JHR 98: 1087-1106 (2025) Bp Sy JOURNAL OR She tentonicatiante doi: 10.3897/jhr.98.170415 RESEARCH ARTICLE () I Tymenopter a 9 https://jhr.pensoft.net The Imarasional Society of Hymenopeeriss, RESEARCH Vibroacoustic signalling of the Maltese Messor ants (Hymenoptera, Formicidae), with geographical insights into M. ibericus Albena Lapeva-Gjonova', Ilia Gjonov', Rumyana Kostova', David Mifsud* I Faculty of Biology, Sofia University, 8 Dragan Tsankov str, 1164 Sofia, Bulgaria 2 Institute of Earth Systems, Division of Rural Sciences and Food Systems, University of Malta, Msida MSD 2080, Malta Corresponding author: Albena Lapeva-Gjonova (gjonova@gmail.com) Academiceditor: Francisco Hita Garcia | Received 28 August 2025 | Accepted 24 October2025 | Published 17 November2025 https./zoobank. org/B3C7233E-6A66-4D 1 E-BC1B-954DAF98404D Citation: Lapeva-Gjonova A, Gjonov I, Kostova R, Mifsud D (2025) Vibroacoustic signalling of the Maltese Messor ants (Hymenoptera, Formicidae), with geographical insights into M. ibericus. Journal of Hymenoptera Research 98: 1087-1106. https://doi.org/10.3897/jhr.98.170415 Abstract This study presents vibroacoustic recordings of the three Messor species in the Maltese archipelago, M. cap- itatus (Latreille, 1798), M. bouvieri Bondroit, 1918, and M. ibericus Santschi, 1925, and their signal characteristics were analysed. The observed patterns are consistent with the hypothesis that acoustic traits in Messor ants are subject to species-specific constraints. To gain further insight into the sound parameters of M. ibericus, geographically isolated populations from Malta, Gozo, Sicily, Crete, and Bulgaria were compared, revealing variation and significant differences at the population level. Additionally, the study provides new occurrence data for M. ibericus (queens and workers), which was previously reported as M. structor (Latreille, 1798) for Malta, along with information on its habitat preferences and activity. Keywords Biogeography, harvester ants, Mediterranean, vibroacoustics Introduction Over the last decades, increasing information has accumulated on the unexpectedly wide distribution of hidden taxonomic diversity in European ants (Seifert 2009; Csdsz et al. 2014; Seifert 2018; Steiner et al. 2018). In this context, one of the emblematic examples in this line are the ants of the genus Messor Forel, 1890, known as harvester Copyright Albena Lapeva-Gjonova et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1088 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) ants, in which intraspecific variability, polymorphism, cases of hybridisation and even xenoparity are well documented (Schlick-Steiner et al. 2006; Steiner et al. 2011, 2018; Orou et al. 2023; Juvé et al. 2025a, 2025b). The application of complementary analyses from the genetics, cuticular hydrocar- bons, ethology and ecology to standard comparative morphology and morphometrics in ants, known as integrative taxonomy, is the most convincing approach to reveal true biodiversity (Seifert 2009). In recent years, acoustic communication studies in a number of ants have shown that they can often be species-specific and has been suc- cessfully used to detect and confirm new species, especially those that are morphologi- cally indistinguishable from other closely related species (Pavan et al. 1997; Ferreira et al. 2010; Pefa Carrillo 2021). Species of Messor are known to possess stridulatory organs that produce vibrational signals by rubbing the tergal portion of the postpetiolus, known as the scraper (or plec- trum), against a finely striated region called the file (or pars stridens), located dorsally to the first metasomal segment (Grasso et al. 1998; Hernandez et al. 2002). However, only in a few species stridulation fields (SF) have been studied and documented by scanning electron microscopy. Even more limited are available records of the acoustic signals produced and their role in behaviour (e.g. Hahn and Maschwitz 1985; Priano et al. 1997; Grasso et al. 2009). Vibroacoustic signals have been analysed in a few species of the genus Messor, including M. barbarus (Linnaeus, 1767), M. capitatus (La- treille, 1798), MZ. minor (André, 1883), M. structor (Latreille, 1798), and M. wasmanni Krausse, 1910 (Grasso et al. 1999, 2000; Gjonov et al. 2025). The majority of species in the genus Messor occur in the Palaearctic, with the Medi- terranean subregion having high species diversity (Borowiec 2014). The historical ac- cumulation of many synonyms and species names makes the genus taxonomically challenging, and recent taxonomic revisions are available for two of the species groups, M. structor and M. semirufus (Steiner et al. 2018; Salata et al. 2023). To date, three species of the genus Messor have been documented from the Maltese archipelago, recorded from a considerable number of localities. These include Messor bouvieri Bondroit, 1918, Messor capitatus, and M. structor. A fourth species of the ge- nus from the island of Comino was reported by Baroni Urbani (1968) and Schembri and Collingwood (1981) and identified as M. meridionalis ssp. wasmanni Krausse and M. caducus (Victor, 1839). In the Mediterranean Region, both taxa are now recognised as M. wasmanni. However, the presence of this species in Malta raise questions about its natural occurrence, particularly given the absence of this species from the islands of Malta, Gozo, and Sicily (Baroni Urbani 1968). This study presents vibroacoustic recordings of all three Messor species typical of the Maltese archipelago, M. capitatus, M. bouvieri, and M. ibericus Santschi, 1925, the latter previously reported as M. structor for this region. In order to gain further insight into the sound parameters of workers of the species M. ibericus, a comparison between geographically isolated populations from Malta, Gozo, Sicily, Crete and Bulgaria was conducted. In addition, new occurrence data of M. ibericus from Malta and Gozo are provided. Notably, a recent discovery indicates that M. ibericus workers are hybrids Vibroacoustic signalling in Maltese Messor ants 1089 derived from queens of this species and males of M. structor (either wild-type or clonal lineages), even though /M. structor itself may be absent from the area, as has been dem- onstrated for Sicily Juvé et al. 2025a). Materials and methods Samples collection Specimens of Messor species were collected by hand throughout the islands of Malta and Gozo to assess species occurrence within the archipelago and were stored in 96% ethanol for morphological examination. They were sampled by the first author in 2017 (November), 2024 (April) and 2025 (March). Identification of the ants follow Schem- bri and Collingwood (1981) and Seifert (2018). Specimens used for vibroacoustic recordings were collected in Malta (April 2024), Crete (September 2023), Bulgaria (May 2024), and Sicily (May 2025). They were kept alive until processing, which was carried out indoors. The studied material is stored in the Zoological collection of Sofia University (BFUS). Data on the occurrence by species and nesting sam- ples of the specimens used for sound recordings are provided below, and additional details on the presence of M. ibericus in Malta are provided in Suppl. material 1. Considering the genetic complexity observed in M. ibericus (including the hybrid origin of workers) by Juvé et al. (2025a), we use this name for the worker caste as well, for consistency. Although we acknowledge that the worker caste represents hybrid individuals (MZ. ibericus x M. structor), our study is not genomic or taxonomic in scope, and therefore the use of the established species name ensures comparability with previous ecological and morphological research. Messor ibericus Santschi, 1925 Fig» 1A, B Sound recordings. Marta — Malta isl. * Gnejna Bay; 35°55.224'N, 14°20.640'E; alt. 9 m; 18 Apr. 2024; sound records: R264, R265, R266 (major workers); — Gozo isl. ¢ Ta’ Pinu; 36°03.630'N, 14°12.546'E; alt. 127 m; 20 Apr. 2024; sound records: R285, R288, R289 (major workers), R287 (queen); Butcaria * Plovdiv; 42°8.820'N, 24°45.096'E; alt. 205 m; 27 May 2024; sound records: R314, R315, R316 (major workers); GREECE ¢ Crete; Thronos; 35°16.188'N, 24°37.086'E; alt. 372 m; 19 Sep. 2023, sound records: R161, R164, R166 (major workers); [tary ¢ Sicily; Vizzini; 37°8.572'N, 14°44.682'E; alt. 395 m; 10 May 2025; sound records: RO1, RO2, ROG (major workers). Messor bouvieri Bondroit, 1918 Fig. 1C Sound recordings. Matta — Malta isl. * Ghadira; 35°58.157'N, 14°20.960'E; 4 m; 19 Apr. 2024; sound records: R276, R278, R279 (major workers). 1090 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) Figure |. Habitus of Maltese Messor ants A Messor ibericus (worker) B M. ibericus (queen) C M. bouvieri (worker) D M. capitatus (worker). Scale bars: 1 mm. Messor capitatus (Latreille, 1798) Fig. 1D Sound recordings. Marta — Malta isl. * Il-Kalanka; 35°49.483'N, 14°33.388'E, 18 m; 16 Apr. 2024; sound records: R242, R243, R248 (major workers). Photographic images Images of the specimens were taken with Nikon DS-Ri2 through Nikon SMZ 1270i stereomicroscope and then aligned and stacked using Helicon Focus 8.3.6. Pro. Stridu- lation fields were imaged by scanning electron microscope JSM-5510 at 10 kW. Sound recording and analysis The terminology in Grasso et al. (2000) was used for signal parameters. The recorded signal (presumed disturbing) consists of trains of chirps, each composed of two parts (forward and backward). The maximum frequency was measured not across the entire chirp, but separately for each part. Since the experimental setup did not allow for cali- brated measurement of absolute amplitude values, the ratio between measured ampli- tudes under constant conditions was used for the purposes of this study. Vibroacoustic signalling in Maltese Messor ants 1091 The following signal parameters were selected for comparing acoustic signals: chirp rate (chirps/s), chirp duration (ms), forward chirp duration (ms), backward chirp dura- tion (ms), maximum frequency of the forward chirp (Hz), maximum frequency of the backward chirp (Hz), and the ratio of the amplitude at maximum frequency between the forward and backward parts of the chirp. Signals were recorded using a DIY piezoelectric contact microphone, following the methodology described by Gjonov et al. (2025), and a Zoom F3 digital recorder. GNU Audacity software was used to process and measure the parameters. Noise in the record- ings was suppressed by 24 dB, using a noise sample taken from parts of the recording where no signal was present. After confirming that no recorded signals were within this frequency range, a high-pass filter at 100 Hz with a roll-off of 24 dB per octave was also applied. For each recording, a section containing a relatively long chirp train was selected. Its duration was measured in seconds, and the chirps were counted to obtain the chirp rate value. The other parameters were measured from three chirps within this section. For each of the three Messor species, across the major worker caste and differ- ent locations, recordings from three specimens were analysed, except for the queen of M. ibericus, for which only a single specimen was available; its measurements are presented but were not included in the analysis. Selected recordings are included in the Suppl. materials 2-5. Sample oscillograms and spectrograms of trains and chirps were prepared for each species, caste, and location. Statistics Principal Component Analysis (PCA) was used to visualise the variance among the samples and to reduce the dimensionality of the dataset. Variables with correlations to the axes greater than r = 0.2 were retained in the analysis. Since most of the measured acoustic variables did not follow a normal distribution, a non-parametric Permutational Multivariate Analysis of Variance (PERMANOVA) was employed, incorporating a Monte Carlo permutation procedure appropriate for small sample sizes. Two sets of PERMANOVAs were conducted to assess both over- all and pairwise differences in acoustic characteristics: (1) between Messor ant species found in Maltese Islands, and (2) among five geographically distant populations of Messor ibericus (Malta and Gozo, Sicily, Crete, Bulgaria). All data were z-score standardised to account for differences in scale and units. Analyses were based on an Euclidean distance matrix, with an unrestricted permutation of raw data— suitable for single-factor designs—as the permutation method. A total of 9999 permuta- tions were performed. To test for differences in variation around group centroids, a homo- geneity of multivariate dispersions test (PERMDISP) was applied, as variation in dispersion can influence PERMANOVA results. All groups showed significant differences (p < 0.01). Nevertheless, as PERMANOVA is robust to heterogeneity in balanced designs (i.e., equal sample sizes), the results were interpreted with caution (Anderson and Walsh 2013). To identify the most influential variables distinguishing the groups and for classifi- cation purposes, a Stepwise Discriminant Analysis (SDA) was performed. At each step, 1092 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) the variable that minimised the overall Wilks’ lambda—the proportion of total vari- ance in discriminant scores not explained by group differences—was included. Vari- ables were entered into the model if their P-value significance was < 0.05 and removed if > 0.10. A cross-validation procedure was also performed. The statistical analyses were conducted using PRIMER w7 (Clarke and Gorley 2015) and SPSS 17 (IBM 2007). Results Sound recordings — stridulation, oscilograms and spectograms, acoustic char- acteristics The stridulatory apparatus of Messor species is positioned on the dorsal surface of the postpetiole and the first metasomal tergite (Fig. 2A—D). The pars stridens, located in the mid-dorsal region of the first metasomal pretergite, is distinctly oval. Except M. bou- vieri, the stridulatory field (SF) is wider than long, with transverse parallel ridges that become denser posteriorly. The surrounding tegument displays an irregular strigulate texture of short transverse ridges, either convex or concave. Well-developed “pillars” 1eku OIE 264m JSM-s5 16 1eakW 1eku Figure 2. Stridulatory fields in Maltese Messor ants A, B MV. ibericus (worker) C Messor bouvieri (worker) D ™. capitatus (worker). Vibroacoustic signalling in Maltese Messor ants 1093 beneath the pretergite margin are clearly visible in SEM images of M. ibericus, while in M. capitatus the pars stridens is sharply delimited from the adjacent integument. When restrained, ants emit stridulatory signals through characteristic dorsoventral abdominal movements, produced either in brief bursts or in extended sequences. ‘The resulting vibrations are transmitted via the pincer. A single stroke of the plectrum across the stridulatory field generates a series of pulses, termed “chirps,” each arising from contact between the plectrum and an individual ridge. In Messor, the sequential passage of the plectrum produces a distinctive disyllabic chirp. Oscillograms and spectrograms of portions of trains and chirps from the vibroa- coustic signals of major workers of Messor bouvieri, M. capitatus, and M. ibericus are presented below (Figs 3-6), and audio files, after noise reduction and high-pass filtering, are also provided (see Suppl. materials 2-5). For M. ibericus, recordings were obtained 0.0 os 1.0 LS Messor ibericus Malta 0.005 A “=e” we” nsec dad fe Nada hh sa ALAAMAAAUIAMD ALLA eeteeeecghhihbbhidt MMMM iii oy eee bial Figure 3. Oscillogram and sonogram of stridulation distress signals in Messor ibericus (worker) A train B chirp. 1094 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) Messor ibericus queen, Malta nS ih eat IR AT Wh sa bbl Aish ¢ a Wi Lik Pinte anna a ma (a a Ihe lh tie chi i oe | CECE TTEPET ETE EE ATTY ited a i“ FRR O RT ial Ua Figure 4. Oscillogram and sonogram of stridulation distress signals in Messor ibericus (queen) A train B chirp. from a queen collected on the island of Gozo (Fig. 4) as well as from major workers sam- pled across different populations, with material from Malta, Sicily, Crete, and Bulgaria. Acoustic characteristics of the three Messor species found in Malta Selected parameters of acoustic signals were used to characterise the three species of the genus Messor found on the Maltese Islands (Table 1). PERMANOVA results indicated significant differences in the acoustic character- istics of the three Maltese Messor species (F = 15.96, p(MC) < 0.001), with post-hoc pairwise tests confirming differences between all species pairs (Suppl. material 6: tables 1, 2). The separation was clearly evident in the PCA plot (Fig. 7), where the first two axes explained 84.7% of the total variation. SDA identified chirp duration, chirp rate, Vibroacoustic signalling in Maltese Messor ants 1095 Messor bouvieri Malta Messor bouvieri Malta wate vi ie —nerapenmanemenganeneceie Tre | Figure 5. Oscillogram and sonogram of stridulation distress signals in Messor bouvieri (worker) A train B chirp. the amplitude ratio between the forward and backward chirp and backward chirp du- ration as the most important discriminative variables. Cross-validation achieved 100% classification accuracy (Suppl. material 6: tables 3, 4). Although the sample size was relatively small and may not capture the full range of variation in population-level acoustic traits, distinct species-specific patterns were apparent. M/. bouvieri exhibited the longest chirp duration (428.22 ms), the slowest chirp rate (2.31 chirps/s), and the highest forward-to-backward chirp amplitude ratio (1.17), followed by M. capitatus and M. ibericus which demonstrated the highest chirp rate of 2.98 chirps/s (Table 1). Both M. bouvieri and M. capitatus had a longer backward chirp, whereas M. iberi- cus showed a longer forward chirp. M. capitatus had the highest frequencies in both chirp parts (forward — 3777.89 Hz and backward — 3723.44 Hz), while MZ. bouvieri and M. ibericus had lower and more similar frequencies (Table 1). The lowest overall 1096 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) | Messor capitatus Malta i 7 | Figure 6. Oscillogram and sonogram of stridulation distress signals in Messor capitatus (worker) A train B chirp. variation in acoustic parameters was observed in M. bouvieri, except for chirp rate, where M. ibericus showed the lowest variability. The greatest variation in the duration of both chirp parts occurred in M. capitatus, whereas M. ibericus displayed the highest variation for all other parameters (Table 1). Acoustic characteristics of the studied Messor ibericus populations The same parameters listed in Table 1 were used to characterise the acoustic signals of Messor ibericus specimens (major workers and a queen) from geographically distant populations from Malta, Gozo, Sicily, Crete, and Bulgaria (Table 2). Acoustic parameters of M. ibericus major workers varied significantly among geographically distant populations (F = 60.76, p(MC) < 0.001), with post-hoc tests confirming differences between all pairs except the Malta-Bulgaria pair (Sup- pl. material 6: tables 5, 6). SDA identified chirp rate, backward frequency peak, Vibroacoustic signalling in Maltese Messor ants 1097 Table |. Descriptives of the studied acoustic parameters of the three Maltese Messor species. Acoustic parameter Species Sample size Range Mean Std. Deviation Std. Error Median Coeff. of (N) variation % Chirp rate (chirp/s) M. bouvieri 3 [2.09,2.48] 2.31 0.2 0.11 2.36 8.66 M. capitatus 3 [2.25,3.01] 2.73 0.42 0.24 2.93 15.38 M. ibericus 6 [2.85,3.15] 2.98 0.12 0.03 2.93 3.90 Chirp duration (ms) M. bouvieri 9 [373,488 428.22 31.44 10.48 430 7.34 M. capitatus 9 328,398 359.78 28.31 9.44 360 7.87 M. ibericus 18 [253,333 295 26.53 De 293 8.99 Forward chirp M. bouvieri 9 155,217 180.56 18.04 6.01 179 9.99 duration (ms) M. capitatus 9 109,227 150.56 50.05 16.68 130 33.24 M. ibericus 18 [151,220 180 22.08 4.82 179 12.28 Backward chirp M. bouvieri 9 [204,258 236.11 18.2 6.07 245 7.71 duration (ms) M. capitatus 9 [101,253 197.67 63.04 21.01 218 31.89 M. ibericus 18 [62,126] 96 16.92 3.69 99 17.58 Forward chirp M. bouvieri 9 3174,3284 3229 39.9 13.3 3246 1.24 frequency peak (Hz) =v. capitatus 9 [3727,3893] 3777.89 68.86 22.95 3732 1.82 M. ibericus 18 [2904,4036 3387 397.02 86.64 3283 11.72 Backward chirp M. bouvieri 9 3167,3249 3223.22 24.35 8.12 3228 0.76 frequency peak (Hz) WV. capitatus 9 [3301,3863] 3723.44 168.83 56.28 3741 4.53 M. ibericus 18 [2900,4039 3432 414.87 90.53 3601 12.09 Forward chirp M. bouvieri 9 -49.6,-44.7 -46.84 1.47 0.49 -47.1 3.13 amplitude M. capitatus 9 [-59.9,-52.3] -56.83 2.66 0.89 -57.6 4.68 M. ibericus 18 [-72,-49.6] -62.04 5.63 1.23 -62.9 14.83 Backward chirp M. bouvieri 9 [-56.1,-52.9 -54.68 0.94 0.31 -55 1.72 amplitude M. capitatus 9 [-64.6,-53.9] -59.42 3.96 1.32 -58.2 6.66 M. ibericus 18 [-75.6,-58.9 -65.45 5.44 1.19 -63.2 14.31 Amplitude forward / M. bouvieri 9 [1.13,1.22] 1.17 0.03 0.01 1.17 2.33 backward chirp ratio VY. capitatus 9 [0.99,1.13] 1.05 0.05 0.02 1.03 4.48 M. ibericus 18 [0.94,1.19] 1.06 0.07 0.01 1.05 6.62 Forward freq. peak Backward freq. peak A Backward duration ifs] 3 OD Arfiplitude ratio } } er duration © PC2 (38.5%) Wi ™. capitatus <M. bouvieri @ ™M. ibericus - Malta A J : a = M. ibericus - Gozo PC1 (46.2%) Figure 7. Principal Component Analysis (PCA) of the acoustic characteristics of Messor species from the Maltese Islands. 1098 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) Table 2. Descriptives of the studied acoustic parameters of the Messor ibericus samples. Acoustic parameter Chirp rate (chirp/s) Chirp duration (ms) Forward chirp duration (ms) Backward chirp duration (ms) Forward chirp frequency peak (Hz) Backward chirp frequency peak (Hz) Forward chirp amplitude Backward chirp amplitude Amplitude forward/back- ward chirp ratio Locality Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Malta Gozo Gozo queen Sicily Crete Bulgaria Sample size (N) iS’) OD 0 O09 WW ODO 0 0 DOD OD WD OD OD D9 OD OD WD OD OD OD OD OD WW OD OO DO OW ODO OO OO OW ODO OO 0 OD WO 0 O09 0 0 WW OO WW WD WO KH WH Range 3.06,3.15 [2.85,2.93 2.86 5.74,6.88 [5.27,5.49 [2.92,3.44 286,333 [253,314 [277,331 153,171 [151,198 [276,340 161,215 [151,220 [151,190 [76,86] [72,91] [167,221] 95,126 [62,111 [90,111 [67,91] 70,100 [82,105 [2904,3307 [3192,4036 [3653,3709 [3068,3288 [2984,3673 [3074,3140 2900,3296 3244,4039 [3601,3678 2950,3227 2966,3661 [2753,3139 [-72,-49.6] [-67.2,-61.1 [-64.6,-59.6 [-75,-57.8] [-55.5,-38.9 [-63.7,-56.8 [-75.6,-59.00] [-73.9,-61.3 [-60.8,-58.9 [-74.1,-60.6 [-66.2,-42.2 [-65.1,-54.3 1.02,1.19 0.96,1.11 [0.94,0.96 0.95,1.16 0.99,1.25 [0.94,1.07 Mean 3.10 2.90 2.86 6.33 5.41 3.19 312.67 276.44 297.67 160.33 169.89 302.78 190.67 173.78 165.67 80.44 81.78 191.33 106.56 84.67 100 76.67 78.56 96.11 3027.78 3646.22 3683.67 3165.44 3266.44 3105.67 3019.89 3779.33 3628.33 3123.11 3218.44 3032.56 -59.90 -64.19 -62 -67.39 -47.31 -59.5 -65.74 -66.96 -60.07 -69.09 -53.19 -59.08 1.10 1.04 0.97 1.03 1.12 0.99 Std. Deviation 0.04 0.03 0 0.49 0.09 0.22 16.97 22.75 29.14 5.81 15.93 21.48 16.93 23.89 21,22 3.17 7 19.97 9.98 17.68 10.54 7.65 10.88 7.04 180.17 328.07 28.38 74.17 311.9 23.43 173.40 241.91 43.09 90.74 329.17 126.58 7.87 2.35 2.51 7.01 5.97 2.67 6.56 4.13 1.02 4.54 9.51 3.78 0.06 0.05 0.03 0.06 0.07 0.04 Std. Error 0.01 0.01 0 0.17 0.03 0.07 5.66 758 16.83 1.94 5.31 7.16 5.65 7.96 12.25 1.06 2.33 6.66 3.33 5.89 6.08 2.55 3.63 239 60.06 109.36 16.38 24.72 103.97 7.81 57.80 80.64 24.88 30.25 109.72 42.19 2.62 0.78 1.45 2.34 1.99 0.89 2.19 1.38 0.59 1.51 3.17 1.26 0.02 0.02 0.02 0.02 0.02 0.01 Median 3.09 22911 0 6.37 5.45 3.22 312 281 285 160 169 301 190 156 Coeff. of Variation (%) 1.32 1.20 0 7.8 1.73 7.02 5.43 8.23 9.79 3.62 9.37 7.09 8.88 B75. 12.81 3.94 8.55 10.44 9.36 20.88 10.54 9.98 13.84 7.33 5.95 9.00 0.77 2.34 9.55 0.75 5.74 6.40 1.19 2.91 10.23 4.17 13.14 3.66 4.04 10.4 12.63 4.49 9.98 6.16 1.7 6.58 17.88 6.4 5.39 4.54 2.96 6.15 6.68 4.04 Forward freq.|peak Backward freq. peak 3 A a A Chirp rate z o a, N Siok : @@ : on uration, @ g Backward duration ‘i oO & a f - o @ Malta ee e A Gozo ¥% & © Crete @ Sicily MH Bulgaria a PC1 (60.0%) Figure 8. Principal Component Analysis (PCA) of the acoustic characteristics of Messor ibericus populations. chirp duration, backward chirp duration, amplitude ratio between the forward and backward chirp, forward chirp duration, and forward frequency peak as discrimi- native variables (Suppl. material 6: table 7). PCA ordination (Fig. 8), consistent with the PERMANOVA results, placed the Bulgarian and Maltese populations in close proximity, reflecting their similarly long total, forward, and backward chirp durations—the longest among all populations (Table 2). However, the Maltese population showed markedly higher chirp frequencies, with signals from Gozo reaching the maximum values (3646.22 Hz in the first part and 3779.33 Hz in the second). In contrast, the Bulgarian population exhibited the lowest frequencies (3105.67 Hz and 3032.56 Hz, respectively). The Sicilian population displayed the highest chirp rate, followed by the Cretan population. The Cretan population also showed the highest ratio of forward- to backward-part amplitude (Table 2). Discussion This study constitutes the first investigation of vibroacoustic signals in ants from Malta and provides a comparative analysis of signals in workers of Messor ibericus across dif- ferent geographical populations. Workers of the three species possess a well-defined stridulatory apparatus, in line with previous reports on Messor ants (e.g., Grasso et al. 1998; Hernandez et al. 2002). Notably, only M. ibericus displays tegumentary foldings, the so-called “pillars”, beneath the pars stridens, which are thought to play an amplifying role (Castro et al. 2015). 1100 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) While several species of the genus Messor have been acoustically examined in previous research (Hahn and Maschwitz 1985; Baroni Urbani et al. 1988; Grasso et al. 1999, 2000), this study presents the first audio recordings of the three Maltese species—M. capitatus, M. bouvieri, and M. ibericus—together with oscillograms and analyses of their signal parameters. It is, however, highly probable that the stridulation data reported by Grasso et al. (2000) for Messor structor actually refer to M. ibericus, as their study predates the taxonomic revision of this species group (Steiner et al. 2018). Although the sound recordings in Grasso et al. (2000) and those obtained in the present study both represent distress signals elicited by immobilization of Messor speci- mens with a thin pincer gripping the thorax, a direct comparison of the measured parameters is not feasible, as the studies employed different set and recording ranges— the ultrasonic spectrum in the former and the audible spectrum in the latter. Our results demonstrate clear acoustic divergence among the three Maltese Messor species, though this should be interpreted cautiously given the small sample sizes. The data further enhances the nature of signal differences in studied species. Messor bouvieri consistently produced the longest chirps at the slowest rate, with the highest forward-to- backward amplitude ratio. Its signals were also the most stereotyped, showing the lowest variability across most parameters. In contrast, /. capitatus was distinguished by higher frequencies in both chirp parts and showed the greatest variability in chirp part dura- tions, reflecting a more flexible or heterogeneous signaling system. M. ibericus exhibited the fastest chirps and shortest durations, with a longer forward part relative to backward one, which is the opposite to the pattern seen in M. bouvieri and M. capitatus. Notably, M. ibericus showed the lowest variability in chirp rate. These findings suggest that each species adopts a distinct signaling strategy. Such divergence may reduces acoustic overlap, especially in sympatric contexts where signal interference could hinder communication. Further geographic comparisons reveal variation and significant differences within M. ibericus, with the exception of one pair—the Maltese and Bulgarian populations. Maltese populations exhibited chirp rates, similar to those of Bulgarian populations, but markedly lower than those of Sicilian and Cretan populations. Similarly, chirp du- rations were longer in the Maltese and Bulgarian ants than in the Sicilian and Cretan ants. Frequency peaks also varied geographically: Sicilian and Bulgarian ants produced lower values, whereas Maltese ants showed higher and more variable peaks, with the Gozo population presenting the highest frequency peak. At the same time, parameters such as chirp rate and chirp duration may depend on various factors, including temperature and the individual’s physiological state, as shown in crickets (e.g. Toms 1992; Ciceran 1994; Hartbauer et al. 2012). Biogeographical and ecological notes on Messor ibericus This study provides new insights into the presence of Messor ibericus in the Maltese Islands, specifically Malta and Gozo, where it was previously considered M. structor, and documents its habitat preferences and activity. Prior to the taxonomic revision of the European Messor structor species group by Steiner et al. (2018), all records, including those from Malta, were attributed to a single Vibroacoustic signalling in Maltese Messor ants 1101 species, MV. structor. Steiner et al. (2018) provided a new approach to species discrimina- tion within the group, based not only on morphological traits, but also on geographical, genetical and ecological studies. ‘To date, M. ibericus has been documented from Spain, France, Germany, Italy, Switzerland, Slovenia, Croatia, Romania, Bulgaria, and Greece, with its northernmost occurrence in Germany (50.05°N) and its southernmost presence in Crete (34.93°N) (Steiner et al. 2019; Salata et al. 2020). The new records from Malta represent some of the southernmost localities within the species’ known distribution. Ac- cording to Schifani et al. (2021), M. ibericus may be the sole representative of this group in Italy. While MZ. structor in southern Europe is the most cold-tolerant member of the group, occurring at elevations up to 1,800 m (Borowiec and Salata 2025), M. ibericus is more typically found at lower altitudes. Unlike the other two more common Messor spe- cies in Malta, M. capitatus and M. bouvieri, which were active during the day, the workers and queens of M. ibericus in Ta Pinu (Gozo) were also active outside their nest at night. New records of Messor ibericus corroborate the species’ habitat preferences as it has been found in a dry valley in Malta (Wied Qirda), xerothermous grassland in Sicily, urban set- tlements in Plovdiv city centre in Bulgaria, garden in Crete and in crevices of sealed urban surfaces in Ta’ Pinu in Gozo. Except for urban sites, all nests were situated beneath stones. Conclusion Although our sample size for stridulation recordings was limited, the observed patterns support the hypothesis that acoustic traits in Messor ants are subject to species-specific and population constraints. Future work with larger and more geographically diverse samples, along with experiments testing behavioral responses, will be essential for de- termining the function of these acoustic signals and clarifying whether population-lev- el differences reflect variations in stridulatory organ morphology, stridulation behavior, or environmental influences on signal production. Acknowledgements We thank the Section Editor Francisco Hita Garcia and two anonymous reviewers for their valuable comments and suggestions, which helped to improve an earlier version of this manuscript. This study was supported by the National Science Fund of the Republic of Bul- garia under grant No. KP-06-N-51/6, dated 11.11.2021. References Anderson MJ, Walsh, DCI (2013) What null hypothesis are you testing? PERMANOVA, ANOSIM and the mantel test in the face of heterogeneous dispersions. Ecological Mono- graph 83: 557-574. https://doi.org/10.1890/12-2010.1 1102 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) Baroni Urbani C (1968) Studi sulla mirmecofauna d'Italia. [V. La fauna mirmecologica delle isole Maltesi ed il suo significato ecologico e biogeografico. Annali del Museo Civico di Storia Naturale “Giacomo Doria” 77: 408-559. Baroni-Urbani C, Buser MW, Schilliger, E (1988) Substrate vibration during recruitment in ant social organization. Insectes Sociaux 35: 241-250. https://doi.org/10.1007/BF02224057 Borowiec L (2014) Catalogue of ants of Europe, the Mediterranean Basin and adjacent regions (Hymenoptera: Formicidae). Genus 25(1—2): 1-340. Borowiec L, Salata S (2025) A monographic review of ants of Greece (Hymenoptera: Formi- cidae) Vol. 2. Review of the subfamily Myrmicinae except the genera Temnothorax and Tetramorium. Part 1: text. Bytom: Natural History Monographs of the Upper Silesian Mu- seum: 1-476. Castro S, Alvarez M, Munguira, ML (2015) Morphology of the stridulatory organs of Iberian myrmicine ants (Hymenoptera: Formicidae). Italian Journal of Zoology 82(3): 387-397. https://doi.org/10.1080/11250003.2015.1038323 Ciceran M, Murray AM, Rowell G (1994) Natural variation in the temporal patterning of calling song structure in the field cricket Gryllus pennsylvanicus: effects of temperature, age, mass, time of day, and nearest neighbour. Canadian Journal of Zoology 72(1): 38-42. https://doi.org/10.1139/z94-006 Clarke KR, Gorley RN (2015) PRIMER v7 User Manual/Tutorial. PRIMER-E, Ltd, 300 pp. Csdsz S, Seifert B, Miller B, Trindl! A, Schulz A, Heinze J (2014) Cryptic diversity in the Mediterranean Temnothorax lichtensteini species complex (Hymenoptera: Formicidae). Or- ganisms Diversity & Evolution 14: 75-88. https://doi.org/10.1007/s13127-013-0153-3 Ferreira RS, Poteaux C, Delabie JHC, Fresneau D, Rybak F (2010) Stridulations reveal cryp- tic speciation in neotropical sympatric ants. PLoS ONE 5(12): e15363. https://doi. org/10.1371/journal.pone.0015363 Gjonov I, Lapeva-Gjonova A, Pramatarova M (2025) Biotremological research using a DIY piezoelectric contact microphone - examples with insects. Biodiversity Data Journal 13: e143481. https://doi.org/10.3897/BDJ.13.e143481 Grasso DA, Mori A, Le Mou §, Giovannotti M, Fanfani A (1998) ‘The stridulatory organ of four Messor ant species (Hymenoptera, Formicidae). Italian Journal of Zoology 65(2): 167-174. https://doi.org/10.1080/11250009809386741 Grasso DA, Mori A, Le Mou F (1999) Recruitment and trail communication in two species of Messor ants (Hymenoptera, Formicidae). Italian Journal of Zoology 66(4): 373-378. https://doi.org/10.1080/11250009909356280 Grasso DA, Priano M, Pavan G, Mori A, Le Moli F (2000) Stridulation in four species of Mes- sor ants (Hymenoptera, Formicidae). Italian Journal of Zoology 67(3): 281-283. https:// doi.org/10.1080/11250000009356325 Hahn M, Maschwitz U (1985) Foraging strategies and recruitment behaviour in the Euro- pean harvester ant Messor rufitarsis (E.). Oecologia 68: 45-51. https://doi.org/10.1007/ BF00379472 Hartbauer M, Stabentheiner A, Romer H (2012) Signalling plasticity and energy saving in a tropical bushcricket. Journal of Comparative Physiology A 198(3): 203-217. https://doi. org/10.1007/s00359-011-0700-3 Vibroacoustic signalling in Maltese Messor ants 1103 Hernandez JM, Martinez MD, Ruiz E (2002) Descripcion del organo estridulador en Messor barbarus (Linneo, 1767) (Hymenoptera, Formicidae). Anales de Biologia 24: 167-174. Juvé Y, Lutrat C, Ha A, Weyna A., Lauroua E, Afonso Silva AC, Roux C, Schifani E, Galkowski C, Lebas C, Allio R, Stoyanov I, Galtier N, Schlick-Steiner BC, Steiner FM, Baas D, Kaufmann B, Romiguier J (2025a). One mother for two species via ob- ligate cross-species cloning in ants. Nature 646: s372-377. https://doi.org/10.1038/ s41586-025-09425-w Juvé Y, Weyna A, Lauroua E, Nidelet S, Khaldi M, Barech G, Lebas C, Rasplus J, Cruaud A, Condamine F, Romiguier J (2025b) Phylogenomics of Messor harvester ants (Hymenop- tera: Formicidae: Stenammini) unravels their biogeographical origin and diversification patterns. Systematic Entomology 1-16. https://doi.org/10.1111/syen.12693 Pavan G, Priano M, De Carli P, Fanfani A, Giovannotti M (1997) Stridulatory organ and ul- trasonic emission in certain species of ponerine ants (genus: Ectatomma and Pachycondyla, Hymenoptera, Formicidae). Bioacoustics 8(3—4): 209-221. https://doi.org/10.1080/095 24622.1997.9753363 Pefa Carrillo KI, Lorenzi MC, Brault M, Devienne P, Lachaud JP, Pavan G, Poteaux C (2021) A new putative species in the Ectatomma ruidum complex (Formicidae: Ectatomminae) produces a species-specific distress call. Bioacoustics 31(3): 332-347. https://doi.org/10.1 080/09524622.2021.1938226 Salata S, Lapeva-Gjonova A, Georgiadis C, Borowiec L (2023) Review of the Messor semiru- us complex (Hymenoptera, Formicidae) in Greece. ZooKeys 1185: 105-142. https://doi. org/10.3897/zookeys.1185.111484 Schembri SP, Collingwood CA (1981) A revision of the myrmecofauna of the Maltese Islands (Hymenoptera, Formicidae). Annali del Museo Civico di Storia Naturale “Giacomo Do- tia’ 83: 417-44). Schifani E, Nalini E, Gentile V, Alamanni F, Ancona C, Caria M, Cillo D, Bazzato E (2021) Ants of Sardinia: an updated checklist based on new faunistic, morphological and biogeo- graphical notes. Redia 104: 21-35. https://doi.org/10.19263/REDIA-104.21.03 Schillinger E, Baroni Urbani C (1985) Morphologie de lorgane de stridulation et sono- grammes comparés chez les ouvriéres de deux espéces de fourmis moissonneuses du genre Messor (Hymenoptera, Formicidae). Bulletin de la Société Vaudoise des Sciences Naturelles 77: 377-383. Schlick-Steiner BC, Steiner FM, Konrad H, Marké B, Csdész S, Heller G, Ferencz B, Sipos B, Christian E, Stauffer C (2006) More than one species of Messor harvester ants (Hymenop- tera: Formicidae) in Central Europe. European Journal of Entomology 103(2): 469-476. https://doi.org/10.14411/eje.2006.060 Seifert B (2009) Cryptic species in ants (Hymenoptera: Formicidae) revisited: we need a change in the alpha-taxonomic approach. Myrmecological News 12:149-166. Seifert B (2018) The Ants of Central and North Europe. lutra Verlags- und Vertriebsgesells- chaft, Tauer, 408 pp. Steiner FM, Seifert B, Grasso DA, Le Moli FE, Arthofer W, Stauffer C, Crozier RH, Schlick- Steiner BC (2011) Mixed colonies and hybridisation of Messor harvester ant species 1104 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) (Hymenoptera: Formicidae). Organisms Diversity & Evolution 11(2): 107-134. https:// doi.org/10.1007/s13127-011-0045-3 Steiner FM, Cs6sz S$, Marké B, Gamisch A, Rinnhofer L, Folterbauer C, Hammerle S, Stauffer C, Arthofer W, Schlick-Steiner BC (2018) Turning one into five: Integrative tax- onomy uncovers complex evolution of cryptic species in the harvester ant Messor “struc- tor’. Molecular Phylogenetics and Evolution 127: 387-404. https://doi.org/10.1016/j. ympev.2018.04.005 Toms RB (1992) Effects of temperature on chirp rates of tree crickets (Orthoptera: Oecanthi- dae). South African Journal of Zoology 27(2): 70-73. https://doi.org/10.1080/02541858 .1992.11448264 Supplementary material | Occurrence data on Messor ants from the study Author: Albena Lapeva-Gjonova Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl1 Supplementary material 2 Audio of stridulation performed by a worker of Messor ibericus Authors: Albena Lapeva-Gjonova, Ilia Gjonov Data type: wav Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl2 Vibroacoustic signalling in Maltese Messor ants 1105 Supplementary material 3 Audio of stridulation performed by a queen of Messor ibericus Authors: Albena Lapeva-Gjonova, Ilia Gjonov Data type: wav Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl3 Supplementary material 4 Audio of stridulation performed by a worker of Messor bouvieri Authors: Albena Lapeva-Gjonova, Ilia Gjonov Data type: wav Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl4 Supplementary material 5 Audio of stridulation performed by a worker of Messor capitatus Authors: Albena Lapeva-Gjonova, Ilia Gjonov Data type: wav Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl5 1106 Albena Lapeva-Gjonova et al. / Journal of Hymenoptera Research 98: 1087-1106 (2025) Supplementary material 6 Results from statistical analysis Author: Rumyana Kostova Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.98.170415.suppl6