e\u003cp\u003e\u003cb\u003eForaging behavior of\u003c/b\u003e \u003cb\u003eM. anderseni\u003c/b\u003e\u003c/p\u003e\u003cp\u003eForaging was defined as commencing when a worker left a nest and did not immediately return, and ending when it either entered an \u003cem\u003eI. reburrus\u003c/em\u003e nest or returned to its own nest. Data were collected on three days in late March and early April by following 49 haphazardly chosen workers from nine colonies. We quantified foraging distance (defined as the absolute farthest distance from their nest, not the total distance travelled), foraging duration, any items returned to the nest, and also noted any seemingly relevant behavioral observations. When an \u003cem\u003eI. reburrus\u003c/em\u003e nest was entered by a worker of \u003cem\u003eM. anderseni\u003c/em\u003e, we determined whether it was the closest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance.\u003c/p\u003e\n\u003ch3\u003eSpeed\u003c/h3\u003e\n\u003cp\u003eThe running speeds of \u003cem\u003eM. anderseni\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e workers were measured on three days in June 2014 at two \u003cem\u003eI. reburrus\u003c/em\u003e nest entrances and four nearby \u003cem\u003eI. reburrus\u003c/em\u003e foraging trails. Videos set at 29.97 frames per second were recorded using an iPhone 5 or an Olympus TG-830 set on a tripod. Individuals were tracked for a minimum distance of 5 cm and maximum of 30 cm. 95 \u003cem\u003eM. anderseni\u003c/em\u003e workers entering and exiting the \u003cem\u003eI. reburrus\u003c/em\u003e nest (including those with and without items) were recorded and compared with 63 \u003cem\u003eI. reburrus\u003c/em\u003e workers entering and exiting their own nest, and those on foraging trails. Videos were analyzed using the distance tracking feature of Kinovea 0.8.15 (Kinovea.org) to determine nest entering and exit speeds and running speeds. For \u003cem\u003eM. anderseni\u003c/em\u003e, nest exit speed was split between workers with and without a stolen item. Running speed for \u003cem\u003eI. reburrus\u003c/em\u003e was the speed of workers on the foraging trails, but because \u003cem\u003eM. anderseni\u003c/em\u003e did not forage on trails and was not practicable to film away from nest entrances, running speed was calculated from the combination of data for all its activities.\u003c/p\u003e\n\u003ch3\u003eChemical Mimicry\u003c/h3\u003e\n\u003cp\u003eTo determine if chemical mimicry was a possible mechanism allowing interspecific nest raiding behavior in this system, we examined worker cuticular hydrocarbon profiles of \u003cem\u003eM. anderseni\u003c/em\u003e, \u003cem\u003eI. reburrus\u003c/em\u003e and other sympatric \u003cem\u003eMelophorus\u003c/em\u003e and \u003cem\u003eIridomyrmex\u003c/em\u003e species that do not display parasitism. Specifically, we sampled workers from the following number of colonies at 3 sites: \u003cem\u003eI. reburrus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2 colonies) and \u003cem\u003eM. anderseni\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;11) from CSIRO, \u003cem\u003eI. reburrus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) and \u003cem\u003eM. anderseni\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) from Melville Island, \u003cem\u003eI. reburrus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eI. anceps\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003eI. pallidus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3) from Darwin, and \u003cem\u003eMelophorus\u003c/em\u003e sp 1. (n\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eMelophorus\u003c/em\u003e sp. 11 (n\u0026thinsp;=\u0026thinsp;1) from Gunlom Falls in Kakadu National Park. We tried to collect other sympatric \u003cem\u003eMelophorus\u003c/em\u003e specimens from Darwin, but we were unable to find any.\u003c/p\u003e\u003cp\u003eSamples were collected live, frozen, and stored at -20C in glass vials filled with drierite desiccant until they were shipped to the University of Illinois, Urbana for analysis. Hydrocarbon extraction was conducted by placing the ants in 300\u0026micro;l of hexane for 5 minutes. The resultant extract was filtered through a glass wool plug in a glass pipette and concentrated down to 10\u0026micro;l, 1\u0026micro;l of which was injected into an Agilent 7890 gas chromatograph (Agilent Technologies, Santa Clara, CA), connected to an Agilent 5977 mass selective detector. The GC injection port and the transfer line were set to 260 \u0026ordm;C. The column temperature was held at 60 \u0026ordm;C for 2 min, increased to 220\u0026ordm;C at 40 \u0026ordm;C/min, and then to 315 \u0026ordm;C at 4 \u0026ordm;C/min and held for 5min. Helium was the carrier gas at 1 ml/min, and samples were injected in splitless mode with a purge time of 0.75 min. Electron impact ionization mass spectra were obtained using 70 eV ionizing voltage, with a source temperature of 230 \u0026ordm;C. Major compounds were identified from a combination of their mass spectra, looking specifically for molecular ions or enhanced ions due to fragmentation on either side of methyl branch points, and by comparison of their retention indices to those of straight-chain hydrocarbons.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis\u003c/h2\u003e\u003cp\u003eFor all analyses of colony establishment and survival, the data of queens from the two nuptial flights were combined. Statistical tests were conducted using R software version 3.1.0. Linear, polynomial (2nd degree), and logarithmic regressions were used to determine the relationships between \u003cem\u003eM. anderseni\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e activity with soil temperature. The models were ranked for suitability using Akaike\u0026rsquo;s Information Criterion (AIC), which favors both model fit and model simplicity (Burnham \u0026amp; Anderson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Foraging duration and distance data of the \u003cem\u003eM. anderseni\u003c/em\u003e workers, and speed of the \u003cem\u003eM. anderseni\u003c/em\u003e workers and \u003cem\u003eI. reburrus\u003c/em\u003e workers were compared using Mann-Whitney U-tests.\u003c/p\u003e\u003cp\u003eFor analysis of chemical profiles, relative compound abundances of each worker were calculated relative to the cumulative total abundance of all compounds within the extract. Non-metric multi-dimensional scaling was used to analyze the similarity of profiles (Primer 6, PRIMER-E Ltd., Ivybridge, UK), with Chord (normalized Euclidean) distances used to calculate the distance matrices. Analysis of Similarity (ANOSIM) was used to test the statistical separation in ordination space of the profiles of \u003cem\u003eM. anderseni\u003c/em\u003e vs \u003cem\u003eI. reburrus\u003c/em\u003e workers and \u003cem\u003eM. anderseni\u003c/em\u003e vs all other \u003cem\u003eIridomyrmex\u003c/em\u003e and \u003cem\u003eMelophorus\u003c/em\u003e workers other than \u003cem\u003eI. reburrus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eColony establishment and survival\u003c/h2\u003e\u003cp\u003eOne hundred and one \u003cem\u003eM. anderseni\u003c/em\u003e queens were located making nuptial nests, 62 at CSIRO and 11 at Humpty Doo on the first flight date, and another 28 at CSIRO on the second. The queens were highly clustered, establishing at CSIRO on the first flight around only five nest entrances of one \u003cem\u003eI. reburrus\u003c/em\u003e colony and one nest entrance of a second colony. At Humpty Doo they established around 3 nest entrances of one colony, and on the second flight at CSIRO they established around five nest entrances of a different colony. Considered together, the mean (\u0026plusmn;\u0026thinsp;SE) distance to the nearest \u003cem\u003eIridomyrmex\u003c/em\u003e nest was 133.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 cm, ranging from 25 to 650 cm. Mean queens per \u003cem\u003eIridomyrmex\u003c/em\u003e nest entrance was 8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9. Mean distance to the nearest \u003cem\u003eM. anderseni\u003c/em\u003e neighbor was 44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 cm, ranging from 10 to 233 cm.\u003c/p\u003e\u003cp\u003eThe first workers were observed emerging from incipient colonies after 56 days on 6 April 2014. Only 78 queens produced workers, and after five months, only 27 (35%) of the nests had signs of activity, being 14 of 36 (38.8%), 11 of 23 (47%), 2 of 7 (28.6%), and 0 of 13 (0%) for the four clusters respectively. The two mature colony clusters had a mean of 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 and 11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 \u003cem\u003eM. anderseni\u003c/em\u003e colonies per \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance and the \u003cem\u003eM. anderseni\u003c/em\u003e nests had a mean distance of 341.6\u0026thinsp;\u0026plusmn;\u0026thinsp;51 cm and 154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;32.4 cm from the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance respectively (range 27\u0026ndash;461 cm).\u003c/p\u003e\u003cp\u003e\u003cem\u003eM. anderseni\u003c/em\u003e in clusters that were more widely separated (greater distance to nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest and to nearest \u003cem\u003eM. anderseni\u003c/em\u003e neighbor) had a lower proportion of estimated queens surviving. The colonies in the cluster with a total estimated 0% surviving queens, commenced with an average distance of 220.4\u0026thinsp;\u0026plusmn;\u0026thinsp;36 cm from the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance and were on average 94.1\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2 cm apart from the nearest neighboring \u003cem\u003eM. anderseni\u003c/em\u003e neighbor, whereas colonies in the cluster with a total estimated 47% of queens surviving had on average 118.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6 cm from the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance and an average of 38.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4 cm apart from the nearest \u003cem\u003eM. anderseni\u003c/em\u003e neighbor. Similarly, in the clusters established prior to the survey, the cluster with an average of 11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 colonies per \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance had colonies that were closer (154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;51 cm) to the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance compared to the cluster with only one (\u0026plusmn;\u0026thinsp;0.52) colony per \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance (341.6\u0026thinsp;\u0026plusmn;\u0026thinsp;32.4 cm from the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance). Although nearest neighbor distances for the established colonies were not measured, the colonies in the second cluster were a meter apart at minimum, which is greater than the estimated nearest neighbor distances observed in the first cluster.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eActivity\u003c/h2\u003e\u003cp\u003eSoil temperatures at \u003cem\u003eM. anderseni\u003c/em\u003e nests and \u003cem\u003eI. reburrus\u003c/em\u003e nests were similar, peaking at approximately midday (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eI. reburrus\u003c/em\u003e activity had a negative relationship with soil temperature (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.52, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with no activity occurring above 50\u0026deg;C, whereas \u003cem\u003eM. anderseni\u003c/em\u003e activity was positively related to temperature (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.35, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with peak activity occurring just above 50\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eM. anderseni\u003c/em\u003e worker activity commenced when soil temperatures exceeded 37.4\u0026deg;C (X\u0026thinsp;=\u0026thinsp;39.2\u0026deg;C). Up to 11 am, the number of \u003cem\u003eM. anderseni\u003c/em\u003e workers exiting their nests exceeded the number of workers entering (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Activity increased until mid-day, after which it declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Activity at peak temperatures (12pm and 1pm) accounted for more than half of all its activity. Activity ceased in the afternoon at temperatures lower than that for activity commencement, on average at 36.7\u0026deg;C (low of 33.2\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eActivity of \u003cem\u003eM. anderseni\u003c/em\u003e at \u003cem\u003eI. reburrus\u003c/em\u003e nests followed that of activity at \u003cem\u003eM. anderseni\u003c/em\u003e nests, rising rapidly following activity initiation and peaking at midday (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). More \u003cem\u003eM. anderseni\u003c/em\u003e workers entered \u003cem\u003eI. reburrus\u003c/em\u003e nests than were exiting up to the last three hours of activity. The mean (\u0026plusmn;\u0026thinsp;SE) number of days each \u003cem\u003eM. anderseni\u003c/em\u003e colony was active in the 30-day period was 9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 days, ranging from one to 22 days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eForaging behavior\u003c/h2\u003e\u003cp\u003eMean \u003cem\u003eM. anderseni\u003c/em\u003e foraging duration was 63\u0026thinsp;\u0026plusmn;\u0026thinsp;9 s and mean foraging distance was 249\u0026thinsp;\u0026plusmn;\u0026thinsp;19 cm. Of the 49 foragers followed, 42 (86%) entered an \u003cem\u003eI. reburrus\u003c/em\u003e nest, and 32 (76.2%) of those entered the closest nest entrance. There was no difference (Mann-Whitney U-test: U\u0026thinsp;=\u0026thinsp;126, Z\u0026thinsp;=\u0026thinsp;0.6, P\u0026thinsp;=\u0026thinsp;0.56) in foraging duration between workers that did or didn\u0026rsquo;t enter an \u003cem\u003eI. reburrus\u003c/em\u003e nest (63 vs 61s respectively), but workers that entered an \u003cem\u003eI. reburrus\u003c/em\u003e nest travelled more than three times farther than those that did not (281 vs 93 cm respectively; Mann-Whitney U-test: U\u0026thinsp;=\u0026thinsp;11, Z\u0026thinsp;=\u0026thinsp;3.9, P\u0026thinsp;=\u0026thinsp;0.0001; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the workers that entered an \u003cem\u003eI. reburrus\u003c/em\u003e nest, there was no difference in foraging duration between workers that entered the nearest or a farther \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance (62 vs 67 s respectively; Mann-Whitney U-test: U\u0026thinsp;=\u0026thinsp;115, Z\u0026thinsp;=\u0026thinsp;1.3, P\u0026thinsp;=\u0026thinsp;0.19), despite workers entering a farther nest entrance travelling 62% further (Mann-Whitney U-test: U\u0026thinsp;=\u0026thinsp;61, Z\u0026thinsp;=\u0026thinsp;2.9, P\u0026thinsp;=\u0026thinsp;0.004). Of the 1,444 observations of \u003cem\u003eM. anderseni\u003c/em\u003e being observed exiting \u003cem\u003eI. reburrus\u003c/em\u003e nests, in 122 (8.4%) they were observed stealing at item, with 117 (95.9%) of these items being brood, and the other five items being seeds or other invertebrates (beetle, caterpillar, spider). Four out of the five non-brood items were abandoned either immediately or soon after exiting the \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SE) foraging duration and distance of \u003cem\u003eM. anderseni\u003c/em\u003e workers that did or did not enter an \u003cem\u003eIridomyrmex\u003c/em\u003e nest, and if it was the nearest \u003cem\u003eIridomyrmex\u003c/em\u003e nest entrance to the forager\u0026rsquo;s nest.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEntered nest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDid not enter nest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEntered nearest entrance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEntered farther entrance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuration (s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e61.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistance (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e281.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e93.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e245.3\u0026thinsp;\u0026plusmn;\u0026thinsp;19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e396.9\u0026thinsp;\u0026plusmn;\u0026thinsp;41.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSpeed\u003c/h2\u003e\u003cp\u003eMean \u003cem\u003eM. anderseni\u003c/em\u003e running speed (9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 cm/s) was faster (P\u0026thinsp;=\u0026thinsp;0.0005) than \u003cem\u003eI. reburrus\u003c/em\u003e running speed (7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The speed of \u003cem\u003eM. anderseni\u003c/em\u003e exiting an \u003cem\u003eI. reburrus\u003c/em\u003e nest without a stolen item was more than twofold the speed of \u003cem\u003eI. reburrus.\u003c/em\u003e An \u003cem\u003eM. anderseni\u003c/em\u003e exiting a nest with a stolen item was also faster than one without a stolen item (12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 cm/s and 9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 cm/s respectively; Mann-Whitney U-test: U\u0026thinsp;=\u0026thinsp;161, Z\u0026thinsp;=\u0026thinsp;4.2, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In the few instances when \u003cem\u003eM. anderseni\u003c/em\u003e were recorded running along the \u003cem\u003eI. reburrus\u003c/em\u003e foraging trails their speeds were as high as 18 cm/s.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SE) \u003cem\u003eM. anderseni\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e speed (cm/s) while foraging, and entering or exiting an \u003cem\u003eI. reburrus\u003c/em\u003e nest, as well as metrics of respective Mann-Whitney U-tests.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eM. anderseni\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eI. reburrus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eU\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eForaging\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e479\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-3.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.0005\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntering\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.0479\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExiting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-6.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eChemical mimicry\u003c/h2\u003e\u003cp\u003eExtracts of \u003cem\u003eI. reburrus\u003c/em\u003e and \u003cem\u003eM. anderseni\u003c/em\u003e worker cuticular hydrocarbon profiles were qualitatively identical in that no compounds were unique to either species in all locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Saturated hydrocarbons with chain lengths between 25 and 29 carbons comprised most hydrocarbons found on the cuticle of both species (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The relative abundances of compounds within the profiles of both species were also similar, such that the NMDS analysis displayed strong clustering of their profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; ANOSIM: Global R\u0026thinsp;=\u0026thinsp;0.014, P\u0026thinsp;=\u0026thinsp;0.412). The two other sympatric \u003cem\u003eIridomyrmex\u003c/em\u003e and \u003cem\u003eMelophorus\u003c/em\u003e species shared very few compounds with \u003cem\u003eI. reburrus\u003c/em\u003e and \u003cem\u003eM. anderseni\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and their profiles separated greatly in ordination space from those of \u003cem\u003eM. anderseni\u003c/em\u003e (ANOSIM: Global R\u0026thinsp;=\u0026thinsp;0.929, P\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCompound identifications corresponding to number labels of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Relative compound abundances given in average (minimum, maximum). Kovat\u0026rsquo;s retention index (RI) and diagnostic ion listed for each compound, when available. Sample sizes: 6 \u003cem\u003eI. reburrus\u003c/em\u003e, 13 \u003cem\u003eM. anderseni\u003c/em\u003e, 1 \u003cem\u003eI. anceps\u003c/em\u003e, 1 \u003cem\u003eM.\u003c/em\u003e sp1.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiagnostic ions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eI. reburrus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eM. anderseni\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eI. anceps\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eM.\u003c/em\u003e sp.1\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePentacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e352\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.14 (1.77, 4.92)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.9 (0, 4.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13-;11-Methylpentacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e351 (M+-15), 197/197; 169/225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.96 (1.23, 5.41)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.12 (1.35, 5.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHexacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.79 (0.52, 1.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.7 (0, 1.79)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14-; 12-Methylhexacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e365 (M+-15), 211/197; 183/225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.46 (0.42, 2.78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.07 (0, 0.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e380\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.87 (4.84, 20.21)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.84 (2.8, 33.47)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13-; 11-Methylheptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e379 (M+-15), 197/225; 169/253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.22 (18.96, 25.26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24.57 (14.28, 29.25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9-; 7-Methylheptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e379 (M+-15), 141/281; 113/309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.84 (2.14, 4.38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.95 (2.05, 22.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3-Methylheptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e379 (M+-15), 57/365\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.36 (4.18, 8.69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.7 (4.24, 12.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5, 15-Dimethylheptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e393 (M+-15), 351/85, 197/239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.29 (1.97, 6.54)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.84 (2.52, 5.68)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOctacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e394\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.52 (0, 1.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.29 (0, 1.65)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3, 11-Dimethylheptacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e393 (M+-15), 379/57, 253/183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.58 (2.35, 5.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.94 (1.31, 6.04)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14, 16-Dimethyloctacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e407 (M+-15), 239/211, 197/253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.18 (1.55, 3.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.97 (0, 5.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNonacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e408\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.97 (0.74, 8.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.44 (0.88, 12.76)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15-; 13-Methylnonacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e407 (M+-15), 225; 197/253;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.14 (3.16, 8.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.92 (5.45, 9.49)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9-Methylnonacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e407 (M+-15), 141/309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4 (3.56, 4.26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.92 (3.06, 5.09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7-Methylnonacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e407 (M+-15), 127/323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.72 (1.86, 3.71)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.99 (1.92, 3.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ex, y-Dimethylnonacosane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.55 (1.99, 8.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.33 (1.04, 8.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eColony establishment and survival\u003c/h2\u003e\u003cp\u003eThe high number of \u003cem\u003eM. anderseni\u003c/em\u003e queens (8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 queens) per \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance, and their close proximity to \u003cem\u003eI. reburrus\u003c/em\u003e nest entrances (133.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 cm), support a pattern of queens attempting to establish their colonies in the vicinity of \u003cem\u003eI. reburrus\u003c/em\u003e nests. Although a direct association was not measured in this study (i.e. we did not map the locations of other ant species), the specialized feeding habits of \u003cem\u003eM. anderseni\u003c/em\u003e make a strong case for association. Nest associations are also seen in other ant species with specialized feeding requirements like \u003cem\u003eMyrmecocystus mexicanus\u003c/em\u003e for dead \u003cem\u003ePogonomyrmex occidentalis\u003c/em\u003e workers in north America, resulting in \u003cem\u003eM. mexicanus\u003c/em\u003e having nest distributions associated with those of \u003cem\u003eP. occidentalis\u003c/em\u003e (Cole et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Notably, no conspecific fights were observed between \u003cem\u003eM. anderseni\u003c/em\u003e workers despite their high colony densities. A lack of aggression has also been reported between workers of \u003cem\u003eM. aeneovirens\u003c/em\u003e (Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) although aggression has been found in \u003cem\u003eM. bagoti\u003c/em\u003e (Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This suggests that competition for resources between neighboring \u003cem\u003eM. anderseni\u003c/em\u003e colonies is low due to the presence of a high value and predictable food source.\u003c/p\u003e\u003cp\u003eOther \u003cem\u003eMelophorus\u003c/em\u003e species appear to have a more widely dispersed pattern of colony distribution compared to that of \u003cem\u003eM. anderseni\u003c/em\u003e. \u003cem\u003eM. bagoti\u003c/em\u003e has been reported to have a density of 3\u0026ndash;13 nests per hectare (Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Schultheiss \u0026amp; Nooten \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and an average of ~\u0026thinsp;20 meter between colonies (Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eM. aeneovirens\u003c/em\u003e has been surveyed with an even lower density; five colonies in one hectare and 42.8 m to the nearest neighbor (Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Another unnamed \u003cem\u003eMelophorus\u003c/em\u003e species had a range between nearest neighboring nests of 10.7 to 51 m (Schultheiss et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, despite the higher colony density of \u003cem\u003eM. anderseni\u003c/em\u003e in localized areas near \u003cem\u003eIridomyrmex\u003c/em\u003e nests soon after nuptial flights are conducted, it is possible that later in the year following the extirpation of many incipient colonies, the average colony density over a large area (i,e, hectares) may not be so dissimilar to other \u003cem\u003eMelophorus.\u003c/em\u003e Regardless, that \u003cem\u003eM. anderseni\u003c/em\u003e colonies had greater apparent survival when spaced closer together than those farther apart indicates that such clustering is important for survival, presumably from attack by \u003cem\u003eI. reburrus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eActivity\u003c/h2\u003e\u003cp\u003eThe thermophilic activity patterns of \u003cem\u003eM. anderseni\u003c/em\u003e were congruent with those of other \u003cem\u003eMelophorus\u003c/em\u003e. Aboveground activity did not occur below temperature at ant height above the soil being 37.4\u0026deg;C, which is close to the lowest temperature activity was recorded for the sympatric \u003cem\u003eM. aeneovirens\u003c/em\u003e (35.2\u0026deg;C) (Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and lower than that of \u003cem\u003eM. bagoti\u003c/em\u003e from the central deserts (\u0026gt;\u0026thinsp;40\u0026deg;C) (Christian \u0026amp; Morton \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Also consistent with all other \u003cem\u003eMelophorus\u003c/em\u003e species studied (Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Schultheiss et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) was that activity is seemingly actively reduced and ceased prematurely despite soil temperature remaining above the initiation threshold.\u003c/p\u003e\u003cp\u003eNotably, \u003cem\u003eM. anderseni\u003c/em\u003e colonies were not active every day (range of 1\u0026ndash;22 out of 30 days) despite suitable above-ground activity temperature being met and exceeded. The reasoning for this cannot be a seasonal influence because of the short timeframe of the study. Instead, we suspect that because of the high water, protein, and lipid content that would be present in stolen brood, foraging is not required every day for colony nutrition, and because of the dangerous nature of brood stealing, not foraging when a colony\u0026rsquo;s nutritional needs are met eliminates unnecessary risk of worker mortality.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eForaging Behavior\u003c/h2\u003e\u003cp\u003e\u003cem\u003eM. anderseni\u003c/em\u003e specializes in foraging inside \u003cem\u003eI. reburrus\u003c/em\u003e nests and almost exclusively steals brood. Notably, its foraging success rate (8.4%) is a lower than that recorded of the other \u003cem\u003eMelophorus\u003c/em\u003e species: \u003cem\u003eM. aeneovirens\u003c/em\u003e (range of 12-23.6%; Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), \u003cem\u003eM. bagoti\u003c/em\u003e (~\u0026thinsp;23%; Muser et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and \u003cem\u003eMelophorus sp\u003c/em\u003e (~\u0026thinsp;20%; Schultheiss et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This low rate is likely due to the high risk involved in stealing brood. How and why there was no difference in foraging duration between workers that entered the nearest or a farther \u003cem\u003eI. reburrus\u003c/em\u003e nest entrance (the 62% difference in distance being statistically significant) remains unclear.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSpeed\u003c/h2\u003e\u003cp\u003eHigh running speed is associated with ants that forage in hot environments (Hurlbert et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zollikofer \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This is apparent in their morphology, with thermophilic ants having longer legs compared to closely related non-thermophilic species (Sommer and Wehner \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). During the analysis of the video footage used to measure speed, \u003cem\u003eM. anderseni\u003c/em\u003e appeared to \u0026ldquo;float\u0026rdquo; over \u003cem\u003eI. reburrus\u003c/em\u003e workers when nest entrances were surrounded by these workers. This observation may not be an exaggeration given what is known about ant locomotion. Zollikofer (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) found that at high running speeds, stride exceeds the maximum span of legs, revealing there is loss of contact with the ground. Besides speed itself being an advantage to avoid aggressive encounters with \u003cem\u003eIridomyrmex\u003c/em\u003e workers, in crowded areas inside the \u003cem\u003eI. reburrus\u003c/em\u003e nests, these brief aerial moments would allow for minimizing contact with \u003cem\u003eI. reburrus\u003c/em\u003e workers. Running speed used as an evasive tactic is found in another parasitic ant, \u003cem\u003ePseudomyrmex nigropilosus\u003c/em\u003e which walks 2.6 times faster than the three species it robs (Amador-Vargas \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, \u003cem\u003eP. nigropilosus\u003c/em\u003e uses speed in combination with erratic direction changes, as well as unpredictable stopping and speeding up to further aid robbing behavior. Although not systematically measured in our study, this type of unpredictable movement is apparent in \u003cem\u003eM. anderseni\u003c/em\u003e, particularly when attempting to enter \u003cem\u003eI. reburrus\u003c/em\u003e nest entrances surrounded by \u003cem\u003eI. reburrus\u003c/em\u003e workers. This could potentially explain why speed was slower for \u003cem\u003eM. anderseni\u003c/em\u003e entering (6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 cm/s) than exiting (9.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 cm/s) an \u003cem\u003eI. reburrus\u003c/em\u003e nest.\u003c/p\u003e\u003cp\u003e\u003cem\u003eM. anderseni\u003c/em\u003e running speed (maximum recorded being 18 cm/s) is slower compared to other \u003cem\u003eMelophorus\u003c/em\u003e and ecologically equivalent species on other continents. For example, \u003cem\u003eM. bagoti\u003c/em\u003e runs at approximately 20 cm/s (Wystrach et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u003cem\u003eOcymyrmex barbiger\u003c/em\u003e up to 38 cm/s (Marsh \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), and \u003cem\u003eCataglyphis bombycina\u003c/em\u003e, can run up to 85.5 cm/s (Pfeffer et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Higher running speeds are associated with higher surface temperatures (Marsh \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Hurlbert et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Zollikofer \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and both \u003cem\u003eM. bagoti\u003c/em\u003e and \u003cem\u003eCataglyphis spp\u003c/em\u003e have higher surface temperatures associated with their maximum activity levels in desert environments than what \u003cem\u003eM. anderseni\u003c/em\u003e experiences in its tropical environment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eChemical Mimicry\u003c/h2\u003e\u003cp\u003eThe worker cuticular hydrocarbon profiles of \u003cem\u003eM. anderseni\u003c/em\u003e match those of \u003cem\u003eI. reburrus\u003c/em\u003e, and profiles of both species are distinct from congeneric \u003cem\u003eMelophorus\u003c/em\u003e and \u003cem\u003eIridomyrmex\u003c/em\u003e species, suggesting chemical mimicry is a likely tactic used by \u003cem\u003eM. anderseni\u003c/em\u003e to gain access to \u003cem\u003eIridomyrmex\u003c/em\u003e nests. Chemical mimicry is a widespread strategy of both cleptoparasites who enter and exploit insect nests as well as social parasites who reside in host nests (Lenior et al. 2001; Bagn\u0026egrave;res \u0026amp; Lorenzi \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Temporary entering and exploiting nest resources is also known to be associated with other chemical strategies: chemical insignificance, when social parasites lack chemical recognition cues (e.g., Uboni et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and incomplete chemical mimicry associated with a non-specific host association (e.g. Quezada-Eu\u0026aacute;n et al \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Neither of these scenarios seem to be used by \u003cem\u003eM. anderseni\u003c/em\u003e, and the degree to which \u003cem\u003eM. anderseni\u003c/em\u003e replicates the chemical profile of \u003cem\u003eI. reburrus\u003c/em\u003e is similar to social parasites that are obligate nest inhabitants (Lenior et al. 2001). Together, the chemical data corroborate the behavioral data suggesting that \u003cem\u003eM. anderseni\u003c/em\u003e is an obligate and exclusive forager on \u003cem\u003eI. reburrus\u003c/em\u003e nests, as their chemical profiles would likely only provide camouflage with \u003cem\u003eI. reburrus\u003c/em\u003e. Whether or not \u003cem\u003eM. anderseni\u003c/em\u003e generates or environmentally acquires a chemical profile matching \u003cem\u003eI. reburrus\u003c/em\u003e is not known. However, hypotheses such as those made by Agosti (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) that \u003cem\u003eM. anderseni\u003c/em\u003e might gain chemical profiles of hosts through interactions with adult workers, remain a distinct possibility. Beyond the worker profiles analyzed in this study, we also anecdotally sampled \u003cem\u003eM. anderseni\u003c/em\u003e queens from some of our study sites. While those samples were not replicated in a way appropriate for presenting in this study, we did observe that both foundress and established queens have an observable hydrocarbon profile with some overlap in compounds reported for workers in this study. A follow up study that investigates the expression of \u003cem\u003eM. anderseni\u003c/em\u003e queen profiles would be fruitful for determining the relative contributions of genetic versus environmental cues in this mimicry.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no financial or non-financial interests that are directly or indirectly related to this work.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eWe acknowledge Sara Hu for conducting this work as part of her Master\u0026rsquo;s thesis (Hu 2017). SH and TM were supported by the International Research Experiences for Students program of the National Science Foundation (OISE-1261015), and the Explorer\u0026rsquo;s Fund of the National Geographic Society.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgosti D (1997) Two new engimatic \u003cem\u003eMelophorus\u003c/em\u003e species (Hymenoptera: Formicidae) from Australia. J NY Entomol Soc 105:161-169\u003c/li\u003e\n\u003cli\u003eAkino T, Knapp JJ, Thomas JA, Elmes, GW (1999) Chemical mimicry and host specificity in the butterfly \u003cem\u003eMaculinea rebeli\u003c/em\u003e, a social parasite of Myrmica ant colonies. Proc R Soc Lond B Biol Sci 266:1419-1426 \u003c/li\u003e\n\u003cli\u003eAls TD, Vila R, Kandul NP, Nash DR, et al. (2004) The evolution of alternative parasitic life histories in large blue butterflies. 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J., \u0026amp; Gordon, D. M. (2012). Nestmate recognition in ants (Hymenoptera: Formicidae): a review. \u003cem\u003eMyrmecol. News\u003c/em\u003e, 16, 101-110.\u003c/li\u003e\n\u003cli\u003eSuarez AV, Scharf HM, Reeve HK, Hauber ME (2020) Signal detection, acceptance thresholds and the evolution of animal recognition systems. Philos Trans R Soc Lond B Biol Sci 375:20190464.\u003c/li\u003e\n\u003cli\u003eTaylor JA, Tulloch D 1985. Rainfall in the wet-dry tropics: Extreme events at Darwin and similarities between years during the period 1870-1983 inclusive. Aust J Ecol 10:281-295\u003c/li\u003e\n\u003cli\u003eUboni A, Bagn\u0026egrave;res A-G, Christid\u0026egrave;s J-P, Lorenzi MC (2012) Cleptoparasites, social parasites and a common host: chemical insignificance for visiting host nests, chemical mimicry for living in. J Insect Physiol 58:1259-1264\u003c/li\u003e\n\u003cli\u003eWystrach A, Philippides A, Aurejac A, Cheng K, Graham P (2014) Visual scanning behaviours and their role in the navigation of the Australian desert ant \u003cem\u003eMelophorus bagoti\u003c/em\u003e. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 200:615-626 \u003c/li\u003e\n\u003cli\u003eZollikofer C (1994) Stepping patterns in ants. \u0026ndash; 2. Influence of body morphology. J Exp Biol 192:107-118\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chemical mimicry, cuticular hydrocarbons, Iridomyrmex reburrus, nest raiding, parasite","lastPublishedDoi":"10.21203/rs.3.rs-7361241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7361241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eMelophorus anderseni\u003c/em\u003e is a thermophilic ant species that nests near colonies of \u003cem\u003eIridomyrmex\u003c/em\u003e on which it conducts brood raids. We examined their colony founding behavior and foraging ecology to evaluate possible mechanisms that may facilitate interspecific nest raiding. Specifically, wee 1) examined colony establishment and survival, 2) patterns of worker activity (walking speed, foraging and raiding behavior), and 3) compared cuticular hydrocarbon profiles of \u003cem\u003eM. anderseni\u003c/em\u003e and \u003cem\u003eI. reburrus\u003c/em\u003e workers.\u003c/p\u003e\u003cp\u003eFollowing nuptial flights, \u003cem\u003eM. anderseni\u003c/em\u003e queens established colonies in clusters around \u003cem\u003eI. reburrus\u003c/em\u003e colonies, with an average distance to the nearest \u003cem\u003eIridomyrmex\u003c/em\u003e nest of 133.1(\u0026plusmn;\u0026thinsp;8.9) cm (range 25 to 650 cm). The average number of \u003cem\u003eM. anderseni\u003c/em\u003e queens per \u003cem\u003eIridomyrmex\u003c/em\u003e nest entrance was 8.4 (\u0026plusmn;\u0026thinsp;1.9), and the average distance to the nearest \u003cem\u003eM. anderseni\u003c/em\u003e neighbor was 44.9 (\u0026plusmn;\u0026thinsp;5.3) cm (range 10 to 233 cm). Queen survival was lower in clusters that were farther from the nearest \u003cem\u003eI. reburrus\u003c/em\u003e nest or to other \u003cem\u003eM. anderseni\u003c/em\u003e queens.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIridomyrmex reburrus\u003c/em\u003e activity was negatively related to soil temperature, with no activity occurring above 50\u0026deg;C. In contrast, \u003cem\u003eM. anderseni\u003c/em\u003e activity was positively related to temperature with peak activity occurring just above 50\u0026deg;C. \u003cem\u003eMelophorus anderseni\u003c/em\u003e worker activity commenced when soil temperatures exceeded 37.4\u0026deg;C, and activity increased until mid-day. Activity ceased in the afternoon at temperatures lower than that for activity commencement, on average at 36.7\u0026deg;C. Activity of \u003cem\u003eM. anderseni\u003c/em\u003e at \u003cem\u003eI. reburrus\u003c/em\u003e nests followed that of activity at \u003cem\u003eM. anderseni\u003c/em\u003e nests.\u003c/p\u003e\u003cp\u003e\u003cem\u003eWorkers of M. anderseni\u003c/em\u003e had an average foraging duration of 63 seconds and a mean foraging distance of 249 cm. Of the 49 foragers followed, 42 (86%) entered an \u003cem\u003eI. reburrus\u003c/em\u003e nest, and 32 (76.2%) of those entered the closest nest entrance. Of the 1,444 observations of \u003cem\u003eM. anderseni\u003c/em\u003e exiting an \u003cem\u003eI. reburrus\u003c/em\u003e nest, they stole an item in 122 (8.4%) instances, with 117 (95.9%) of these items being brood. Workers of \u003cem\u003eM. anderseni\u003c/em\u003e had faster running speeds (mean 9.72 cm/s, peak 18 cm/s) than \u003cem\u003eI. reburrus\u003c/em\u003e workers (7.05 cm/s). On average, the speed of \u003cem\u003eM. anderseni\u003c/em\u003e exiting an \u003cem\u003eI. reburrus\u003c/em\u003e nest was more than twice the speed of \u003cem\u003eI. reburrus.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eExtracts of \u003cem\u003eI. reburrus\u003c/em\u003e and \u003cem\u003eM. anderseni\u003c/em\u003e worker cuticular hydrocarbon profiles were qualitatively identical in that no compounds were unique to either species. Saturated hydrocarbons with chain lengths between 25 and 29 carbons comprised the majority of hydrocarbons found on the cuticle of both species. The relative abundances of compounds within the profiles of both species were also similar. Two other sympatric \u003cem\u003eIridomyrmex\u003c/em\u003e and \u003cem\u003eMelophorus\u003c/em\u003e species shared very few compounds with \u003cem\u003eI. reburrus\u003c/em\u003e and \u003cem\u003eM. anderseni\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eOur results support that \u003cem\u003eM. anderseni\u003c/em\u003e specializes on brood raiding from \u003cem\u003eI. reburrus\u003c/em\u003e nests. Both behavior (running speed) and chemical mimicry are likely used in combination to facilitate this specialized foraging. Additional research is still needed to determine the source (e.g. genetic versus environmental) of \u003cem\u003eM. anderseni\u0026rsquo;s\u003c/em\u003e hydrocarbon profiles, and if geographic variation in host use and chemical mimicry exists.\u003c/p\u003e","manuscriptTitle":"Ecology and mechanisms of parasitism of Iridomyrmex by the thermophilic ant, Melophorus anderseni","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 07:06:04","doi":"10.21203/rs.3.rs-7361241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-10-19T08:36:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-03T08:05:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T06:13:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T09:25:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2025-08-13T01:59:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"379c204e-426e-48cc-af56-0fd242d0254c","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T03:37:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 07:06:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7361241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7361241","identity":"rs-7361241","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}