Chemical antlers: sexual dimorphism in salivary and lacrimal glands of house mouse subspecies

Chemical antlers: sexual dimorphism in salivary and lacrimal glands of house mouse subspecies | 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 Chemical antlers: sexual dimorphism in salivary and lacrimal glands of house mouse subspecies Barbora Vošlajerová Bímová, Miloš Macholán, Denisa Buchtová, Vodičková Kepková Kateřina, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8423338/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sexual dimorphism (SD), the systematic difference in phenotype between males and females of the same species, can arise through sexual and natural selection. Although SD is traditionally associated with conspicuous traits such as body size or colouration, it may also occur in cryptic characteristics such as chemical signalling. In mammals, where olfactory communication plays a central role, SD may be reflected in differences in the size or morphology of scent glands, as well as in the abundance and composition of their secretions. Here, we investigate sexual dimorphism in the size, histology, and protein content of the submandibular and lacrimal glands in two house mouse subspecies, Mus musculus musculus and M. m. domesticus . We showed remarkable dimorphism in both glands, with males of both subspecies exhibiting larger glands, including a higher proportion of granular convoluted tubules (GCTs) in the submandibular gland. Subspecies-specific differences in gland size were detected only in the submandibular gland, which was larger in M. m. musculus . In contrast, SD was more pronounced in the lacrimal gland in both subspecies and was strongest in M. m. domesticus . Furthermore, we found subspecies-specific differences in tear protein content and odour cue preference, suggesting mate recognition systems may be more divergent between these closely related taxa than previously assumed. By integrating data from wild animals and wild-derived strains, we provide a comprehensive assessment of sex-specific morphological and biochemical divergence in these exocrine glands. Our findings underscore the evolutionary significance of cryptic sexual dimorphism in mammalian olfactory signalling systems. sexual dimorphism sexual preferences chemical communication reproductive isolation scent glands house mouse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sexual dimorphism, defined as the systematic difference in phenotype between males and females of the same species, typically reflects the action of sexual selection, although natural selection may also contribute. Sometimes, it is even difficult to distinguish between these two evolutionary forces: territory defence, for example, is important for the protection of food resources (natural selection) but can also serve females to evaluate the individual quality of a male as a potential mate (sexual selection). The interplay between these forces is central to understanding the evolution of sexually dimorphic traits and their behavioural functions. In mammals, the extent of sexual dimorphism varies greatly depending on the social structure, the mating system, and the ecological requirements. The highest sexual dimorphism in external traits is, therefore, typical for species that are polygynous, diurnal, and inhabitants of open habitats, while monogamous and nocturnal species are usually monomorphic (Huxley 1938; Wade 1979; Andersson 1994; Vanpé et al. 2008; McPherson and Chenoweth 2012). However, this distinction is probably oversimplified, as some apparently monomorphic species can exhibit only facultative monogamy or are promiscuous (Kleiman 1977; Ostfeld and Heske 1993; Isaac 2005; Kokko and Rankin 2006) or adopted alternative strategies to maximize male mating success (Dunham and Rudolf 2009). Nevertheless, dimorphism may be subtler or related to traits that are less obvious than body size, colouration, and exaggerated structures, such as peacock's tail, deer antlers, beetle horns, or the monstrous eye span of stalk-eyed flies. It is well known that many mammals use chemical cues rather than visual communication. Such species, often thought to show little sexual dimorphism, may, in fact, be extremely sexually dimorphic (Eisenberg and Kleiman 1972; Fan 1987; Blaustein 1981; Arnold and Houck 1982). Sexual dimorphism may therefore be related to differences in the size of scent glands and other organs involved in odour signalling, as well as the abundance and repertoire of scent produced (Stoddart 1974; Blaustein 1981; Luzynski et al. 2021). Indeed, there is growing evidence that various traits associated with chemical communication exhibit sexual dimorphism in many mammalian species previously considered to be sexually monomorphic (Jannett 1986; Maico et al. 2001; Rosell and Schulte 2004; Macdonald and Herrera 2013; Spence-Aizenberg et al. 2018; Muñoz-Romo et al. 2021). This phenomenon has remained largely overlooked, partly because many of these species are nocturnal and thus challenging to study. Moreover, scent-related tissues and odours are often hidden for humans. Typical examples of animals displaying such cryptic sexual dimorphism are small rodents, including one of the most commonly used model organisms – the house mouse ( Mus musculus ). When considering usual sexually dimorphic traits, male house mice are generally reported to be heavier than females (Eisen and Legates 1966; Nomaguchi and Sakurai 1993; Slábová and Frynta 2007; Piálek et al. 2008; Haisová-Slábová et al. 2010; Ruff et al. 2017; Panti-May et al. 2018; The Jackson Laboratory 2024), although the weight differences are not always significant. In addition, no sexual size dimorphism in cranial and dental variables has been observed in this species (Macholán 1996; Sans-Fuentes et al. 2009; Csanády and Mošanský 2018). In contrast, mice exhibit substantial intersexual differences in behaviour, much of which is related to odours and olfaction, such as aggression, mating, and parental care (Kimchi et al. 2007; Stowers and Liberles 2016; Ishii and Touhara 2019). In this context, it is startling that sexual dimorphism of olfactory organs has received little attention. A growing body of research suggests that sexually dimorphic behaviours are more likely to be associated with dimorphism in olfactory signals, whereas the expression of receptors in relevant olfactory tissues shows minimal differences between males and females (Holy et al. 2000; Kimchi et al. 2007; Kimoto et al. 2007; Ben-Shaul et al. 2010; Ibarra-Soria et al. 2014; Kuntová et al. 2018). Mouse odours are complex mixtures of volatile and non-volatile chemicals found in skin secretions, urine, tears, saliva and faeces. They are known to differ considerably between males and females in their quantities, chemical composition and proportions of individual components (Laukaitis et al. 2005; Mucignat-Caretta et al. 2010; Karn and Laukaitis 2011, 2015; Karn et al. 2014; Stopka et al. 2016; Stopková et al. 2016, 2017, 2023; Kuntová et al. 2018; Matejková et al. 2024) with some signals being reported as sexually dimorphic or even sex-specific (Kimoto et al. 2005, 2007; Roberts et al. 2010, 2012; Karn and Laukaitis 2015; Karn et al. 2014; Stopková et al. 2016; Stopková et al. 2023). Although the main source of odour cues in mice is undoubtedly urine (Hurst et al. 2001), we have focused on tears and saliva because, as products of glands in the orofacial region, they are important sources of signals in close contact communication (Luo et al. 2003; Bímová et al. 2009). Peptide pheromones from these fluids are dropped on the ground or remain on the body surface allowing signal transmission between consubspecifics exclusively by direct contact (Cavalier et al 2014). Interestingly, 16–21% of salivary proteins and 15% of tear proteins are sexually dimorphic (Karn and Laukaitis 2011; Stopka et al. 2016; Stopková et al. 2017). Similar sexual dimorphism was also found at the transcriptional level (Stopková et al. 2016; Kuntová et al. 2018). Among these sexually dimorphic compounds, lipocalins (such as MUPs, OBPs, and ESPs) and the androgen-binding protein subfamily of secretoglobins are the most abundant. Some of the urinary and salivary signals also differ qualitatively and/or quantitatively between different house mouse subspecies and have been proposed as important cues for subspecies-specific mate recognition (Laukaitis et al. 1997; Bímová et al. 2005; Stopková et al. 2007) and thus as significant contributors to the establishment and maintenance of reproductive barriers between house mouse subspecies (Vošlajerová Bímová et al. 2011; Laukaitis and Karn 2012). In this context, the potential role of tear-based signals in subspecies-specific communication remains largely unexplored. In this study, we investigated sexual dimorphism in size, histology, and overall protein excretion in submandibular and lacrimal glands, as well as the behaviour associated with their products. To capture potential intraspecies differentiation, we compared two house mouse subspecies, Mus musculus musculus and M. m. domesticus (see Baird and Macholán 2012, for a review). The two taxa differ not only genetically, but also in several behavioural strategies, including male aggression (Thuessen 1977; van Zegeren and van Oortmerssen 1981; Ďureje et al. 2011; Latour and Ganem 2017), mate choice (Smadja et al. 2004; Bímová et al. 2005; Vošlajerová Bímová et al. 2011; Ganem 2012), dispersal (Hiadlovská et al. 2012, 2013; Vošlajerová Bímová et al. 2016) and establishment of social structure (Hiadlovská et al. 2015, 2021; Mikula et al. 2022; Macholán et al. 2023; Bendová et al. 2024). We employed both wild animals and inbred wild-derived strains representing the two subspecies. Our study adds another piece of puzzle to the underlying mechanisms and ecological implications that determine the morphological divergence between male and female individuals in these glandular structures. Specifically, we test the hypothesis that morphological and chemical sexual dimorphism in exocrine glands is associated with subspecies-specific variation in mate recognition systems, potentially contributing to reproductive isolation between taxa. By investigating sexual dimorphism within this specific anatomical and behavioural context, we aim to enhance our understanding of its role in the mechanisms driving evolutionary divergence. Materials and Methods House mice Wild mice Wild mice of two subspecies, western European Mus musculus domesticus and eastern European M. m. musculus , were collected during three trapping sessions (June 2019, October 2019, October 2022) in northeastern Bavaria (Germany) and the western part of the Czech Republic (Supplementary Table S1, Figure 1B). All sampling sites were located more than 30 km away from the centre of the hybrid zone between the two subspecies (Macholán et al. 2007; Baird and Macholán 2012; Ďureje et al. 2012). The mice were sacrificed and dissected in a field laboratory next day after capture. All animals were also genotyped with diagnostic molecular markers (Macholán et al. 2007) to confirm their subspecific status. Hybrids, juvenile individuals (body weight < 10 g), and pregnant females were excluded from subsequent analyses. In total, we analysed 98 wild mice from 18 localities: 56 M. m. domesticus (31 males, 25 females; 8 localities) and 42 M. m. musculus (26 males, 16 females; 10 localities) (Table S1). Wild-derived strains of mice (WDS) We also used inbred mice of 11 WDS representing both subspecies (Piálek et al. 2025, https://housemice.cz/cs), purchased from the Institute of Vertebrate Biology, Czech Academy of Sciences, Studenec (Supplementary Table S2, Figure 1A). Mice were weaned at 20 days, singly housed, and sacrificed at adulthood (85–150 days), following the same protocol as in wild mice. In total, we examined 174 mice: 80 individuals (42 males, 38 females) of five strains derived from M. m. domesticus and 94 individuals (48 males, 46 females) of six strains derived from M. m. musculus . Sample collection Before dissection, saliva and tears were collected for the protein content analysis and as signals in subsequent behavioural experiments. The samples were gathered in the native state without sedation or stimulation using mouthwash and eyewash with 20 µl of normal saline. Mouthwash was carried out by pipetting 10 µl of saline into the oral cavity of the mouse, rinsing gently, and aspirating back into the pipette. This procedure was repeated twice. In the case of eyewash, 10 µl of saline was instilled into the lateral canthus, the eye was gently stimulated to induce lacrimation, and the fluid was aspirated back into the pipette (Petznik et al. 2011). The procedure was repeated for the other eye. All saliva samples with visible food or blood residues were excluded from further analyses. The samples were immediately stored at -80 °C. The mice were then sacrificed by cervical dislocation and dissected; both right and left parts of the submandibular salivary glands (SMG) and exorbital lacrimal glands (LAC) were collected free of visible adipose tissue and ligaments, and weighed with an accuracy of 0.0001 g. The relative mass was calculated for each gland as the ratio of the gland mass and total body mass (measured with an accuracy of 0.01 g). Data normality was tested using Kolmogorov-Smirnov tests, followed by factorial ANOVA to test the effects of sex and/or subspecies and their interaction on the relative mass of SMG and LAC. Calculations were performed using STATISTICA 6.0. Histology of salivary glands For the SMG gland histology, we used four WDSs: domesticus -derived STRA and DROS, and musculus -derived BUSNA and MBK, with four animals per sex. In each individual, we randomly (but in a balanced design) sampled either the left or right part of the paired gland. The tissues were fixed in 4% buffered formalin for 24 hours at room temperature and then stored in 70% ethanol until further processing (up to 2 weeks). After embedding in paraffin, the 5-μm sections were prepared from the central part of the gland. From each gland, 6 sections (25 μm apart, keeping every 5th section) were stained with haematoxylin/eosin. Three high-quality microscopic fields, preferably chosen from each odd-numbered section, were photographed using the Olympus BX51 microscope and DP71 camera. After training on granular convoluted tubule (GCT) histology (Mori et al. 2011; Scudamore 2014), we quantified the GCT tissue percentage in each image using the FiJi biological image analysis software (Schindelin et al. 2012). We calculated the mean percentage of the GCT area for each individual based on average measures acquired from three different sections. Data normality was tested using Kolmogorov-Smirnov tests, followed by factorial ANOVAto test for the effect of sex and/or subspecies and their interaction on relative GCT proportion in the SMG gland. The calculations were carried out with the STATISTICA 6.0 software. The LAC histology was not examined because the extraorbital lacrimal gland in mice consists predominantly of acinar cells and myoepithelial cells, with excretory tubules, but the tubular structure of this gland is not as differentiated in the mouse as SMG (Scudamore 2014; Makarenkova et al. 2015). Protein content analysis We quantified the total protein content in tear and saliva samples from four WDS: STRA, DROS ( domesticus -derived) and BUSNA, MBK ( musculus -derived). We collected samples from up to 6 animals per sex and strain, totaling 39 saliva samples and 48 tear samples. Protein quantification was performed with the micro-bicinchoninic acid (BCA) reaction and spectrophotometry (Smith et al. 1985; Noble and Bailey 2009; Kielkopf et al. 2020) using the BCA Pierce microassay kit (Thermo Fisher 23235). Initially, we roughly estimated the total protein concentration in each sample (Kielkopf et al. 2020) by measuring UV absorbance at 280 nm using the Take3 Micro-Volume Plate on the BioTek Synergy HTX reader and the Gen5 software v. 2.09. Based on these measurements, we adjusted the subsequent dilution of the samples for the micro-BCA reaction so that the expected concentration fell above the quantification limit of the microplate format of the BCA microassay kit (2 µg/ml). This corresponded to 3.5 µl of the samples in 150 µl of saline. Protein standards ranging from 1 to 200 µg/ml (Supplementary Material Table S3) were prepared using the Protein Assay standard BSA set (Thermofisher 23208). The BCA microassay was performed according to the manufacturer’s protocol. All samples and standards were analyzed in duplicates. Absorbance at 562 nm was measured using the BioTek Synergy HTX reader with Gen5 2.09. The protein concentrations were calculated using the dilution curve of the known standards. The data were log-transformed to ensure their normality and then tested with the Kolmogorov-Smirnov test. A factorial ANOVA was performed to examine the influence of sex and/or subspecies on total protein concentrations in the samples of saliva and tears. STATISTICA 6.0 was used for statistical analyses, with the significance level set to 0.05 for all tests performed. When significant interactions or main effects were detected, Tukey’s HSD post-hoc tests were carried out to determine specific group differences. Test of discrimination and preferences of tear cues To test the behavioural relevance of the potential role of tears as signals in mouse communication, we used 42 mice from two WDSs as signal donors and recipients: DROS ( domesticus -derived; 10 males and 10 females) and MBK ( musculus -derived; 11 males and 11 females) (Fig. 1 A). The animals were subject to a simple two-way discrimination/preference test using a Y-maze. All tested mice were adults, at least 90 days old, weaned at 20 days of age, kept either separately or with one same-sex littermate, and later isolated at the age of 55–60 days. The experimental setup consisted of a habituating box connected to the stem of the Y-maze with one-way air circulation (for more details, see Talley et al. 2001; Bímová et al. 2005, 2009). Each tested individual was allowed to choose between a pair of signal cues consisting, respectively, of 10 µl of normal saline (control) and 10 µl of eyewash representing a cue with tears as a potential signal. We used opposite-sex stimuli; both signal donors and tested individuals came from the same WDS. As the cue, we used the 10 µl aliquot mixture of eyewash taken from 3–6 adult individuals of the same strain and sex. The use of the mixture eliminated the influence of individual odour (age, physical condition) while preserving potential information about sex and subspecies. The collected samples were stored at −80 °C, thawed and mixed just before use, and kept on ice throughout the experiment. Prior to testing, 10 µl of each sample was pipetted on the sterile filter paper and placed at the end of the arms of the Y-maze. The tested mouse was habituated for 15 min in the habituation box separated by a perforated door from the maze. Then the door was opened, and the mouse was allowed to enter. The experiment, lasting for 5 min after the mouse reached the zone of decision in the central part of the Y-maze, was video-recorded and further analysed. All experiments were carried out during the light phase of the day. The ability of each individual to discriminate and prefer tear cues was assessed according to Smadja and Ganem (2002; see also Bímová et al. 2005; Vošlajerová Bímová et al. 2011). We tested the first choice of the signal as the first cue the mouse had chosen to investigate. In addition, we calculated the coefficient of preference as the relative difference of times spent sniffing one of the cues, T tears or T saline : R signal = (T tears – T saline )/(T tears + T saline ). We thus recognize no preference if R signal = 0; saline was considered as preferred when R signal was significantly negative (-1–0), while negative values (0–1) suggested tear preference. The difference of R signal from 0 was tested with a t-test (H 0 : μ = 0); the χ 2 test was used for comparison of the first chosen signals. The data were tested for normality using the Kolmogorov–Smirnov test and processed using STATISTICA 6.0. Ethical note The WDS stocks used in this study are part of an extensive WDS collection of the Institute of Vertebrate Biology of the Czech Academy of Sciences in Studenec (Piálek et al. 2025; www.housemice.cz), where behavioural experiments were carried out. Mice were kept in plastic cages (VELAZ, 43 × 30 × 22 cm) at an average temperature of 22 °C under the 14:10 light:dark regime (lights on at 7 am and off at 9 pm), with shavings litter and nesting material available. Food and water were supplied ad libitum . The facility is accredited for breeding (48389/2020-MZE-18134, 2021–2026) and use (62065/2017-MZE-17214, 2017–2022) of experimental animals. The animals were handled by authorized persons only (Licenses No. ZH: CZ 0127; BVB: CZ01293 and DB as a university student under supervision). This study was performed in accordance with Czech law implementing all corresponding EU regulations and approved by the IVB Ethical Committee. Results Gland size Wild mice As locality had no significant effect within subspecies (nested ANOVA, locality nested within sex and subspecies; SMG: F (1,28) = 0.736, p = 0.814; LAC: F (1,28) = 1.167, p = 0.298), data were pooled by subspecies for subsequent analyses. Males had significantly higher relative (gland-to-body) weights than females for both the SMG and LAC (factorial ANOVA: SMG: F( 1,94 ) = 4.073, p = 0.046; LAC: F( 1,94 ) = 19.004, p < 0.001). The magnitude of sexual dimorphism differed between subspecies, with a stronger effect observed in M. m. domesticus , which was significant for the LAC gland (Fig. 2; Supplementary Table S4). We also detected significant differences in SMG size between the two subspecies, with both males and females of M. m. musculus exhibiting larger glands than those of M. m. domesticus (factorial ANOVA: F( 1,94 ) = 27.710, p < 0.001). In contrast, no intersubspecific difference was detected in LAC size (factorial ANOVA: F( 1,94 ) = 0.478, p = 0.492; see Supplementary Tables S1 and S4). WDS mice In WDSs, the pattern was similar to that observed in wild mice: males of both subspecies had significantly larger SMG and LAC glands than females (factorial ANOVA: SMG: F( 1,170 ) = 26.919, p < 0.001; LAC: F( 1,168 ) = 109.914, p < 0.001). Again, sexual dimorphism was more pronounced in domesticus -derived strains than in musculus -derived strains for both glands (Fig. 3, Supplementary material Table S5). As in wild mice, musculus -derived WDSs tended to have slightly larger glands than domesticus -derived WDSs, although these differences were not statistically significant (factorial ANOVA: SMG: F( 1,170 ) = 2.772, p = 0.098; LAC: F( 1,168 ) = 1.067, p = 0.303). To facilitate comparison between WDSs and wild mice, strains were pooled within subspecies. However, substantial variation among individual stocks was observed (Supplementary Fig. S1), partly reflecting differences in sample size among strains (see Supplementary Table S2 for details). Histology Histological analysis was consistent with the patterns of sexual dimorphism inferred from gland mass measurements. The relative amount of granular convoluted tubular tissue (GCT) in the SMG differed between sexes and subspecies (factorial ANOVA: sex: F( 1,28 ) = 197.085, p < 0.001; subspecies: F( 1,28 ) = 55.99, p < 0.001; subspecies × sex interaction: F( 1,28 ) = 9.695, p = 0.004). The proportion of GCT was significantly higher in males than in females of both subspecies, and musculus -derived strains of both sexes exhibited higher proportion of GCT than domesticus -derived strains (Fig. 4, Supplementary Table S6). Greater development of sexual dimorphism in the proportion of GCT was found in the domesticus strains, corresponding to trends observed in relative gland size analysis (Tukey HSD test, Supplementary Table S6). We also analysed SMG glands from three males and three females of the classical laboratory strain C57BL/6J (hereafter BL6), which has well-described gland histology and established patterns of GCT in both sexes (Brown et al. 2020; Chung et al. 2017; Jayasinghe et al. 1990), and used these animals as a control group. As expected, the relative GCT amount in BL6 mice was similar to that observed in domesticus -derived strains (not shown in Fig. 4), consistent with the prevailing genetic background of this strain (Yang et al. 2011). This result supports the validity of our method (Supplementary Fig. S2). Protein content Total protein content in saliva and tears did not differ significantly between males and females in either subspecies (factorial ANOVA: saliva: F( 1,35 ) = 0.098, p = 0.756; tears: F( 1,44 ) = 1.213, p = 0.277; Supplementary Table S7). When comparing the two subspecies, total protein content in tear samples was significantly higher in M. m. domesticus than in M. m. musculus in both sexes, whereas no significant difference was detected in saliva (factorial ANOVA: saliva: F( 1,35 ) = 0.165, p = 0.687; tears: F( 1,44 ) = 16.565, p < 0.001; Fig. 5; Supplementary Table S7). Tear-based discrimination and preference In the two-way choice test using tears and saline as olfactory cues, a statistically significant preference for tear cues was detected in the domesticus -derived DROS strain. This effect was observed in both females and males when preference was quantified using the coefficient of preference (R_signal > 0; t-test against zero: females: t = 3.174, df = 9, p = 0.011; males: t = 2.350, df = 9, p = 0.043; Fig. 6; Supplementary Table S8). In contrast, no significant preference for tears was detected in the musculus -derived MBK strain. Discussion Sexual dimorphism in salivary and lacrimal gland morphology In this study, we examined sexual dimorphism in the submandibular salivary and exorbital lacrimal glands of two house mouse subspecies using wild mice and wild-derived strains. We focused on gland size, SMG histology, and total protein content of glandular secretions. We also tested whether tear-derived cues may contribute to chemical communication in the two mouse subspecies. Our results showed that males of both subspecies, in both wild mice and WDSs, possess larger salivary and lacrimal glands than females. In addition, males and females of M. m. musculus tended to have larger glands than those of M. m. domesticus , although this difference was statistically significant only for the SMG in wild mice. Histological analysis confirmed a strong male-biased sexual dimorphism in the proportion of granular convoluted tubules (GCT) in the SMG (Chrètien 1977; Gresik 1980; Gresik et al. 1996). This represents one of the best-studied examples of sexual dimorphism in mouse organs involved in chemical communication. GCT has been examined using a wide range of approaches, from neuroanatomical analyses to the detection of enzymes present in salivary secretions (reviewed in Gresik 1994; Pinkstaff 1998). However, most previous studies have relied on classical inbred laboratory strains such as BL6. To our knowledge, the present study is the first to address this aspect using wild mice and wild-derived strains. Thus, our results move beyond classical laboratory models and document GCT sexual dimorphism in the house mouse as a biological species, including natural variation between subspecies that is not represented in standard inbred strains (Guénet and Bonhomme 2003; Didion and Pardo-Manuel de Villena 2013). By comparing two distinct subspecies, we incorporated intraspecific variation, allowing the results to be interpreted in an evolutionary context. GCT is a unique, rodent-specific structure of SMG (Amano et al. 2012), interspersed with the striated and intercalated ducts. In males, GCT is markedly hypertrophic and characterized by large secretory granules. This hypertrophy is primarily driven by the influence of adrenocortical, thyroid, and sex hormones (Gresik 1994; Gresik et al. 1996; Señorale-Pose et al. 1998; Kurabuchi et al. 1999, 2009, 2019). Especially in adulthood, with 40% more GCT in males than in females (Jayasinghe et al. 1990; Señorale-Pose et al.1998, Tandler et al. 2001), the dimorphism is rather striking. Our study revealed similar results in the WDS males under study. Importantly, larger SMG glands with more strongly hypertrophied GCTs were found in WDSs representing M. m. musculus , a subspecies previously shown to exhibit consistently higher testosterone levels (Hiadlovská et al. 2015). These results are consistent with sex-specific differences in SMG gene expression reported by Mukaibo et al. (2019), further supporting the role of these glands in the production of sexual signals (Brown et al. 2020). Inclusion of the well-characterized classical laboratory strain BL6 as a control validated the accuracy of our results, indicating that the observed patterns are not artifacts of specific strains studied (Phifer-Rixey and Nachman 2015; Piálek et al. 2025). Although the fundamental function of the salivary glands is the production of primary saliva, which lubricates food, enables mastication, and participates in food digestion (Junqueira et al. 1964), GCT cells also synthesize a large variety of biologically active polypeptides, including epidermal growth factor (EGF), nerve growth factor (NGF), renin, and kallikreins (Kurabuchi et al. 1999; 2002; 2019; Tandler et al. 2001). Particularly, kallikreins have been suggested to play a role in chemical communication (Karn and Laukaitis 2011). Due to the internal connection of the nasolacrimal duct, tear proteins can enter the oral cavity (Ruberte et al. 2017) and both salivary and tear proteins can be transferred to fur and nesting material through grooming and cleaning behaviours (Laukaitis et al. 1997; Stopka et al. 2016; Stopková et al. 2017; Barabas et al. 2019, 2022). Barabas et al. (2022) identified several groups of salivary and tear proteins in the nest material that modulate aggressive and prosocial behaviours. These findings support the idea that both tear and salivary proteins can, at least in part, function as olfactory cues even outside direct physical contact and reinforce the notion that glandular dimorphism may contribute to sexual selection and mate signalling (McPherson and Chenoweth, 2012), acting as ‘chemical antlers’ – functional analogues of conspicuous sexually dimorphic traits such as deer’s antlers. Subspecies differences in chemical signalling strategies While many salivary and tear proteins show quantitative differences in overall abundance and expression profiles between sexes (Mucignat Caretta and Caretta 2014; Stopka et al. 2016; Stopková et al. 2016, 2017, 2023; Chung et al. 2017; Kuntová et al. 2017), there are also marked qualitative differences in the protein composition (Roberts et al. 2010, 2012; Kimoto et al. 2005, 2007; Karn and Laukaitis 2014; Stopka et al. 2016; Stopková et al. 2016, 2017; Chung et al. 2017; Karn et al. 2021). Together, these findings indicate that both qualitative and quantitative differences are important to understanding sexual dimorphism in these exocrine glands and their secretions. In contrast, the significantly higher tear protein content observed in WDSs representing one of the subspecies ( M. m. domesticus ) in both sexes suggests that overall protein excretion may be influenced more strongly by subspecies-specific factors than by sex alone (Rollins et al. 2017). In light of generally larger SMG and LAC and a higher proportion of GCT in males than in females, the absence of sexual dimorphism in the total protein content of saliva and tears is notable. Moreover, despite the similar size of lacrimal glands in the two subspecies, we found a significantly higher total tear protein content in M. m. domesticus than in M. m. musculus . These observations suggest that the morphological differences may not directly translate to differences in overall protein output, potentially due to yet unidentified factors regulating protein concentration (Stopka et al. 2016; Proctor et al. 2021; Stopková et al. 2023). However, this apparent discordance may have another, perhaps simpler, explanation. Stopka et al. (2016) and Barabas et al. 2022 detected secretoglobins and other proteins in saliva, that are expressed exclusively in the lacrimal gland, indicating that tears and tear proteins can be transported to the oral cavity, where they mix with saliva. Under this scenario, a higher protein content in the tears of M. m. domesticus , particularly in males (Fig. 5A), could partially offset the higher production of salivary proteins in M. m. musculus associated with larger SMG size (Figs. 2–3) and higher proportion of GCT (Fig. 4). Our behavioural tests revealed a preference for tears as olfactory cues relative to saline, which was statistically significant only in the domesticus -derived WDS. This finding suggests that tear-derived cues may contribute to social interactions and mate-related behaviours, consistent with previous studies demonstrating the behavioural relevance of lacrimal gland products (Kimoto et al. 2005, 2007; Stopková et al. 2016). The observed divergence between the WDSs may therefore reflect differences in the relative contribution of tear cues to behavioural strategies and sexual communication in the two taxa. While many semiochemical proteins are known to differ between the two subspecies in olfactory-related secretions, the functional significance of this divergence beyond subspecies recognition remains incompletely understood (Stopková et al. 2007; Vošlajerová Bímová et al. 2011; Hurst et al. 2017). In this context, the absence of significant preference to tears over saline in the musculus -derived WDS may indicate a reduced reliance on lacrimal olfactory cues in this subspecies under the tested conditions. Similar subspecies- or species-specific differences in the use of olfactory cues have also been reported in studies of scent-marking behaviour and olfactory strategies in other mammals (Mykytowycz and Goodrich 1974; Barja and de Miguel, 2010; Becker et al. 2018). Evolutionary implications of sexually dimorphic chemical signalling Our study represents the first comparison of sexual dimorphism in the morphology of glands involved in olfactory communication between two closely related taxa. This allows our findings to be discussed within the broader context of species divergence, which in house mice has been documented across multiple behavioural, physiological, and chemical traits. The differences in gland sizes and especially sexual size dimorphism, consistently observed both in wild and wild-derived strains of M. m. musculus and M. m. domesticus , are likely shaped by a combination of genetic, physiological, and ecological factors (Badayaev et al. 2002; Williams and Caroll 2009). Although the social structure of house mice can vary depending on the ecological context (Berry 1981; Sage 1981; Singleton and Krebs 2007), the two subspecies largely share the same synanthropic (commensal) niche (Sage 1981; Boursot et al. 1993; Macholán et al. 2012). Nevertheless, they differ markedly in several social and behavioural characteristics that are relevant to chemical communication and results of this study. For example, M. m. domesticus exhibits higher levels of aggression, more strongly substructured populations and more rapid establishment of social hierarchies than M. m. musculus (Thuessen 1977; van Zegeren and van Oortmerssen 1981; Ďureje et al. 2011; Hiadlovská et al. 2015; Latour and Ganem 2017; Vošlajerová Bímová et al. 2020; Hiadlovská et al. 2021; Mikula et al. 2022; Macholán et al. 2023; Bendová et al. 2024). Rapid establishment of social rank, supported by effective social communication, can reduce stress and contribute to the stability of the group (Koolhaas et al. 1999; Parmigiani et al. 1999; Hurst and Beynon 2004; Mucignat Caretta and Caretta 2014; Barabas et al. 2021, 2022; Yu et al. 2024). Differences in social organization and competitive interactions may therefore influence the selective pressures acting on communication systems. Our findings suggest that M. m. domesticus may experience stronger sexual selection pressures than M. m. musculus , possibly driven by more intense competition for mates or more rigid, hierarchy-dependent social interactions. Thus these taxa may face different social pressures that drive the evolution of more specialized communication mechanisms and more pronounced sexual dimorphism in glandular structures (Isaak 2005; Kokko and Rankin 2006). This is corroborated, besides higher aggression and tighter social organization, by larger differences between males and females revealed for several analysed traits in M. m. domesticus (Figs. 2–4). In this context, our results are consistent with the idea that the two subspecies differ in the relative emphasis placed on distinct chemical signalling channels. Previous work has shown that M. m. musculus produces a higher amount of major urinary proteins than M. m. domesticus and exhibits greater diversity and sexual dimorphism in urinary signalling (Stopková et al. 2007; Stopka et al. 2012; Hurst et al. 2017; Sheehan et al 2019, Penn et al 2022). Furthermore, the former subspecies possesses larger submandibular glands and larger GCT, whereas the latter showed a higher volume of tear proteins and a preference for tears over saline (this study). These findings are consistent with higher choosiness of M. m. musculus males and females based on urine (Smadja and Ganem 2002; Smadja et al. 2004; Bímová et al. 2005; Vošlajerová Bímová et al. 2011) and saliva (Vošlajerová Bímová et al. 2011) used as signals. Altogether, this leads us to assume higher importance of tears for M. m. domesticus , in contrast to M. m. musculus , which relies more heavily on urine and saliva (though urine is still a primary source of information for both subspecies, especially when mice are not in close contact). The question of why, then, M. m. musculus has also slightly larger lacrimal glands than M. m. domesticus, even though the difference was not found to be significant in our data, contrary to SMG difference, is yet to be answered. However, it should be pointed out that proteins only constitute 0.3–2% of the total tear volume (depending on the physiological context; Van Haeringen 1981). Finally, we can hypothesize that while musculus males and females are choosier based on urinary and salivary cues, M. m. domesticus may display assortative preference when tears are involved. We want to emphasise that the lacrimal signals were never tested as cues in mate choice in mice. This prediction can be easily tested using a simple Y-maze experiment contrasting heterosubspecific cues in the same way as in all previous studies (see Vošlajerová Bímová et al. 2011). Our results thus highlight how even subtle differences in mating systems and social structures can shape the evolution of sexual dimorphism and divergence of olfactory communication pathway and contribute to understanding how chemical signalling traits may diverge during subspecies differentiation, potentially playing a role in reproductive isolation. Cryptic sexual dimorphism and chemical communication in mammals Our study highlights the importance of integrating morphological, histological, physiological, and behavioural data to understand sexual dimorphism in chemical communication systems. As noted by Eisenberg and Kleiman (1972), the role of chemical signals in mammalian communication can be subtle yet significant, influencing both intra- and inter-sexual interactions. Sexual dimorphism emerging as a consequence of these interactions, shaped by natural and/or sexual selection, can then be rather cryptic. Nevertheless, these ‘chemical antlers’, as we call them, can be as important as (or even more significant than) widely known conspicuous sexually dimorphic structures of many vertebrate and invertebrate species, especially in macrosmatic organisms like house mice. Indeed, a number of studies have documented that species – often considered to show little or no sexual dimorphism – are, in fact, sexually dimorphic (Eisenberg and Kleiman 1972; Fan 1987; Blaustein 1981; Arnold and Houck 1982; Jannett 1986; Maico et al. 2001; Rosell and Schulte 2004; Macdonald and Herrera 2013; Spence-Aizenberg et al. 2018; Muñoz-Romo et al. 2021; this study). Therefore, the size and morphology of the secretory organs can provide a valuable insight into sexual dimorphism, especially when supplemented with other approaches. The scent gland sexual dimorphism is widely known in other rodents (Rosell and Schulte 2004; Macdonald and Herrera 2013; Muñoz-Romo et al. 2021; Rodriguez et al. 2023) but in mice, it has been so far largely neglected, with focus being rather on the chemical nature of the excretions (Hurst and Beynon 2004; Chung et al. 2017) or neurological pathways (Bergan et al. 2014; van der Linden et al. 2018). However, the link between sexual dimorphism and olfaction has been recently reported in a study examining more than 100 mammal species, including the mouse (Tombak et al. 2024). There is a growing body of evidence, that the molecular underpinnings of sexual dimorphism have mainly centred on expression of genes (Loire et al. 2017) that exhibit rapid evolution (Harissin et al. 2015). High levels of dimorphism are shown to correlate with expansion of gene families enriched in olfactory sensory perception (Van der Linen et al. 2018; Padilla-Morales et al. 2024), suggesting a relationship between intense sexual selection and alterations in gene family size, illustrating the complex interplay between sexual dimorphism, gene family size evolution, and their roles in mammalian genome evolution (Van der Linen et al. 2018; Padilla-Morales et al. 2024). The sexual dimorphism observed in the submandibular and lacrimal glands of house mice thus provides valuable insights into the broader mechanisms of sexual selection and communication in mammals. Conclusion In this study, we provide a comprehensive characterization of sexual dimorphism in two exocrine glands involved in chemical communication in the house mouse, using both wild mice and wild-derived strains. Males of both subspecies exhibited larger glands and a higher proportion of granular convoluted tubules, while the subspecies differed in the relative magnitude of glandular dimorphism, protein output of glandular secretions, and behavioural responses. Employing two closely related taxa places our findings in the context of subspecies divergence and the emergence of reproductive barriers between nascent species. Our findings demonstrate that closely related subspecies can differ not only in the chemical composition of olfactory signals but also in the relative contribution of distinct secretory organs to chemical communication. We further show that even subtle differences in mating systems and social structures can shape the evolution of sexual dimorphism and divergence of olfactory communication pathways. Our results highlight the importance of cryptic sexual dimorphism and glandular morphology as integral, yet previously underappreciated, components of sexual dimorphism in mammalian olfactory signalling systems. Declarations Acknowledgements: We thank J. Piálek for providing WDS mice and breeding facilities, Ľ. Ďureje for help with mouse trapping and preparation of histological samples, L. Rousková for additional tear and saliva sample collection, and the technical staff of the breeding facility of the Institute of Vertebrate Biology, Czech Academy of Sciences, in Studenec for mouse care. We are grateful to two anonymous colleagues for their valuable comments and revisions during manuscript preparation. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project no. Inter-Excellence, Inter-Action, LTAUSA18). Competing Interests: The authors declare that they have no competing interests. Data Availability Statement: All data supporting the results of this study are provided in the article and its supplementary materials. Additional raw data (e.g., individual measurements) are available from the corresponding author upon request. Author contributions: BVB designed the experiments, contributed to mouse trapping, histological analyses and data collection, managed the BCA and Take3 proteomic analyses, statistical analyses, and led manuscript writing. MM was project leader, contributed to experiment design, mouse trapping and dissections, manuscript writing, and provided financial support. DB performed behavioral analyses of sexual preferences and data analysis under supervision of ZH and later BVB. KVK designed and funded proteomic analyses, analyzed proteomic data, and contributed to manuscript writing. KD assisted with mouse trapping and dissections. ZH conceived the study idea, designed experiments, contributed to trapping, dissections, sample collection, data and statistical analyses, and manuscript writing. Funding: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project no. Inter-Excellence, Inter-Action, LTAUSA18). Ethics Approval All animal procedures were approved by the Ethical Committee of the Institute of Animal Physiology and Genetics, Czech Academy of Sciences, and were conducted in accordance with national and EU legislation. 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08:13:45","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":286703,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/30957f219232a941425b93e0.html"},{"id":100372415,"identity":"8b1d45aa-64b3-4c4a-8df4-273ab5c94cb3","added_by":"auto","created_at":"2026-01-16 08:12:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465080,"visible":true,"origin":"","legend":"\u003cp\u003eThe geographic distribution of the inbred wild-derived strains (WDSs) of house mice of both subspecies, \u003cem\u003eMus musculus musculus \u003c/em\u003e(red dots)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eMus musculus domesticus \u003c/em\u003e(blue dots) in Europe (A); and position of sampling localities of wild mice (B; shown as a yellow rectangle in A). The violet line (solid in A, dashed in B) depicts the hybrid zone between the two subspecies. The list of wild mice and WDS mice are given in Supplementary Material Tables 1 and 2, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/f7fd3f14eb12590bf8c1e6fe.png"},{"id":100372458,"identity":"e47b0bf8-9c90-4bcc-834d-b576d563ddae","added_by":"auto","created_at":"2026-01-16 08:12:26","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":225586,"visible":true,"origin":"","legend":"\u003cp\u003eSexual size dimorphism in the submandibular (A) and lacrimal (B) glands in wild mice. Mean relative gland sizes ± 95% CI for males (dark boxes) and females (light boxes), for M. m. domesticus (blue) and M. m. musculus (red).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/8d056381ace85da6bbde9cae.jpeg"},{"id":100270085,"identity":"f2d99880-247f-42d1-ad54-3b410108f593","added_by":"auto","created_at":"2026-01-14 19:40:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77541,"visible":true,"origin":"","legend":"\u003cp\u003eSexual size dimorphism in the submandibular (A) and lacrimal (B) gland in WDSs. Mean relative gland sizes ± 95% CI for males (dark boxes) and females (light boxes), for \u003cem\u003eM. m. domesticus\u003c/em\u003e (blue) and \u003cem\u003eM. m. musculus\u003c/em\u003e (red).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/405172861013ab64905bffce.jpeg"},{"id":100270089,"identity":"e144c80f-b673-4653-adda-36ff9900c36d","added_by":"auto","created_at":"2026-01-14 19:40:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":651094,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of the submandibular gland in wild-derived strains of both subspecies: BUSNA, MBK (\u003cem\u003eM. m. musculus\u003c/em\u003e, red) and STRA, DROS (\u003cem\u003eM. m. domesticus\u003c/em\u003e, blue). A) Mean values of the relative amount of GCT (+/- 95 CI) are indicated separately for males (dark boxes) and females (light boxes). B) Examples of the histological sections with a graphic representation of GCT analyses in the FiJi Image (inserts): GCTs are lighter, surrounded by darker acinar tissues.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/08ca4be9f84700823bb47b9c.jpeg"},{"id":100372823,"identity":"2f74f15c-ed02-48e4-ba82-4522ac97b72b","added_by":"auto","created_at":"2026-01-16 08:13:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72040,"visible":true,"origin":"","legend":"\u003cp\u003eTotal protein content in saliva (A) and tears (B) in wild-derived strains of both subspecies: BUSNA, MBK (\u003cem\u003eM. m. musculus\u003c/em\u003e) and STRA, DROS (\u003cem\u003eM. m. domesticus\u003c/em\u003e); mean values (+/- 95 CI) are indicated for \u003cem\u003edomesticus\u003c/em\u003e-derived (blue boxes) and \u003cem\u003emusculus\u003c/em\u003e-derived (red boxes) WDSs, separately for males (dark) and females (light boxes).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/31e30620ea2ec3b063344550.jpeg"},{"id":100372423,"identity":"ab527a95-f4e3-457d-885d-4458a55ee00d","added_by":"auto","created_at":"2026-01-16 08:12:21","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":300103,"visible":true,"origin":"","legend":"\u003cp\u003eThe coefficient of preference R\u003csub\u003esignal\u003c/sub\u003e as the relative difference of times spent sniffing tears vs. normal saline [R\u003csub\u003esignal\u003c/sub\u003e = (T\u003csub\u003etears\u003c/sub\u003e – T\u003csub\u003esaline\u003c/sub\u003e)/(T\u003csub\u003etears\u003c/sub\u003e + T\u003csub\u003esaline\u003c/sub\u003e)] in the Y-maze discrimination/preference test. The coefficient is represented separately per sex (males - dark boxes and females - light boxes) and strain (subspecies; \u003cem\u003edomesticus\u003c/em\u003e (blue), \u003cem\u003emusculus\u003c/em\u003e (red)), for more details see Supplementary Table S8.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/38c2b154cd00ce6bedfc3ab0.jpeg"},{"id":107704443,"identity":"3a8142ab-1b66-4ab0-a487-82219bd00dec","added_by":"auto","created_at":"2026-04-24 08:45:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2070070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/eab5571b-6cc8-4d2f-9e1f-1917ddd0d9ec.pdf"},{"id":100373226,"identity":"d861665b-4291-4b46-bd31-595ce0029b8f","added_by":"auto","created_at":"2026-01-16 08:13:51","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":349801,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLE1.xls","url":"https://assets-eu.researchsquare.com/files/rs-8423338/v1/306454c018aceee1a96dc107.xls"}],"financialInterests":"","formattedTitle":"Chemical antlers: sexual dimorphism in salivary and lacrimal glands of house mouse subspecies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSexual dimorphism, defined as the systematic difference in phenotype between males and females of the same species, typically reflects the action of sexual selection, although natural selection may also contribute. Sometimes, it is even difficult to distinguish between these two evolutionary forces: territory defence, for example, is important for the protection of food resources (natural selection) but can also serve females to evaluate the individual quality of a male as a potential mate (sexual selection). The interplay between these forces is central to understanding the evolution of sexually dimorphic traits and their behavioural functions. In mammals, the extent of sexual dimorphism varies greatly depending on the social structure, the mating system, and the ecological requirements. The highest sexual dimorphism in external traits is, therefore, typical for species that are polygynous, diurnal, and inhabitants of open habitats, while monogamous and nocturnal species are usually monomorphic (Huxley 1938; Wade 1979; Andersson 1994; Vanp\u0026eacute; et al. 2008; McPherson and Chenoweth 2012). However, this distinction is probably oversimplified, as some apparently monomorphic species can exhibit only facultative monogamy or are promiscuous (Kleiman 1977; Ostfeld and Heske 1993; Isaac 2005; Kokko and Rankin 2006) or adopted alternative strategies to maximize male mating success (Dunham and Rudolf 2009).\u003c/p\u003e\n\u003cp\u003eNevertheless, dimorphism may be subtler or related to traits that are less obvious than body size, colouration, and exaggerated structures, such as peacock\u0026apos;s tail, deer antlers, beetle horns, or the monstrous eye span of stalk-eyed flies. It is well known that many mammals use chemical cues rather than visual communication. Such species, often thought to show little sexual dimorphism, may, in fact, be extremely sexually dimorphic (Eisenberg and Kleiman 1972; Fan 1987; Blaustein 1981; Arnold and Houck 1982). Sexual dimorphism may therefore be related to differences in the size of scent glands and other organs involved in odour signalling, as well as the abundance and repertoire of scent produced (Stoddart 1974; Blaustein 1981; Luzynski et al. 2021). Indeed, there is growing evidence that various traits associated with chemical communication exhibit sexual dimorphism in many mammalian species previously considered to be sexually monomorphic (Jannett 1986; Maico et al. 2001; Rosell and Schulte 2004; Macdonald and Herrera 2013; Spence-Aizenberg et al. 2018; Mu\u0026ntilde;oz-Romo et al. 2021). This phenomenon has remained largely overlooked, partly because many of these species are nocturnal and thus challenging to study. Moreover, scent-related tissues and odours are often hidden for humans. Typical examples of animals displaying such cryptic sexual dimorphism are small rodents, including one of the most commonly used model organisms \u0026ndash; the house mouse (\u003cem\u003eMus musculus\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eWhen considering usual sexually dimorphic traits, male house mice are generally reported to be heavier than females\u0026nbsp;(Eisen and Legates 1966; Nomaguchi and Sakurai 1993; Sl\u0026aacute;bov\u0026aacute;\u0026nbsp;and\u0026nbsp;Frynta 2007; Pi\u0026aacute;lek et al. 2008; Haisov\u0026aacute;-Sl\u0026aacute;bov\u0026aacute; et al. 2010; Ruff et al. 2017; Panti-May et al. 2018; The Jackson Laboratory 2024), although the weight differences are not always significant. In addition, no sexual size dimorphism in\u0026nbsp;cranial and dental\u0026nbsp;variables has been\u0026nbsp;observed\u0026nbsp;in this species (Machol\u0026aacute;n 1996; Sans-Fuentes et al. 2009; Csan\u0026aacute;dy and Mo\u0026scaron;ansk\u0026yacute; 2018). In contrast, mice exhibit substantial intersexual differences in behaviour, much of which is related to odours and olfaction, such as aggression, mating, and parental care (Kimchi et al. 2007; Stowers and Liberles 2016; Ishii and Touhara 2019). In this context, it is startling that sexual dimorphism of olfactory organs has received little attention. A\u0026nbsp;growing body of research suggests that sexually dimorphic behaviours are more likely to be associated with dimorphism in olfactory signals, whereas the expression of receptors in relevant olfactory tissues shows minimal differences between males and females (Holy et al. 2000; Kimchi et al. 2007; Kimoto et al. 2007; Ben-Shaul et al. 2010; Ibarra-Soria et al. 2014;\u0026nbsp;Kuntov\u0026aacute; et al. 2018).\u003c/p\u003e\n\u003cp\u003eMouse odours are complex mixtures of volatile and non-volatile chemicals found in skin secretions, urine, tears, saliva and faeces.\u0026nbsp;They are known to differ\u0026nbsp;considerably between males and females in their quantities, chemical composition and proportions of individual components\u0026nbsp;(Laukaitis et al. 2005; Mucignat-Caretta et al. 2010; Karn and Laukaitis 2011, 2015; Karn et al. 2014; Stopka et al. 2016; Stopkov\u0026aacute; et al. 2016, 2017, 2023; Kuntov\u0026aacute; et al. 2018; Matejkov\u0026aacute; et al. 2024) with some signals being reported as sexually dimorphic or even sex-specific (Kimoto et al. 2005, 2007; Roberts et al. 2010, 2012; Karn and Laukaitis 2015; Karn et al. 2014; Stopkov\u0026aacute; et al. 2016; Stopkov\u0026aacute; et al. 2023).\u0026nbsp;Although the main source of odour cues in mice is undoubtedly urine (Hurst et al. 2001), we have focused on tears and saliva because, as products of glands in the orofacial region, they are important sources of signals in close contact communication (Luo et al. 2003; B\u0026iacute;mov\u0026aacute; et al. 2009). Peptide pheromones from these fluids are dropped on the ground or remain on the body surface allowing signal transmission between consubspecifics exclusively by direct contact (Cavalier et al 2014). Interestingly, 16\u0026ndash;21% of salivary proteins and 15% of tear proteins are sexually dimorphic (Karn and Laukaitis 2011; Stopka et al. 2016; Stopkov\u0026aacute; et al. 2017). Similar sexual dimorphism was also found at the transcriptional level (Stopkov\u0026aacute; et al. 2016; Kuntov\u0026aacute; et al. 2018). Among these sexually dimorphic compounds, lipocalins (such as MUPs, OBPs, and ESPs) and the androgen-binding protein subfamily of secretoglobins are the most abundant. Some of the urinary and salivary signals also differ qualitatively and/or quantitatively between different house mouse subspecies and have been proposed as important cues for subspecies-specific mate recognition (Laukaitis et al. 1997; B\u0026iacute;mov\u0026aacute; et al. 2005; Stopkov\u0026aacute; et al. 2007) and thus as significant contributors to the establishment and maintenance of reproductive barriers between house mouse subspecies (Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011; Laukaitis and Karn 2012). In this context, the potential role of tear-based signals in subspecies-specific communication remains largely unexplored.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated sexual dimorphism in size, histology, and overall protein excretion in submandibular and lacrimal glands, as well as the behaviour associated with their products. To capture potential intraspecies differentiation, we compared two house mouse subspecies, \u003cem\u003eMus musculus musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e (see Baird and Machol\u0026aacute;n 2012, for a review). The two taxa differ not only genetically, but also in several behavioural strategies, including male aggression (Thuessen 1977; van Zegeren and van Oortmerssen 1981; Ďureje et al. 2011; Latour and Ganem 2017), mate choice (Smadja et al. 2004; B\u0026iacute;mov\u0026aacute; et al. 2005; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011; Ganem 2012), dispersal (Hiadlovsk\u0026aacute; et al. 2012, 2013; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2016) and establishment of social structure (Hiadlovsk\u0026aacute; et al. 2015, 2021; Mikula et al. 2022; Machol\u0026aacute;n et al. 2023; Bendov\u0026aacute; et al. 2024). We employed both wild animals and inbred wild-derived strains representing the two subspecies. Our study adds another piece of puzzle to the underlying mechanisms and ecological implications that determine the morphological divergence between male and female individuals in these glandular structures. Specifically, we test the hypothesis that morphological and chemical sexual dimorphism in exocrine glands is associated with subspecies-specific variation in mate recognition systems, potentially contributing to reproductive isolation between taxa. By investigating sexual dimorphism within this specific anatomical and behavioural context, we aim to enhance our understanding of its role in the mechanisms driving evolutionary divergence.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eHouse mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWild mice\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWild mice of two subspecies, western European \u003cem\u003eMus musculus domesticus\u003c/em\u003e and eastern European \u003cem\u003eM. m. musculus\u003c/em\u003e, were collected during three trapping sessions (June 2019, October 2019, October 2022) in northeastern Bavaria (Germany) and the western part of the Czech Republic (Supplementary Table S1, Figure 1B). All sampling sites were located more than 30 km away from the centre of the hybrid zone between the two subspecies (Machol\u0026aacute;n et al. 2007; Baird and Machol\u0026aacute;n 2012; Ďureje et al. 2012). The mice were sacrificed and dissected in a field laboratory next day after capture. All animals were also genotyped with diagnostic molecular markers (Machol\u0026aacute;n et al. 2007) to confirm their subspecific status. Hybrids, juvenile individuals (body weight \u0026lt; 10 g), and pregnant females were excluded from subsequent analyses. In total, we analysed 98 wild mice from 18 localities: 56\u0026nbsp;\u003cem\u003eM. m. domesticus\u003c/em\u003e (31 males, 25 females; 8 localities) and 42\u0026nbsp;\u003cem\u003eM. m. musculus\u003c/em\u003e (26 males, 16\u0026nbsp;females; 10 localities) (Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWild-derived strains of mice (WDS)\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWe also used inbred mice of 11 WDS representing both subspecies (Pi\u0026aacute;lek et al. 2025, https://housemice.cz/cs), purchased from the Institute of Vertebrate Biology, Czech Academy of Sciences, Studenec (Supplementary Table S2, Figure 1A). Mice were weaned at 20 days, singly housed, and sacrificed at adulthood (85\u0026ndash;150 days), following the same protocol as in wild mice. In total, we examined 174 mice: 80 individuals (42 males, 38 females) of five strains derived from \u003cem\u003eM. m. domesticus\u003c/em\u003e and 94 individuals (48 males, 46 females) of six strains derived from\u0026nbsp;\u003cem\u003eM. m. musculus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore dissection, saliva and tears were collected for the protein content analysis and as signals in subsequent behavioural experiments. The samples were gathered in the native state without sedation or stimulation using mouthwash and eyewash with 20 \u0026micro;l of normal saline. Mouthwash was carried out by pipetting 10 \u0026micro;l of saline into the oral cavity of the mouse, rinsing gently, and aspirating back into the pipette. This procedure was repeated twice. In the case of eyewash, 10 \u0026micro;l of saline was instilled into the lateral canthus, the eye was gently stimulated to induce lacrimation, and the fluid was aspirated back into the pipette (Petznik et al. 2011). The procedure was repeated for the other eye. All saliva samples with visible food or blood residues were excluded from further analyses. The samples were immediately stored at -80 \u0026deg;C. The mice were then sacrificed by cervical dislocation and dissected; both right and left parts of the submandibular salivary glands (SMG) and exorbital lacrimal glands (LAC) were collected free of visible adipose tissue and ligaments, and weighed with an accuracy of 0.0001 g. The relative mass was calculated for each gland as the ratio of the gland mass and total body mass (measured with an accuracy of 0.01 g).\u003c/p\u003e\n\u003cp\u003eData normality was tested using Kolmogorov-Smirnov tests, followed by factorial ANOVA to test the effects of sex and/or subspecies and their interaction on the relative mass of SMG and LAC. Calculations were performed using STATISTICA 6.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology of salivary glands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the SMG gland histology, we used four WDSs: \u003cem\u003edomesticus\u003c/em\u003e-derived STRA and DROS, and \u003cem\u003emusculus\u003c/em\u003e-derived BUSNA and MBK, with four animals per sex. In each individual, we randomly (but in a balanced design) sampled either the left or right part of the paired gland. The tissues were fixed in 4% buffered formalin for 24 hours at room temperature and then stored in 70% ethanol until further processing (up to 2 weeks). After embedding in paraffin, the 5-\u0026mu;m sections were prepared from the central part of the gland. From each gland, 6 sections (25 \u0026mu;m apart, keeping every 5th section) were stained with haematoxylin/eosin. Three high-quality microscopic fields, preferably chosen from each odd-numbered section, were photographed using the Olympus BX51 microscope and DP71 camera. After training on granular convoluted tubule (GCT) histology (Mori et al. 2011; Scudamore 2014), we quantified the GCT tissue percentage in each image using the FiJi biological image analysis software (Schindelin et al. 2012). We calculated the mean percentage of the GCT area for each individual based on average measures acquired from three different sections. Data normality was tested using Kolmogorov-Smirnov tests, followed by factorial ANOVAto test for the effect of sex and/or subspecies and their interaction on relative GCT proportion in the SMG gland. The calculations were carried out with the STATISTICA 6.0 software. The LAC histology was not examined because the extraorbital lacrimal gland in mice consists predominantly of acinar cells and myoepithelial cells, with excretory tubules, but the tubular structure of this gland is not as differentiated in the mouse as SMG (Scudamore 2014; Makarenkova et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein content analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe quantified the total protein content in tear and saliva samples from four WDS: STRA, DROS (\u003cem\u003edomesticus\u003c/em\u003e-derived) and BUSNA, MBK (\u003cem\u003emusculus\u003c/em\u003e-derived). We collected samples from up to 6 animals per sex and strain, totaling 39 saliva samples and 48 tear samples. Protein quantification was performed with the micro-bicinchoninic acid (BCA) reaction and spectrophotometry (Smith et al. 1985; Noble and Bailey 2009; Kielkopf et al. 2020) using the BCA Pierce microassay kit (Thermo Fisher 23235). Initially, we roughly estimated the total protein concentration in each sample (Kielkopf et al. 2020) by measuring UV absorbance at 280 nm using the Take3 Micro-Volume Plate on the BioTek Synergy HTX reader and the Gen5 software v. 2.09. Based on these measurements, we adjusted the subsequent dilution of the samples for the micro-BCA reaction so that the expected concentration fell above the quantification limit of the microplate format of the BCA microassay kit (2 \u0026micro;g/ml). This corresponded to 3.5 \u0026micro;l of the samples in 150 \u0026micro;l of saline. Protein standards ranging from 1 to 200 \u0026micro;g/ml (Supplementary Material Table S3) were prepared using the Protein Assay standard BSA set (Thermofisher 23208). The BCA microassay was performed according to the manufacturer\u0026rsquo;s protocol. All samples and standards were analyzed in duplicates. Absorbance at 562 nm was measured using the BioTek Synergy HTX reader with Gen5 2.09. The protein concentrations were calculated using the dilution curve of the known standards. The data were log-transformed to ensure their normality and then tested with the Kolmogorov-Smirnov test. A factorial ANOVA was performed to examine the influence of sex and/or subspecies on total protein concentrations in the samples of saliva and tears. STATISTICA 6.0 was used for statistical analyses, with the significance level set to 0.05 for all tests performed. When significant interactions or main effects were detected, Tukey\u0026rsquo;s HSD post-hoc tests were carried out to determine specific group differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest of discrimination and preferences of tear cues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the behavioural relevance of the potential role of tears as signals in mouse communication, we used 42 mice from two WDSs as signal donors and recipients: DROS (\u003cem\u003edomesticus\u003c/em\u003e-derived; 10 males and 10 females) and MBK (\u003cem\u003emusculus\u003c/em\u003e-derived; 11 males and 11 females) (Fig. 1 A). The animals were subject to a simple two-way discrimination/preference test using a Y-maze. All tested mice were adults, at least 90 days old, weaned at 20 days of age, kept either separately or with one same-sex littermate, and later isolated at the age of 55\u0026ndash;60 days. The experimental setup consisted of a habituating box connected to the stem of the Y-maze with one-way air circulation (for more details, see Talley et al. 2001; B\u0026iacute;mov\u0026aacute; et al. 2005, 2009). Each tested individual was allowed to choose between a pair of signal cues consisting, respectively, of 10 \u0026micro;l of normal saline (control) and 10 \u0026micro;l of eyewash representing a cue with tears as a potential signal. We used opposite-sex stimuli; both signal donors and tested individuals came from the same WDS.\u003c/p\u003e\n\u003cp\u003eAs the cue, we used the 10 \u0026micro;l aliquot mixture of eyewash taken from 3\u0026ndash;6 adult individuals of the same strain and sex. The use of the mixture eliminated the influence of individual odour (age, physical condition) while preserving potential information about sex and subspecies. The collected samples were stored at \u0026minus;80 \u0026deg;C, thawed and mixed just before use, and kept on ice throughout the experiment. Prior to testing, 10 \u0026micro;l of each sample was pipetted on the sterile filter paper and placed at the end of the arms of the Y-maze. The tested mouse was habituated for 15 min in the habituation box separated by a perforated door from the maze. Then the door was opened, and the mouse was allowed to enter. The experiment, lasting for 5 min after the mouse reached the zone of decision in the central part of the Y-maze, was video-recorded and further analysed. All experiments were carried out during the light phase of the day.\u003c/p\u003e\n\u003cp\u003eThe ability of each individual to discriminate and prefer tear cues was assessed according to Smadja and Ganem (2002; see also B\u0026iacute;mov\u0026aacute; et al. 2005; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011). We tested the first choice of the signal as the first cue the mouse had chosen to investigate. In addition, we calculated the coefficient of preference as the relative difference of times spent sniffing one of the cues, T\u003csub\u003etears\u003c/sub\u003e or T\u003csub\u003esaline\u003c/sub\u003e: R\u003csub\u003esignal\u003c/sub\u003e = (T\u003csub\u003etears\u003c/sub\u003e \u0026ndash; T\u003csub\u003esaline\u003c/sub\u003e)/(T\u003csub\u003etears\u003c/sub\u003e + T\u003csub\u003esaline\u003c/sub\u003e). We thus recognize no preference if R\u003csub\u003esignal\u003c/sub\u003e = 0; saline was considered as preferred when R\u003csub\u003esignal\u003c/sub\u003e was significantly negative (-1\u0026ndash;0), while negative values (0\u0026ndash;1) suggested tear preference. The difference of R\u003csub\u003esignal\u003c/sub\u003e from 0 was tested with a t-test (H\u003csub\u003e0\u003c/sub\u003e: \u0026mu; = 0); the\u0026nbsp;\u0026chi;\u003csup\u003e2\u003c/sup\u003e test\u0026nbsp;was used for comparison of the first chosen signals.\u0026nbsp;The data were tested for normality using the Kolmogorov\u0026ndash;Smirnov test and\u0026nbsp;processed using STATISTICA 6.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WDS stocks used in this study are part of an extensive WDS collection of the Institute of Vertebrate Biology of the Czech Academy of Sciences in Studenec (Pi\u0026aacute;lek et al. 2025; www.housemice.cz), where behavioural experiments were carried out. Mice were kept in plastic cages (VELAZ, 43 \u0026times; 30 \u0026times; 22 cm) at an average temperature of 22 \u0026deg;C under the 14:10 light:dark regime (lights on at 7 am and off at 9 pm), with shavings litter and nesting material available. Food and water were supplied \u003cem\u003ead libitum\u003c/em\u003e. The facility is accredited for breeding (48389/2020-MZE-18134, 2021\u0026ndash;2026) and use (62065/2017-MZE-17214, 2017\u0026ndash;2022) of experimental animals. The animals were handled by authorized persons only (Licenses No. ZH: CZ 0127; BVB: CZ01293 and DB as a university student under supervision). This study was performed in accordance with Czech law implementing all corresponding EU regulations and approved by the IVB Ethical Committee.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGland size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWild mice\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAs locality had no significant effect within subspecies (nested ANOVA, locality nested within sex and subspecies; SMG: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,28)\u003c/sub\u003e = 0.736, \u003cem\u003ep\u003c/em\u003e = 0.814; LAC: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,28)\u003c/sub\u003e\u0026nbsp; = 1.167, \u003cem\u003ep\u003c/em\u003e = 0.298), data were pooled by subspecies for subsequent analyses. Males had significantly higher relative (gland-to-body) weights than females for both the SMG and LAC (factorial ANOVA: SMG: F(\u003csub\u003e1,94\u003c/sub\u003e) = 4.073, p = 0.046; LAC: F(\u003csub\u003e1,94\u003c/sub\u003e) = 19.004, p \u0026lt; 0.001). The magnitude of sexual dimorphism differed between subspecies, with a stronger effect observed in \u003cem\u003eM. m. domesticus\u003c/em\u003e, which was significant for the LAC gland (Fig. 2; Supplementary Table S4). We also detected significant differences in SMG size between the two subspecies, with both males and females of \u003cem\u003eM. m. musculus\u003c/em\u003e exhibiting larger glands than those of \u003cem\u003eM. m. domesticus\u003c/em\u003e (factorial ANOVA: F(\u003csub\u003e1,94\u003c/sub\u003e) = 27.710, p \u0026lt; 0.001). In contrast, no intersubspecific difference was detected in LAC size (factorial ANOVA: F(\u003csub\u003e1,94\u003c/sub\u003e) = 0.478, p = 0.492; see Supplementary Tables S1 and S4).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWDS mice\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eIn WDSs, the pattern was similar to that observed in wild mice: males of both subspecies had significantly larger SMG and LAC glands than females (factorial ANOVA: SMG: F(\u003csub\u003e1,170\u003c/sub\u003e) = 26.919, p \u0026lt; 0.001; LAC: F(\u003csub\u003e1,168\u003c/sub\u003e) = 109.914, p \u0026lt; 0.001). Again, sexual dimorphism was more pronounced in \u003cem\u003edomesticus\u003c/em\u003e-derived strains than in \u003cem\u003emusculus\u003c/em\u003e-derived strains for both glands (Fig. 3, Supplementary material Table S5). As in wild mice, \u003cem\u003emusculus\u003c/em\u003e-derived WDSs tended to have slightly larger glands than \u003cem\u003edomesticus\u003c/em\u003e-derived WDSs, although these differences were not statistically significant (factorial ANOVA: SMG: F(\u003csub\u003e1,170\u003c/sub\u003e) = 2.772, p = 0.098; LAC: F(\u003csub\u003e1,168\u003c/sub\u003e) = 1.067, p = 0.303). To facilitate comparison between WDSs and wild mice, strains were pooled within subspecies. However, substantial variation among individual stocks was observed (Supplementary Fig. S1), partly reflecting differences in sample size among strains (see Supplementary Table S2 for details).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological analysis was consistent with the patterns of sexual dimorphism inferred from gland mass measurements. The relative amount of granular convoluted tubular tissue (GCT) in the SMG differed between sexes and subspecies (factorial ANOVA: sex: F(\u003csub\u003e1,28\u003c/sub\u003e) = 197.085, p \u0026lt; 0.001; subspecies: F(\u003csub\u003e1,28\u003c/sub\u003e) = 55.99, p \u0026lt; 0.001; subspecies \u0026times; sex interaction: F(\u003csub\u003e1,28\u003c/sub\u003e) = 9.695, p = 0.004). The proportion of GCT was significantly higher in males than in females of both subspecies, and \u003cem\u003emusculus\u003c/em\u003e-derived strains of both sexes exhibited higher proportion of GCT than \u003cem\u003edomesticus\u003c/em\u003e-derived strains (Fig. 4, Supplementary Table S6). Greater development of sexual dimorphism in the proportion of GCT was found in the \u003cem\u003edomesticus\u003c/em\u003e strains, corresponding to trends observed in relative gland size analysis (Tukey HSD test, Supplementary Table S6). We also analysed SMG glands from three males and three females of the classical laboratory strain C57BL/6J (hereafter BL6), which has well-described gland histology and established patterns of GCT in both sexes (Brown et al. 2020; Chung et al. 2017; Jayasinghe et al. 1990), and used these animals as a control group. As expected, the relative GCT amount in BL6 mice was similar to that observed in \u003cem\u003edomesticus\u003c/em\u003e-derived strains (not shown in Fig. 4), consistent with the prevailing genetic background of this strain (Yang et al. 2011). This result supports the validity of our method (Supplementary Fig. S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein content in saliva and tears did not differ significantly between males and females in either subspecies (factorial ANOVA: saliva: F(\u003csub\u003e1,35\u003c/sub\u003e) = 0.098, p = 0.756; tears: F(\u003csub\u003e1,44\u003c/sub\u003e) = 1.213, p = 0.277; Supplementary Table S7). When comparing the two subspecies, total protein content in tear samples was significantly higher in \u003cem\u003eM. m. domesticus\u003c/em\u003e than in \u003cem\u003eM. m. musculus\u003c/em\u003e in both sexes, whereas no significant difference was detected in saliva (factorial ANOVA: saliva: F(\u003csub\u003e1,35\u003c/sub\u003e) = 0.165, p = 0.687; tears: F(\u003csub\u003e1,44\u003c/sub\u003e) = 16.565, p \u0026lt; 0.001; Fig. 5; Supplementary Table S7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTear-based discrimination and preference\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the two-way choice test using tears and saline as olfactory cues, a statistically significant preference for tear cues was detected in the \u003cem\u003edomesticus\u003c/em\u003e-derived DROS strain. This effect was observed in both females and males when preference was quantified using the coefficient of preference (R_signal \u0026gt; 0; t-test against zero: females: t = 3.174, df = 9, p = 0.011; males: t = 2.350, df = 9, p = 0.043; Fig. 6; Supplementary Table S8). In contrast, no significant preference for tears was detected in the \u003cem\u003emusculus\u003c/em\u003e-derived MBK strain.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eSexual dimorphism in salivary and lacrimal gland morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we examined sexual dimorphism in the submandibular salivary and exorbital lacrimal glands of two house mouse subspecies using wild mice and wild-derived strains. We focused on gland size, SMG histology, and total protein content of glandular secretions. We also tested whether tear-derived cues may contribute to chemical communication in the two mouse subspecies. Our results showed that males of both subspecies, in both wild mice and WDSs, possess larger salivary and lacrimal glands than females. In addition, males and females of \u003cem\u003eM. m. musculus\u003c/em\u003e tended to have larger glands than those of \u003cem\u003eM. m. domesticus\u003c/em\u003e, although this difference was statistically significant only for the SMG in wild mice. Histological analysis confirmed a strong male-biased sexual dimorphism in the proportion of granular convoluted tubules (GCT) in the SMG (Chr\u0026egrave;tien 1977; Gresik 1980; Gresik et al. 1996). This represents one of the best-studied examples of sexual dimorphism in mouse organs involved in chemical communication. GCT has been examined using a wide range of approaches, from neuroanatomical analyses to the detection of enzymes present in salivary secretions (reviewed in Gresik 1994; Pinkstaff 1998). However, most previous studies have relied on classical inbred laboratory strains such as BL6. To our knowledge, the present study is the first to address this aspect using wild mice and wild-derived strains. Thus, our results move beyond classical laboratory models and document GCT sexual dimorphism in the house mouse as a biological species, including natural variation between subspecies that is not represented in standard inbred strains (Gu\u0026eacute;net and Bonhomme 2003; Didion and Pardo-Manuel de Villena 2013). By comparing two distinct subspecies, we incorporated intraspecific variation, allowing the results to be interpreted in an evolutionary context.\u003c/p\u003e\n\u003cp\u003eGCT is a unique, rodent-specific structure of SMG (Amano et al. 2012), interspersed with the striated and intercalated ducts. In males, GCT is markedly hypertrophic and characterized by large secretory granules. This hypertrophy is primarily driven by the influence of adrenocortical, thyroid, and sex hormones (Gresik 1994; Gresik et al. 1996; Se\u0026ntilde;orale-Pose et al. 1998; Kurabuchi et al. 1999, 2009, 2019). Especially in adulthood, with 40% more GCT in males than in females (Jayasinghe et al. 1990; Se\u0026ntilde;orale-Pose et al.1998, Tandler et al. 2001), the dimorphism is rather striking. Our study revealed similar results in the WDS males under study. Importantly, larger SMG glands with more strongly hypertrophied GCTs were found in WDSs representing \u003cem\u003eM. m. musculus\u003c/em\u003e, a subspecies previously shown to exhibit consistently higher testosterone levels (Hiadlovsk\u0026aacute; et al. 2015). These results are consistent with sex-specific differences in SMG gene expression reported by Mukaibo et al. (2019), further supporting the role of these glands in the production of sexual signals (Brown et al. 2020). Inclusion of the well-characterized classical laboratory strain BL6 as a control validated the accuracy of our results, indicating that the observed patterns are not artifacts of specific strains studied (Phifer-Rixey and Nachman 2015; Pi\u0026aacute;lek et al. 2025).\u003c/p\u003e\n\u003cp\u003eAlthough the fundamental function of the salivary glands is the production of primary saliva, which lubricates food, enables mastication, and participates in food digestion (Junqueira et al. 1964), GCT cells also synthesize a large variety of biologically active polypeptides, including epidermal growth factor (EGF), nerve growth factor (NGF), renin, and kallikreins (Kurabuchi et al. 1999; 2002; 2019; Tandler et al. 2001). Particularly, kallikreins have been suggested to play a role in chemical communication (Karn and Laukaitis 2011). Due to the internal connection of the nasolacrimal duct, tear proteins can enter the oral cavity (Ruberte et al. 2017) and both salivary and tear proteins can be transferred to fur and nesting material through grooming and cleaning behaviours (Laukaitis et al. 1997; Stopka et al. 2016; Stopkov\u0026aacute; et al. 2017; Barabas et al. 2019, 2022). Barabas et al. (2022) identified several groups of salivary and tear proteins in the nest material that modulate aggressive and prosocial behaviours. These findings support the idea that both tear and salivary proteins can, at least in part, function as olfactory cues even outside direct physical contact and reinforce the notion that glandular dimorphism may contribute to sexual selection and mate signalling (McPherson and Chenoweth, 2012), acting as \u0026lsquo;chemical antlers\u0026rsquo; \u0026ndash; functional analogues of conspicuous sexually dimorphic traits such as deer\u0026rsquo;s antlers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubspecies differences in chemical signalling strategies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile many salivary and tear proteins show quantitative differences in overall abundance and expression profiles between sexes (Mucignat Caretta and Caretta 2014; Stopka et al. 2016; Stopkov\u0026aacute; et al. 2016, 2017, 2023; Chung et al. 2017; Kuntov\u0026aacute; et al. 2017), there are also marked qualitative differences in the protein composition (Roberts et al. 2010, 2012; Kimoto et al. 2005, 2007; Karn and Laukaitis 2014; Stopka et al. 2016; Stopkov\u0026aacute; et al. 2016, 2017; Chung et al. 2017; Karn et al. 2021). Together, these findings indicate that both qualitative and quantitative differences are important to understanding sexual dimorphism in these exocrine glands and their secretions. In contrast, the significantly higher tear protein content observed in WDSs representing one of the subspecies (\u003cem\u003eM. m. domesticus\u003c/em\u003e) in both sexes suggests that overall protein excretion may be influenced more strongly by subspecies-specific factors than by sex alone (Rollins et al. 2017).\u003c/p\u003e\n\u003cp\u003eIn light of generally larger SMG and LAC and a higher proportion of GCT in males than in females, the absence of sexual dimorphism in the total protein content of saliva and tears is notable. Moreover, despite the similar size of lacrimal glands in the two subspecies, we found a significantly higher total tear protein content in \u003cem\u003eM. m. domesticus\u003c/em\u003e than in \u003cem\u003eM. m. musculus\u003c/em\u003e. These observations suggest that the morphological differences may not directly translate to differences in overall protein output, potentially due to yet unidentified factors regulating protein concentration (Stopka et al. 2016; Proctor et al. 2021; Stopkov\u0026aacute; et al. 2023). However, this apparent discordance may have another, perhaps simpler, explanation. Stopka et al. (2016) and Barabas et al. 2022 detected secretoglobins and other proteins in saliva, that are expressed exclusively in the lacrimal gland, indicating that tears and tear proteins can be transported to the oral cavity, where they mix with saliva. Under this scenario, a higher protein content in the tears of \u003cem\u003eM. m. domesticus\u003c/em\u003e, particularly in males (Fig. 5A), could partially offset the higher production of salivary proteins in \u003cem\u003eM. m. musculus\u003c/em\u003e associated with larger SMG size (Figs. 2\u0026ndash;3) and higher proportion of GCT (Fig. 4).\u003c/p\u003e\n\u003cp\u003eOur behavioural tests revealed a preference for tears as olfactory cues relative to saline, which was statistically significant only in the \u003cem\u003edomesticus\u003c/em\u003e-derived WDS. This finding suggests that tear-derived cues may contribute to social interactions and mate-related behaviours, consistent with previous studies demonstrating the behavioural relevance of lacrimal gland products (Kimoto et al. 2005, 2007; Stopkov\u0026aacute; et al. 2016). The observed divergence between the WDSs may therefore reflect differences in the relative contribution of tear cues to behavioural strategies and sexual communication in the two taxa. While many semiochemical proteins are known to differ between the two subspecies in olfactory-related secretions, the functional significance of this divergence beyond subspecies recognition remains incompletely understood (Stopkov\u0026aacute; et al. 2007; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011; Hurst et al. 2017). In this context, the absence of significant preference to tears over saline in the\u003cem\u003e musculus\u003c/em\u003e-derived WDS may indicate a reduced reliance on lacrimal olfactory cues in this subspecies under the tested conditions. Similar subspecies- or species-specific differences in the use of olfactory cues have also been reported in studies of scent-marking behaviour and olfactory strategies in other mammals (Mykytowycz and Goodrich 1974; Barja and de Miguel, 2010; Becker et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvolutionary implications of sexually dimorphic chemical signalling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study represents the first comparison of sexual dimorphism in the morphology of glands involved in olfactory communication between two closely related taxa. This allows our findings to be discussed within the broader context of species divergence, which in house mice has been documented across multiple behavioural, physiological, and chemical traits. The differences in gland sizes and especially sexual size dimorphism, consistently observed both in wild and wild-derived strains of \u003cem\u003eM. m. musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e, are likely shaped by a combination of genetic, physiological, and ecological factors (Badayaev et al. 2002; Williams and Caroll 2009). \u003c/p\u003e\n\u003cp\u003eAlthough the social structure of house mice can vary depending on the ecological context (Berry 1981; Sage 1981; Singleton and Krebs 2007), the two subspecies largely share the same synanthropic (commensal) niche (Sage 1981; Boursot et al. 1993; Machol\u0026aacute;n et al. 2012). Nevertheless, they differ markedly in several social and behavioural characteristics that are relevant to chemical communication and results of this study. For example, \u003cem\u003eM. m. domesticus\u003c/em\u003e exhibits higher levels of aggression, more strongly substructured populations and more rapid establishment of social hierarchies than \u003cem\u003eM. m. musculus \u003c/em\u003e(Thuessen 1977; van Zegeren and van Oortmerssen 1981; Ďureje et al. 2011; Hiadlovsk\u0026aacute; et al. 2015; Latour and Ganem 2017; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2020; Hiadlovsk\u0026aacute; et al. 2021; Mikula et al. 2022; Machol\u0026aacute;n et al. 2023; Bendov\u0026aacute; et al. 2024). Rapid establishment of social rank, supported by effective social communication, can reduce stress and contribute to the stability of the group (Koolhaas et al. 1999; Parmigiani et al. 1999; Hurst and Beynon 2004; Mucignat Caretta and Caretta 2014; Barabas et al. 2021, 2022; Yu et al. 2024). Differences in social organization and competitive interactions may therefore influence the selective pressures acting on communication systems.\u003c/p\u003e\n\u003cp\u003eOur findings suggest that \u003cem\u003eM. m. domesticus\u003c/em\u003e may experience stronger sexual selection pressures than \u003cem\u003eM. m. musculus\u003c/em\u003e, possibly driven by more intense competition for mates or more rigid, hierarchy-dependent social interactions. Thus these taxa may face different social pressures that drive the evolution of more specialized communication mechanisms and more pronounced sexual dimorphism in glandular structures (Isaak 2005; Kokko and Rankin 2006). This is corroborated, besides higher aggression and tighter social organization, by larger differences between males and females revealed for several analysed traits in \u003cem\u003eM. m. domesticus\u003c/em\u003e (Figs. 2\u0026ndash;4). In this context, our results are consistent with the idea that the two subspecies differ in the relative emphasis placed on distinct chemical signalling channels. \u003c/p\u003e\n\u003cp\u003ePrevious work has shown that \u003cem\u003eM. m. musculus\u003c/em\u003e produces a higher amount of major urinary proteins than \u003cem\u003eM. m. domesticus\u003c/em\u003e and exhibits greater diversity and sexual dimorphism in urinary signalling (Stopkov\u0026aacute; et al. 2007; Stopka et al. 2012; Hurst et al. 2017; Sheehan et al 2019, Penn et al 2022). Furthermore, the former subspecies possesses larger submandibular glands and larger GCT, whereas the latter showed a higher volume of tear proteins and a preference for tears over saline (this study). These findings are consistent with higher choosiness of \u003cem\u003eM. m. musculus\u003c/em\u003e males and females based on urine (Smadja and Ganem 2002; Smadja et al. 2004; B\u0026iacute;mov\u0026aacute; et al. 2005; Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011) and saliva (Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011) used as signals. Altogether, this leads us to assume higher importance of tears for \u003cem\u003eM. m. domesticus\u003c/em\u003e, in contrast to \u003cem\u003eM. m. musculus\u003c/em\u003e, which relies more heavily on urine and saliva (though urine is still a primary source of information for \u003cem\u003eboth\u003c/em\u003e subspecies, especially when mice are not in close contact). The question of why, then, \u003cem\u003eM. m. musculus\u003c/em\u003e has also slightly larger lacrimal glands than \u003cem\u003eM. m. domesticus, \u003c/em\u003eeven though the difference was not found to be significant in our data, contrary to SMG difference, is yet to be answered. However, it should be pointed out that proteins only constitute 0.3\u0026ndash;2% of the total tear volume (depending on the physiological context; Van Haeringen 1981). \u003c/p\u003e\n\u003cp\u003eFinally, we can hypothesize that while \u003cem\u003emusculus\u003c/em\u003e males and females are choosier based on urinary and salivary cues, \u003cem\u003eM. m. domesticus\u003c/em\u003e may display assortative preference when tears are involved. We want to emphasise that the lacrimal signals were never tested as cues in mate choice in mice. This prediction can be easily tested using a simple Y-maze experiment contrasting heterosubspecific cues in the same way as in all previous studies (see Vo\u0026scaron;lajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. 2011). Our results thus highlight how even subtle differences in mating systems and social structures can shape the evolution of sexual dimorphism and divergence of olfactory communication pathway and contribute to understanding how chemical signalling traits may diverge during subspecies differentiation, potentially playing a role in reproductive isolation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryptic sexual dimorphism and chemical communication in mammals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study highlights the importance of integrating morphological, histological, physiological, and behavioural data to understand sexual dimorphism in chemical communication systems. As noted by Eisenberg and Kleiman (1972), the role of chemical signals in mammalian communication can be subtle yet significant, influencing both intra- and inter-sexual interactions. Sexual dimorphism emerging as a consequence of these interactions, shaped by natural and/or sexual selection, can then be rather cryptic. Nevertheless, these \u0026lsquo;chemical antlers\u0026rsquo;, as we call them, can be as important as (or even more significant than) widely known conspicuous sexually dimorphic structures of many vertebrate and invertebrate species, especially in macrosmatic organisms like house mice. Indeed, a number of studies have documented that species \u0026ndash; often considered to show little or no sexual dimorphism \u0026ndash; are, in fact, sexually dimorphic (Eisenberg and Kleiman 1972; Fan 1987; Blaustein 1981; Arnold and Houck 1982; Jannett 1986; Maico et al. 2001; Rosell and Schulte 2004; Macdonald and Herrera 2013; Spence-Aizenberg et al. 2018; Mu\u0026ntilde;oz-Romo et al. 2021; this study).\u003c/p\u003e\n\u003cp\u003eTherefore, the size and morphology of the secretory organs can provide a valuable insight into sexual dimorphism, especially when supplemented with other approaches. The scent gland sexual dimorphism is widely known in other rodents (Rosell and Schulte 2004; Macdonald and Herrera 2013; Mu\u0026ntilde;oz-Romo et al. 2021; Rodriguez et al. 2023) but in mice, it has been so far largely neglected, with focus being rather on the chemical nature of the excretions (Hurst and Beynon 2004; Chung et al. 2017) or neurological pathways (Bergan et al. 2014; van der Linden et al. 2018). However, the link between sexual dimorphism and olfaction has been recently reported in a study examining more than 100 mammal species, including the mouse (Tombak et al. 2024). There is a growing body of evidence, that the molecular underpinnings of sexual dimorphism have mainly centred on expression of genes (Loire et al. 2017) that exhibit rapid evolution (Harissin et al. 2015). High levels of dimorphism are shown to correlate with expansion of gene families enriched in olfactory sensory perception (Van der Linen et al. 2018; Padilla-Morales et al. 2024), suggesting a relationship between intense sexual selection and alterations in gene family size, illustrating the complex interplay between sexual dimorphism, gene family size evolution, and their roles in mammalian genome evolution (Van der Linen et al. 2018; Padilla-Morales et al. 2024). The sexual dimorphism observed in the submandibular and lacrimal glands of house mice thus provides valuable insights into the broader mechanisms of sexual selection and communication in mammals.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we provide a comprehensive characterization of sexual dimorphism in two exocrine glands involved in chemical communication in the house mouse, using both wild mice and wild-derived strains. Males of both subspecies exhibited larger glands and a higher proportion of granular convoluted tubules, while the subspecies differed in the relative magnitude of glandular dimorphism, protein output of glandular secretions, and behavioural responses. Employing two closely related taxa places our findings in the context of subspecies divergence and the emergence of reproductive barriers between nascent species. Our findings demonstrate that closely related subspecies can differ not only in the chemical composition of olfactory signals but also in the relative contribution of distinct secretory organs to chemical communication. We further show that even subtle differences in mating systems and social structures can shape the evolution of sexual dimorphism and divergence of olfactory communication pathways. Our results highlight the importance of cryptic sexual dimorphism and glandular morphology as integral, yet previously underappreciated, components of sexual dimorphism in mammalian olfactory signalling systems. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank J. Pi\u0026aacute;lek for providing WDS mice and breeding facilities, Ľ. Ďureje for help with mouse trapping and preparation of histological samples, L. Rouskov\u0026aacute; for additional tear and saliva sample collection, and the technical staff of the breeding facility of the Institute of Vertebrate Biology, Czech Academy of Sciences, in Studenec for mouse care. We are grateful to two anonymous colleagues for their valuable comments and revisions during manuscript preparation. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project no. Inter-Excellence, Inter-Action, LTAUSA18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the results of this study are provided in the article and its supplementary materials. Additional raw data (e.g., individual measurements) are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBVB designed the experiments, contributed to mouse trapping, histological analyses and data collection, managed the BCA and Take3 proteomic analyses, statistical analyses, and led manuscript writing. MM was project leader, contributed to experiment design, mouse trapping and dissections, manuscript writing, and provided financial support. DB performed behavioral analyses of sexual preferences and data analysis under supervision of ZH and later BVB. KVK designed and funded proteomic analyses, analyzed proteomic data, and contributed to manuscript writing. KD assisted with mouse trapping and dissections. ZH conceived the study idea, designed experiments, contributed to trapping, dissections, sample collection, data and statistical analyses, and manuscript writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project no. Inter-Excellence, Inter-Action, LTAUSA18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Ethical Committee of the Institute of Animal Physiology and Genetics, Czech Academy of Sciences, and were conducted in accordance with national and EU legislation. The facility at the Institute of Vertebrate Biology, Czech Academy of Sciences, where the experiments were performed, is accredited for breeding (48389/2020-MZE-18134, 2021\u0026ndash;2026) and use (62065/2017-MZE-17214, 2017\u0026ndash;2022) of experimental animals. The animals were handled by authorized persons only (Licenses No. ZH: CZ 0127; BVB: CZ01293 and DB as a university student under supervision).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAmano O, Mizobe K, Bando Y, Sakiyama K (2012) Anatomy and histology of rodent and human major salivary glands- Overview of the Japan Salivary Gland Society-Sponsored Workshop. Acta Histochem. Cytochem. 45:241\u0026ndash;250.\u003c/li\u003e\n \u003cli\u003eAndersson MB (1994) Sexual selection, Princeton University Press, Princeton.\u003c/li\u003e\n \u003cli\u003eArnold SJ, Houck LD (1982) sCourtship pheromones: Evolution by natural and sexual selection, in: Biochemical Aspects of Evolutionary Biology, M. H. Nitecki, ed., University of Chicago Press, Chicago.\u003c/li\u003e\n \u003cli\u003eBadyaev AV, Hill GE, Beck ML, Dervan AA, Duckworth RA, McGraw KJ, Nolan PM LA Whittingham (2002) Sex-biased hatching order and adaptive population divergence in a passerine bird. 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Behav Processes. 216:105008. https://doi.org/10.1016/j.beproc.2024.105008.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sexual dimorphism, sexual preferences, chemical communication, reproductive isolation, scent glands, house mouse","lastPublishedDoi":"10.21203/rs.3.rs-8423338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8423338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSexual dimorphism (SD), the systematic difference in phenotype between males and females of the same species, can arise through sexual and natural selection. Although SD is traditionally associated with conspicuous traits such as body size or colouration, it may also occur in cryptic characteristics such as chemical signalling. In mammals, where olfactory communication plays a central role, SD may be reflected in differences in the size or morphology of scent glands, as well as in the abundance and composition of their secretions. Here, we investigate sexual dimorphism in the size, histology, and protein content of the submandibular and lacrimal glands in two house mouse subspecies, \u003cem\u003eMus musculus musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e. We showed remarkable dimorphism in both glands, with males of both subspecies exhibiting larger glands, including a higher proportion of granular convoluted tubules (GCTs) in the submandibular gland. Subspecies-specific differences in gland size were detected only in the submandibular gland, which was larger in \u003cem\u003eM. m. musculus\u003c/em\u003e. In contrast, SD was more pronounced in the lacrimal gland in both subspecies and was strongest in \u003cem\u003eM. m. domesticus\u003c/em\u003e. Furthermore, we found subspecies-specific differences in tear protein content and odour cue preference, suggesting mate recognition systems may be more divergent between these closely related taxa than previously assumed. By integrating data from wild animals and wild-derived strains, we provide a comprehensive assessment of sex-specific morphological and biochemical divergence in these exocrine glands. Our findings underscore the evolutionary significance of cryptic sexual dimorphism in mammalian olfactory signalling systems.\u003c/p\u003e","manuscriptTitle":"Chemical antlers: sexual dimorphism in salivary and lacrimal glands of house mouse subspecies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 19:40:47","doi":"10.21203/rs.3.rs-8423338/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae88b183-b317-4d33-ac4b-dd09c3c5947b","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T11:27:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 19:40:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8423338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8423338","identity":"rs-8423338","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}