Territorial animals are expected to adjust their response to intruders according to the perceived threat-level. One of the factors that drives threat-level is the identity of the intruder. The dear enemy phenomenon theory postulates that individuals should respond with lower intensity to neighbors, already possessing a territory, than to strangers that may fight to evict them. In social species, the hierarchical status of the intruder might also mediate this response. Such behavioral adjustments presuppose a capacity to discriminate between individuals posing different threat levels. Here, we tested the behavioral response of Alpine marmots to territorial intrusions in a wild population. We compared both dominant females and males responses to scents from neighbor and stranger dominant males (dear enemy phenomenon) and to dominant and subordinate stranger males (social status-specific response). In addition, we tested for any covariance between male scents and social status. We showed that female and male dominant marmots do not adjust the intensity of their behavioral responses to whether the intruder’s territory is bordering or not (neighbors or strangers) or to the intruder’s social status, even though dominant and subordinate males are thought to pose different threats and social status is encoded in scents. Thus, we did not find support for the dear enemy phenomenon and conclude instead that, in dominant Alpine marmots, no intruder should enter a foreign territory. Research taking a more holistic approach of the evolution and maintenance of territoriality is required to understand the flexibility of responses to intruders in group-living species.

Behavioral experiments

Experimental set-up

All behavioral experiments consisted of two-way choice trials where two vertical wooden sticks covered by glass tubes impregnated with scents were placed approximately 50 cm away from the main burrow entrance to maximize the chances to be encountered by marmots and 50 cm apart so that tested individuals could discriminate the scents on both tubes. Two clean tubes were placed on each territory at the beginning of the field season to avoid reactions to the introduction of a foreign object in the territory during each trial. We replaced these tubes by experimental tubes, i.e. tubes with scent-marks, at the beginning of each trial.

General procedure

For each trial, we randomly designated scent-marked tubes to the wooden sticks to avoid any bias due to individual preference for one side or the other. Once the tubes installed, between one and three observers continuously monitored the experimental set-up with 10x42 binoculars and/or 20x60 telescopes in order to identify individuals interacting with the tubes. As soon as a dominant female or male was in close proximity to the experimental set-up (approximately 50 cm), its identity and sex were registered and one observer recorded its behavior with a digital video camera (Sony® Handycam model DCR-DVD650 or JVC® digital video model GZ-E 209) until it moved away from the set-up (more than 5 m or complete disappearance in the burrow). A given trial was considered a success when a dominant individual interacted (i.e. smelled and/or marked) with at least one tube and was considered a failure when no dominant individual approached the experimental set-up within four hours of the installation (Cross et al. 2013), or if a subordinate interacted with at least one of the tubes before a dominant individual. In case of failure, the trial was aborted and repeated later with new scent samples.

Dear enemy behavioral experiment

This behavioral experiment was conducted from 2012 to 2015 and in 2017 and consisted of three different experimental settings: 'stranger vs. control' (SC, N = 53), 'neighbor vs. control' (NC, N = 33) and 'stranger vs. neighbor' (SN, N = 41). The first two experimental settings (SC and NC) were meant to test for a difference in behavioral response of dominant individuals between scent-marked tubes and tubes without marmot scent (i.e. 'control' tubes). This was meant to check that responses towards scent-marked tubes were due to a scent recognition and not due to the presence of a new object in the territory. The third experimental setting (SN) was meant to test for a difference in intensity of the behavioral response of dominant individuals between ‘strangers’ (i.e. tubes scent-marked by a dominant male residing within a territory that has no common boundary with the focal individual) and ‘neighbors’ (i.e. tubes scent-marked by a dominant male residing within a territory that has a common boundary with the focal individual).

Subordinate vs. dominant behavioral experiment

This behavioral experiment was conducted in 2017 and consisted in one experimental setting ('subordinate vs. dominant' (SD)) to test for a difference in the behavioral response of dominant individuals to subordinate and dominant stranger male scent-marks. We performed 16 trials to test whether unknown sexually mature male subordinates that do not yet have a territory (hypothesized as highly threatening) elicit a stronger response than unknown dominant individuals which already have one. For each trial, we used two tubes: one scent-marked by a dominant male and the other by a sexually mature subordinate male, both strangers to the focal individual. In all trials, we used subordinate and dominant individuals of the same family to limit the differences between the two individuals other than their social status (e.g. group scent signature).

Measures of response

Video recordings were displayed in Microsoft Windows Media Player (Microsoft®) in slow motion (x 0.5) to ensure an accurate identification of behaviors as well as to score their duration with an accuracy of 0.5 s. Both the time the focal dominant marmot spent smelling and marking each tube were recorded as well as the number of marks.

Chemical characterization of orbital gland secretion

Orbital gland scent collection

To analyze the chemical composition of Alpine marmot orbital gland scents, we collected orbital gland secretions from 43 male Alpine marmots of two years and older (up to 11 years old; 18 dominant individuals and 25 subordinate individuals), living in 24 different territories between 12^th May and 9^th July 2016. In addition, we re-sampled six of the 25 subordinate individuals in 2017 after they reached dominance. We collected orbital gland secretions with a 5 µL glass capillary wearing clean nitrile gloves to avoid contamination. Once collected, the secretion was then placed into a 1.5 mL opaque chromatographic glass vial filled in advance with 200 μL of dichloromethane solvent (HiPerSolv CHROMANORM for HPLC; VWR, Center Valley, PA, USA). In each 200 μL we added an internal standard, biphenyl (molecular weight, 154.21 g.mol^-1, 99.5%; Sigma Aldrich, St Louis, MO, USA) at a concentration of 0.2 g.L^-1. Several “field control samples” (i.e. vials without marmot secretions and only with solvent) were collected using the same protocol, to control for possible contamination related to the collection protocol. Finally, all samples were sealed with a Teflon-lined cap and stored at -20°C in the field and at -80°C in the laboratory until GC-MS analysis.

GC-MS analysis

We transferred all scent samples in 0.3 mL inserts in new, clean vials to enable their injection in an interfaced Hewlett-Packard 6890 GC system equipped with a non-polar DB-5 MS column (30 m long x 0.25 mm ID x 0.25 µm film thickness, Agilent technologies) coupled with an HP 5973 MSD (Mass Selective Detector) mass spectrometer (Agilent technologies, Palo Alto, CA, USA). Helium was used as a carrier gas at a flow rate of 1 mL.min^-1 and an electron impact ionisation of 70 eV was applied. After having vortexed all vials to homogenize scent samples, 2 µL of sample was injected automatically in splitless mode. The temperature of injection was set to 300 °C and the oven temperature program started with 4 min at 90 °C, then increased by 12 °C.min^-1 up to 210 °C, and then increased again at 5 °C.min^-1 up to 310 °C and finally was held at 310 °C for 5 min. We also ran blank samples containing dichloromethane only every 7 samples. These controls allow an estimation of the potential noise related to the potential accumulation of some compounds along the column or of the instrument drift over time, for example.

Chromatographic data processing

Scent secretions of individuals were characterized by several peaks (i.e. a scent profile) and each of them represents one compound (defined by a specific retention time and mass spectrum). For each sample, we acquired the area of each peak by automatic integration with Agilent MassHunter Qualitative Analysis software (B.07.01 version) and manual check. Furthermore, the internal standard (biphenyl) was used to control instrument drift over time. Three compounds were found in field control samples that were considered as contaminants and were removed from analysis. We further removed all compounds present in less than 5% of the individuals of the two groups (i.e. subordinate and dominant individuals) because their rarity meant they were unlikely to make any contribution to the discrimination of the social status in subsequent analysis. Then, we converted each single peak area into a percentage of the sum of all compounds’ area to obtain the relative abundance of each compound. Finally, we removed peaks with a relative low abundance (< 0.05%) to exclude background noise (Drea et al. 2013)⁠, and took the square-root of the final data set to reduce the impact of the most abundant compounds upon our analyses (Clarke and Warwick 2001).

For chemical data the columns correspont to:

1) the individual ID and year

2) the social status

3 to 30) retention times (in seconds) of each compound