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Article

Multiple Chemical Signals in Male Rock Lizards: Femoral Gland Secretions and Feces May Provide Information on Body Size but Using Different Compounds

by
José Martín
*,
Gonzalo Rodríguez-Ruiz
and
Pilar López
Departamento de Ecología Evolutiva, Museo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Científicas (CSIC), C/José Gutiérrez Abascal 2, 28006 Madrid, Spain
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(7), 858; https://doi.org/10.3390/d15070858
Submission received: 17 June 2023 / Revised: 12 July 2023 / Accepted: 14 July 2023 / Published: 15 July 2023
(This article belongs to the Special Issue Ecology and Evolution of Chemical Communication in Lizards)
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Abstract

:
Chemosensory communication in lizards may be based on multiple compounds coming from multiple sources. Both secretions from external epidermal glands, and internal cloacal glands and feces are known to convey information (sex, familiarity, body size, etc.) for conspecifics. However, although some compounds in femoral gland secretions have been characterized and their function examined, there is very little information on potential semiochemicals in cloacal products and feces. More importantly, it is not well-known whether the compounds and information from multiple sources are redundant, complementary, or different. We analyzed the lipids in femoral gland secretions and feces of male Carpetan rock lizards (Iberolacerta cyreni) and examined which compounds might predict body size. We found many compounds in feces, mainly steroids, alkanes, and branched alkanes, while the main compounds in femoral secretions were steroids and fatty acids. The body size of males was related to the proportions of some alkanes, alkenes, and terpenoids in feces, while it is related to the proportions of some steroids and fatty acids in femoral secretions. There were also differences in the chemical profiles of feces of males and females, which may allow sex recognition. Therefore, femoral secretions and feces may both inform on males’ sex and body size, but the chemical bases of this information are different depending on the source.

1. Introduction

Chemical intraspecific communication is widespread and prominent in lizards [1,2,3,4,5], and this is based on chemical cues arising from different sources [6]. For example, compounds with the function of semiochemicals can be secreted by the skin or by large specialized external holocrine epidermal glands (femoral, preanal, or precloacal glands) [1,7,8], but semiochemicals can also come from internal cloacal glands and be secreted onto the surface of feces or scats, as feces are deposited by the lizard [9,10,11,12,13,14,15,16,17]. Semiochemicals from different sources can provide detailed information on conspecifics, and many species can use two or more of these sources to produce multiple chemical signals. For example, fecal chemicals may proportionate similar or complementary information to femoral gland secretions to female Psammodromus algirus lizards, which can discriminate between young (subadult sneakers) and old (territorial) males based on chemical cues from either femoral secretions or from the feces of males [18]. Tokay geckos (Gekko gecko) can discriminate between their own skin and fecal chemicals, and those of unfamiliar conspecifics of the same sex [19]. However, self-recognition in male Lioalemus tenuis lizards is based on fecal chemicals, but not on precloacal secretions [6].
Several studies have analyzed the chemical composition of femoral, precloacal, or related gland secretions of several lizard species [7,8,20] and examined the relationships between some compounds and the morphological and physiological characteristics of the producer [21,22]. A few studies have also tested the activity of some specific compounds as potential semiochemicals [21,23,24,25,26]. However, in spite of the known role of feces in the communication of lizards, specific chemicals in feces with properties of semiochemicals have not been identified. Chemical signals in feces might probably be a combination of several lipids, as some lizards respond equally to the entire feces or to feces extracts using organic solvents (dichloromethane), while further fractionation of the feces with different solvents (pentane and methanol) led to the loss of the unique signals needed for chemosensory recognition [6,13]. Therefore, it is unknown whether, within a given lizard species that use multiple types of chemical signals, the information provided by fecal chemicals is similar and based on the same compounds than those secreted by the femoral or precloacal glands. This is important because the evolution of multiple sexual signals is one of the least understood features of sexual selection; in many cases, it is not clear whether multiple traits may either signal different characteristics or be redundant as a way to reinforce the reliability of signals, or be directed to different intended receivers [27,28,29,30,31]. Moreover, this topic has been mainly studied in visual sexual signals, e.g., [32,33,34], but is poorly understood with respect to multiple chemical signals, mainly referring to multiple compounds but coming from the same source [6,35,36,37,38].
The Carpetan rock lizard, Iberolacerta cyreni (formerly Lacerta monticola cyreni), is a small lacertid lizard found in the rocky habitats of high mountains of the center of the Iberian Peninsula. Males produce abundant femoral gland secretions during the mating season, which, as in other lizards, may reliably convey chemical information about a male’s characteristics [21,39,40,41]. Females may use this information to choose prospective mates of high quality [20,21,25], while males use these chemical signals in intrasexual agonistic relationships, assessing the fighting potential of rivals [42,43,44]. Femoral gland secretions of male I. cyreni are mainly composed of lipids (mainly cholesterol, carboxylic acids, and minor components such as alcohols, squalene, etc.) [45] and proteins [46]. Some of these lipids may have an important role in communication [22,25,35], although proteins alone also allow at least self-recognition [46]. On the other hand, male I. cyreni lizards deposit fecal pellets on prominent high locations on rocks, acting as a composite visual and chemical signal [11]. Similarly to femoral secretions, uncharacterized chemicals in these feces allow sexual discrimination, self-recognition, discrimination of familiar vs. unfamiliar males, and signaling of male body size [11,15]. However, as in other lizards, it is unknown which specific compounds from feces may function as semiochemicals and the information that they can provide.
Here, using gas chromatography–mass spectrometry (GC-MS), (1) we analyzed the lipidic chemical composition of femoral gland secretions and fecal pellets of the same individual I. cyreni male lizards. Then, (2) we examined the potential relationships between body size and proportions of the main lipidic compounds in both types of chemical cues, and (3) tested whether the chemical basis of such relationships was similar or based on different compounds. We also (4) analyzed compounds in the feces of females (females do not produce femoral secretions and only have vestigial femoral pores) to test for intersexual differences that may allow sex recognition.

2. Materials and Methods

2.1. Study Animals and Sampling Procedures

During June 2021, we captured by lassoing male and female I. cyreni lizards at “Alto del Telégrafo” (Guadarrama Mountains, Central Spain). This is a high treeless mountain area (1930 m asl) where the habitat consists of extensive rock gravels mixed with low shrubs (Juniperus communis, Cytisus oromediterraneus) and grassy meadows. To prevent the same individuals from being sampled repeatedly, lizards were captured in several well-separated areas.
Immediately after capture, we first collected fresh fecal pellets of each individual (males and females). This was favored because most lizards usually defecated while being handled as a defensive response. If required, we forced the expulsion of feces by gently compressing the bellies of lizards. Feces were collected directly from the cloaca of lizards using 1.1 mL total recovery chromatography glass vials (ref. V2275, Análisis Vínicos S.L., Tomelloso, Spain). If defecation did not occur in a short time, we stopped handling to minimize disturbance. Thereafter, we also collected femoral gland secretions of males by gently pressing with forceps around the hindlimb femoral pores. The extracted secretions were directly collected into other identical glass vials. Females only have vestigial femoral pores that not produce visible femoral secretions. Finally, we also prepared in the field blank control vials following the same handling procedures used with the lizards, but without collecting feces or secretion. Vials were closed using Teflon-lined stoppers and kept in the field inside a portable refrigerator with ice, and, in the day, stored at -20 °C in a freezer until analyses (one month after).
After collecting samples, we used a metallic ruler to measure to the nearest 1 mm the snout-to-vent length (SVL; males: mean ± SE = 75 ± 1 mm; range = 70–79 mm, n = 19; females: mean ± SE = 76 ± 1 mm; range = 69–86 mm, n = 10) and tail length of each individual. We also measured with a digital scale (to the nearest 0.1 g) body mass (males: mean ± SE = 7.1 ± 0.3 g; range = 5.1–9.2 g; females: mean ± SE = 5.4 ± 0.2 g; range = 4.1–6.6 g). After ensuring that lizards were in good condition, they were immediately released at their capture sites within 5 min after capture.

2.2. Chemical Analyses

In the laboratory, we thawed the vials with fecal samples and added to each vial 250 μL of n-hexane (capillary GC grade, Sigma-Aldrich Chemical Co., Saint Louis, MO, USA). The vial was closed and the solution mixed for 10 min with a vortex. Then, we kept the vial for 30 min inside a fridge so that the solid material that did not dissolve precipitated at the bottom of the vial. The supernatant clear liquid phase was then extracted using a glass syringe, transferred to a clean vial, sealed with a Teflon-lined stopper, and then analyzed (see below). Vials with femoral secretions were also thawed; we added 200 μL of n-hexane as above, and the vial was vortexed, closed with Teflon-lined stopper, and analyzed directly. We followed the same two procedures to prepare the control vials for each type of samples, respectively.
Samples were analyzed with a gas chromatograph (Agilent 7890A, Santa Clara, CA, USA) equipped with a Agilent HP5-MS column programmed (45 °C for 10 min, 45 to 280 °C at 5 °C/min, and 280 °C for 20 min), coupled to a mass spectrometer (Agilent 5973 Mass Selective Detector, MSD) (for details of similar analytical conditions, see [47]). We identified chemicals tentatively by comparison of mass spectra in the NIST/EPA/NIH 2002 library, and later confirmed them, when possible, with authentic standards (from Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and using information from previous studies of femoral secretions of this lizard species [21,22,45].

2.3. Statistical Analyses of Data

From the chromatograms, we calculated, using the Xcalibur software (Finningan Co., Ipswich, UK), the percentage of the total ion current (TIC) to determine the relative amount of each compound [48]. To address the issue of the non-independence between proportions, we transformed the relative proportions of each compound for statistical analysis using the formula: ln[(proportion)/(1 − proportion)] [48,49].
To explore the relationships between compounds in femoral secretions or feces of males and their body size, the transformed areas of all the compounds in femoral secretions or of the 39 most abundant compounds in feces (i.e., with relative proportions greater than 0.5%, which together accounted for more than 87%) were used to make two separated principal component analyses (PCAs). Then, we used stepwise general regression models (GRMs) to test wheter the factor scores of the extracted principal components (PCs) may be used as potential predictors of body size (log10-transformed SVL or body mass). Backward and forward stepwise analyses produced the same results. Statistical analyses were made using Statistica 7.0 software (StatSoft Inc., Tulsa, OK, USA).
To test for intersexual differences in the chemical profiles of feces, we used the software PRIMER V6.1.13 [50] and PERMANOVA+ V1.0.3 [51]. Analyses were made twice, considering all the compounds found in both sexes or restricted to the 20 most abundant compounds (i.e., with the highest mean relative proportions, considering the average abundance across all individuals), which were shared by both sexes. A resemblance matrix, the foundation for subsequent analysis, was created by calculating the Euclidean distances between each pair of individual samples. Then, to test for intersexual differences in the chemical profiles, we conducted permutational multivariate variance analysis (PERMANOVA, based on the resemblance matrix and employing 999 permutations) [52] and canonical analyses of principal co-ordinates (CAPs) [53].

3. Results

3.1. Chemicals in Feces of Male Lizards

We found a total of 140 lipophilic compounds in fresh feces collected directly from adult male lizards (Table S1). However, the number of compounds detected in a single individual was much lower, ranging between 35 and 92 (mean ± SD = 63 ± 14 compounds/fecal sample). Most of the major compounds were found in most of the samples (i.e., 25 compounds appeared in more than 80% of samples with a mean proportion of 2.91%), whereas minor compounds were found only occasionally (i.e., 85 compounds appeared in less than half of the samples with a mean proportion of 0.14%) (Table S1).
The major chemical classes of compounds that were more abundant (accounting together for more than a 90% relative proportion of the TIC area) were 35 steroids, 18 linear alkanes between n-C15 and n-C35, and 39 branched alkanes (Table 1 and Table S1). In addition, we also found other classes of compounds less abundant, such as five alcohols, eight carboxylic acids between n-C12 and n-C18, four alkenes, one terpenoid (squalene), seventeen methyl and ethyl esters of carboxylic acids between n-C16 and n-C20, two waxy esters, four aldehydes, two tocopherols, three ketones, one aromatic heterocyclic compound, and cyclic octa-atomic sulfur (Table 1). On average, pooling all males, the five most abundant compounds were cholesterol (13.8%), sitosterol (11.3%), heptacosane (9.8%), nonacosane (5.6%), and pentacosane (4.1%) (Table 2 and Table S1).

3.2. Chemicals in Femoral Gland Secretions of Male Lizards

Femoral gland secretions were mainly composed of 16 steroids (%TIC, mean ± SD = 90.7 ± 0.2%), followed by 4 carboxylic acids between n-C16 and n-C18 (9.1 ± 0.1%), 1 terpenoid (squalene) (0.1 ± 0.2%), and 1 alcohol (0.1 ± 0.1%). The most abundant compound was cholesterol (78%), followed by campesterol (5%), hexadecanoic acid (4%), 9-octadecenoic acid (3%), and octadecanoic acid (2%) (Table 3).

3.3. Relationships between Chemicals in Feces and Body Size of Male Lizards

The PCA for the 39 most abundant compounds (TIC > 0.5%) in the feces of males produced four feces principal components (fPCs) with eigenvalues greater than three (Table 2), which together accounted for 61.5% of variance. The SVL of male lizards was negatively and significantly correlated (stepwise GRM: R2 = 0.29, F1,17 = 7.13, p = 0.016) with fPC-3 (β = −0.54, t = −2.67, p = 0.016), describing chemicals in their feces (Figure 1a). A similar result was obtained considering body mass (stepwise GRM: R2 = 0.23, F1,17 = 5.06, p = 0.038; fPC-3, β = −0.48, t = −2.25, p = 0.038) (Figure 1b). Thus, according to factor loadings (Table 2), larger and heavier males had feces with high proportions of squalene and hentriacontane but a low proportion of pentacosane, heptacosene, and nonacosene.

3.4. Relationships between Chemicals in Femoral Gland Secretions and Body Size of Male Lizards

The PCA for the relative proportions of all the 22 compounds found in femoral gland secretion of males produced four femoral-gland principal components (gPCs) with eigenvalues greater than two (Table 3), which together accounted for 73.2% of variance. The SVL of male lizards was negatively and significantly correlated (stepwise GRM: R2 = 0.54, F2,17 = 8.58, p = 0.0014) with gPC-1 (β = −0.64, t = −3.86, p = 0.0012) and gPC-4 scores (β = −0.36, t = −2.21, p = 0.0012) describing chemicals in their femoral gland secretions (Figure 2a,b). Similarly, body mass was significantly correlated (stepwise GRM: R2 = 0.61, F2.17 = 13.33, p = 0.0003) negatively with gPC-1 (β = −0.71, t = −4.67, p = 0.0002) and positively with gPC-3 scores (β = 0.33, t = 2.20, p = 0.042) (Figure 2c,d). Thus, according to factor loadings (Table 3), males with a greater body size had femoral secretions with a high proportion of unsaturated carboxylic acids (hexadecenoic and octadecenoic acids) and some steroids, such as ergosterol and campesterol, but low proportions of cholesterol and saturated carboxylic acids (hexadecanoic and octadecanoic acids).

3.5. Intersexual Differences in the Chemical Profiles of Feces

Females had a total of 167 lipophilic compounds in fresh feces (Table S1), with the number of compounds in an individual ranging between 26 and 104 (mean ± SD = 78 ± 25 compounds/fecal sample). This number was not significantly different between sexes (GLM on log10-transformed numbers: F1,27 = 1.86, p = 0.18). As in males, most of the major compounds in the feces of females were found in most of the samples (i.e., 32 compounds appeared in more than 80% of samples with a mean proportion of 2.26%), whereas minor compounds were found less frequently (i.e., 105 compounds appeared in less than half of the samples with a mean proportion of 0.11%) (Table S1).
Males and females had similar types of lipophilic major compounds, and although the rank order of the proportions of major classes of compounds was not identical, there were not significant overall differences between sexes in these proportions (Pearson’s χ2 = 4.63, p = 0.46) (Table 1). In females, the main major classes of compounds (more than 90% of relative proportion) were 66 branched alkanes, 30 steroids, and 15 linear alkanes between n-C19 and n-C35 (Table 1 and Table S1). Other less abundant classes of compounds were nine alcohols, nine carboxylic acids between n-C10 and n-C22, one terpenoid (squalene), sixteen methyl and ethyl esters of carboxylic acids between n-C16 and n-C20, four alkenes, five aldehydes, five waxy esters, two tocopherols, three ketones, one furanone, and one aromatic heterocyclic compounds (Table 1).
The most abundant compounds were similar in males and females, although there were some differences in the rank order of the most abundant ones (Table S1). On average, pooling all females, the five most abundant compounds were cholesterol (11.8%), heptacosane (8.8%), nonacosane (7.4%), sitosterol (7.1%), and 13-methylnonacosane (4.7%) (Table S1).
Males and females shared 120 from a total of 187 compounds found in feces, representing 63.7% of total TIC area (pooling all individuals), but 20 compounds were exclusively found in males and 47 compounds were exclusively found in females. The overall profiles of the proportion of all compounds in feces differed significantly between males and females (PERMANOVA, pseudo F1.27 = 2.22, p = 0.003), and the CAP analysis assigned 96.5% of the chemical profiles into the correct sex using the Euclidean distances between samples (permutational test, δ29 = 0.95, p = 0.001, using leave-one-out cross-validation and m = 15 axis). However, similar analyses restricted to the 20 most abundant compounds, which were shared by both sexes, did not show significant differences between sexes (PERMANOVA, pseudo F1.27 = 0.81, p = 0.61; CAP, 55.2% correct assignations; permutational test, δ29 = 0.67, p = 0.33, m = 17 axis).

4. Discussion

Previous studies have shown that, as with many other lizards, male Rock lizards I. cyreni are able to discriminate between classes of conspecific males, and estimate their body size, using chemicals from both femoral gland secretions or feces [11,15,42,43]. The current results show that some of the compounds found in femoral gland secretions of I. cyreni, and that have been identified as important in intraspecific communication, e.g., [22,26,54], are also found in the feces of this lizard. This could lead us to think that lizards showed similar discriminatory abilities to scents from femoral secretions or feces simply because lizards would be detecting and responding to the same compounds, although coming from different sources. However, we also found here that the compounds in feces, the variations of which might be potentially used as predictors of body size, were different from those with a potential similar function in femoral gland secretions.
Our chemical analyses showed the occurrence of a high diversity of lipophilic compounds, mainly steroids and alkanes, in the feces of male I. cyreni. Some specific compounds are consistently present and abundant in feces, which may suggest that they are not simply the remains of the prey (i.e., this lizard feeds on a diversity of insects and other arthropods) [55], which could change across individuals depending on the prey types found in each fecal pellet. In contrast, some of these compounds that exhibit a similar pattern across individuals might be released by internal cloacal glands and mixed with feces prior to being deposited, as it has been suggested in skinks and other lizards [6,12,13,14]. Moreover, in Egernia striolata skinks, changes in the diet of the producer of fecal pellets do not affect the chemosensory responses of conspecifics to their feces [13]. Some of these compounds in the feces of male I. cyreni might have a role as semiochemicals in intraspecific communication, as suggested by the previous experiments that examined the behavioral responses of lizards to the scent from feces [11,15]. Unfortunately, there is a lack of information on the chemical composition of feces of other lizard species, even for those where the role of feces in communication is clear and prominent, and further chemical analyses of representatives of different groups are needed.
The major compounds found in the feces of I. cyreni were steroids, especially cholesterol, which coincides with the overall composition of femoral gland secretions of this and many other lizards [7,8,20]. However, in contrast to femoral secretions, lineal and branched alkanes, mainly long-chained ones, were also very abundant in feces. This is, however, similar to the composition of lipids in the feces of the amphisbaenian Trogonophis wiegmanni [47], which also can discriminate between conspecific classes based on scent from feces [56]. Long-chain hydrocarbons are also found functioning as semiochemicals in the skin of snakes [57] and in the cuticle of many insects, e.g., [58]. Further research is necessary to determine whether the mixtures of long-chain alkanes discovered in I. cyreni feces have a similar role in sex identification and even in familiar recognition and reproductive activity.
Interestingly, the relationships between SVL and body mass of male I. cyreni lizards and the proportions of some compounds in their femoral secretions and feces suggest that body size might be potentially inferred from the variations in these compounds, but different ones depending of the source of scent. Thus, in femoral secretions of male I. cyreni, variations in proportions of some steroids and fatty acids vary with body size, and likely age classes. This lizard has two ontogenetic social–spatial reproductive strategies [59], with larger/older males being dominant and more active in defending aggressively large home ranges from other males, while smaller/younger males are subordinate and less active, and adopt a sneaker strategy. For a large male, scent marking of territories to signal his quality to competitor males and to potential mates may be more important than for small males. This could explain why these large males allocate more “costly” compounds to secretions. Among these, for example, ergosterol has alternative important functions in the immune system and their proportions in secretions are also related to the health state of an individual [21,22]. Campesterol (a phytosterol) and octadecenoic (oleic) acid may be directly related to the diet quality or the amount of body fat reserves of a male [54]. The condition-dependent costs of allocating these compounds to scent marks may confer reliability to the signal [41]. In contrast, small males predominantly allocate cholesterol or saturated fatty acids to secretions, which may be less costly, as for these males signaling their quality through scent marking may not be so important.
Body size might also be related to some compounds in feces, but these are different types of compounds than those found in femoral secretions with a similar size-related variation. Interestingly, variations in squalene and some alkanes and alkenes in feces seem related to body size. Also, in the amphisbaenians T. wiegmanni and Blanus cinereus, squalene was more abundant in the scent of larger males and this compound alone triggers aggressive responses from conspecific males [47,60]. The proportions of squalene in the skin of garter snakes are also considered to allow sex recognition [57,61]. That squalene may be a signal of a male with a larger body size and dominance might be explained by the fact that squalene is a biochemical precursor to steroid hormones like testosterone, and the fact that there is probably some sort of metabolic interaction between the levels of these two substances in the body. Also, it might be possible that steroids and fatty acids in feces were not useful in the communication system because these compounds may be altered or transformed by intestinal and cloacal bacteria to other compounds (e.g., cholesterol to cholestanol, or fatty acids to aldehydes) [62,63]. These alterations might hide possible direct relationships of the initial proportion of these compounds with body size, while proportions of some hydrocarbons and squalene might be less affected by bacteria degradation.
Finally, the feces of male and female I. cyreni lizards had similar types of compounds. However, there were intersexual differences in the overall chemical profiles of feces. These differences might probably be based on minor compounds, exclusives of each sex, as analyses restricted to the most abundant and shared main compounds did not show differences between males and females. Because there are not differences in diet between sexes [55], it is unlikely that the presence of different chemical compounds in the feces of males and females were exclusively the consequence of differences in diet. Lizards may be able to discriminate between these differences to assess the sex of the producer of a fecal pellet, which should have important consequences for the social organization (i.e., males may discriminate between feces of other competitor males and those of females), but also on the reproductive behavior of this lizard, because females lack femoral gland secretions. Further studies should examine whether males might use compounds in the feces of females to obtain information on female traits such as reproductive state or potential future reproductive output (clutch size, etc.).
Within the context of multiple sexual signals [11,27,64], it seems that information regarding body size from feces and femoral gland secretions may be considered initially to be redundant and similar. However, the function of different signals may be complementary or used in different contexts. It is likely that fecal pellets deposited on conspicuous sites may first act as visual signals attracting a lizard [9,10,11,64] and later provide a first quick chemical information on the sex and size of the producer. Further, in species with femoral gland secretions, if this information was considered “interesting”, it might induce a lizard to look for additional detailed and probably more reliable information from nearby, but less conspicuous, substrate scent marks produced with these femoral secretions.
Although responses of lizards to some of the compounds identified in this study as potential semiochemicals have already been partially examined in I. cyreni lizards [22,25,35], future studies should continue testing the chemosensory and behavioral responses of this and other lizard species to single compounds, mixes of them in different proportions, or manipulations of the original scent marks or feces. These studies, together with chemical analyses, would help to reveal and understand the potential role of compounds in feces as semiochemicals providing reliable information in intraspecific communication in lizards.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15070858/s1, Table S1. Compounds in femoral gland secretions of male and female I. cyreni lizards. Mean (±SD) relative proportion (%) of the total ion current (TIC) area of compounds (ordered by major chemical classes and by retention times—RT), and frequency (%) of individuals presenting a particular compound.

Author Contributions

Conceptualization, J.M. and P.L.; methodology, J.M. and P.L.; validation, J.M.; formal analysis, J.M.; investigation, J.M., G.R.-R. and P.L.; writing—original draft preparation, J.M.; writing—review and editing, J.M., G.R.-R. and P.L.; funding acquisition, J.M. and P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Spanish Ministerio de Ciencia e Innovación project PID2021-122358NB-I00 (MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe).

Institutional Review Board Statement

The captures enforced all the present Spanish laws and were performed under a license granted by the “Dirección General de Biodiversidad y Recursos Naturales”, Comunidad Autónoma de Madrid (Spain) (Ref. 10/170740.9/21). The study followed ASAB (2020) guidelines for the ethical treatment of animals in behavioral research, and was carried out in accordance with the national animal welfare standards and protocols, which were supervised by the “Comisión Ética de Experimentación Animal (CEEA)” of the Museo Nacional de Ciencias Naturales, CSIC.

Data Availability Statement

The data presented in this study are openly available in FigShare at https://figshare.com/articles/dataset/Multiple_Chemical_Signals_in_Male_Rock_Lizards/23669145 (accessed on 13 June 2023).

Acknowledgments

We thank the two anonymous reviewers for their helpful comments and the “El Ventorrillo” MNCN-CSIC Field Station for the use of their facilities.

Conflicts of Interest

The authors declare no conflict of interest.

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Diversity 15 00858 g001 550
Figure 1. Body-size-related variation in compounds in feces of male lizards I. cyreni. Relationships between (a) log SVL or (b) log body mass of males and the fPC-3 scores from a PCA describing the profile of compounds found in their feces. The arrows indicate the correlations of the fPC-scores with the relative proportions of determined compounds.
Figure 1. Body-size-related variation in compounds in feces of male lizards I. cyreni. Relationships between (a) log SVL or (b) log body mass of males and the fPC-3 scores from a PCA describing the profile of compounds found in their feces. The arrows indicate the correlations of the fPC-scores with the relative proportions of determined compounds.
Diversity 15 00858 g001
Diversity 15 00858 g002a 550Diversity 15 00858 g002b 550
Figure 2. Body-size-related variation in compounds in femoral gland secretions of male lizards I. cyreni. Relationships between log SVL of males and the (a) gPC-1 or (b) gPC-3 scores from a PCA describing the profile of compounds found in their femoral gland secretions or between log body mass and (c) gPC-1 or (d) gPC-4 scores. The arrows indicate the correlations of the gPC-scores with the relative proportions of determined compounds.
Figure 2. Body-size-related variation in compounds in femoral gland secretions of male lizards I. cyreni. Relationships between log SVL of males and the (a) gPC-1 or (b) gPC-3 scores from a PCA describing the profile of compounds found in their femoral gland secretions or between log body mass and (c) gPC-1 or (d) gPC-4 scores. The arrows indicate the correlations of the gPC-scores with the relative proportions of determined compounds.
Diversity 15 00858 g002aDiversity 15 00858 g002b
Table
Table 1. Relative proportions (population mean ± SD% of the TIC area) of the major chemical classes of lipidic compounds in feces of male and female I. cyreni lizards.
Table 1. Relative proportions (population mean ± SD% of the TIC area) of the major chemical classes of lipidic compounds in feces of male and female I. cyreni lizards.
Chemical ClassMalesFemales
Steroids38.61 ± 20.84%26.93 ± 12.04%
Linear alkanes26.92 ± 10.51%26.50 ± 9.96%
Branched alkanes25.15 ± 15.95%36.64 ± 14.14%
Alcohols2.95 ± 4.70%3.38 ± 3.28%
Carboxyilic acids2.60 ± 6.28%3.20 ± 5.14%
Alkenes1.64 ± 2.10%1.31 ± 2.85%
Terpenoids0.76 ± 0.72%0.69 ± 0.45%
Esters of carboxylic acids0.56 ± 1.16%0.59 ± 0.67%
Waxy esters0.30 ± 0.66%0.21 ± 0.22%
Aldehydes0.30 ± 0.17%0.35 ± 0.10%
Tocopherols0.16 ± 0.26%0.11 ± 0.20%
Ketones0.03 ± 0.08%0.08 ± 0.17%
Aromatic heterocyclic0.02 ± 0.04%0.01 ± 0.01%
Cyclic octa-atomic sulfur0.01 ± 0.02%0
Furanones00.01 ± 0.04%
Table
Table 2. Main compounds in feces of male I. cyreni lizards. Mean (±SD) relative proportion (%) of the total ion current (TIC) area of compounds (presented in order of abundance) and factor loadings from a PCA (i.e., correlations of each compound with each PC). Correlations between the compounds and the principal components (PC) that were significant at p < 0.001 are marked in bold. Retention times (RT; min) are shown for unidentified (Unid.) compounds.
Table 2. Main compounds in feces of male I. cyreni lizards. Mean (±SD) relative proportion (%) of the total ion current (TIC) area of compounds (presented in order of abundance) and factor loadings from a PCA (i.e., correlations of each compound with each PC). Correlations between the compounds and the principal components (PC) that were significant at p < 0.001 are marked in bold. Retention times (RT; min) are shown for unidentified (Unid.) compounds.
CompoundMean ± SDfPC-1fPC-2fPC-3fPC-4
Cholesterol13.85 ± 7.060.71−0.30−0.230.35
Sitosterol11.34 ± 9.280.92−0.06−0.17−0.09
Heptacosane9.78 ± 5.76−0.68−0.030.40−0.33
Nonacosane5.63 ± 3.680.000.74−0.04−0.20
Pentacosane4.15 ± 3.18−0.270.030.81−0.07
13-Methylheptacosane3.47 ± 5.89−0.610.31−0.17−0.42
Hentriacontane2.80 ± 3.300.250.55−0.510.38
13-Methylnonacosane2.76 ± 3.12−0.68−0.15−0.080.42
Campesterol2.74 ± 2.030.850.01−0.230.02
Unid. Branched Alkane at RT = 55.552.55 ± 2.37−0.680.50−0.02−0.19
Docosanol2.37 ± 3.790.010.170.130.51
Stigmasta-5,24(28)-dien-3-ol1.93 ± 2.170.580.010.40−0.11
Unid. Branched Alkane at RT = 58.181.65 ± 2.01−0.670.01−0.100.39
9-Octadecenoic acid1.53 ± 4.08−0.230.450.200.32
Unid. Branched Alkane at RT = 56.801.37 ± 1.61−0.410.17−0.02−0.62
Octacosane1.29 ± 1.12−0.800.08−0.100.19
Unid. Branched Alkane at RT = 53.971.24 ± 2.31−0.59−0.440.36−0.16
Unid. Branched Alkane at RT = 55.411.14 ± 1.37−0.830.010.040.17
15,19-Dimethyl-pentatriacontane?1.09 ± 1.030.000.32−0.010.16
Tricosane1.07 ± 0.950.080.480.290.09
Unid. Steroid at RT = 68.051.03 ± 1.590.610.300.30−0.19
Unid. Branched Alkane at RT = 55.030.97 ± 1.85−0.210.580.020.21
Unid. Branched Alkane at RT = 58.420.89 ± 0.740.080.730.30−0.36
Stigmasterol0.89 ± 0.850.800.120.000.24
Unid. Branched Alkane at RT = 55.110.80 ± 1.10−0.590.46−0.08−0.28
Ergosta-5,24-dien-3-ol0.76 ± 1.170.75−0.060.410.11
Squalene0.76 ± 0.72−0.62−0.18−0.570.19
Heptacosene0.75 ± 1.68−0.31−0.340.640.07
Hexacosane0.75 ± 0.74−0.70−0.270.33−0.01
Hexadecanoic acid0.71 ± 2.21−0.250.410.340.36
Unid. Branched Alkane at RT = 52.040.65 ± 1.08−0.430.310.050.63
Unid. Branched Alkane at RT = 52.630.62 ± 0.65−0.630.180.330.29
Unid. Steroid at RT = 73.770.62 ± 1.780.310.260.400.13
Stigmast-7-en-3-ol0.61 ± 0.680.460.100.56−0.23
Tritriacontane0.57 ± 0.810.320.810.060.09
Stigmastanol0.57 ± 0.910.42−0.060.17−0.16
Cholesta-5,7-dien-3-ol0.56 ± 0.520.68−0.080.320.28
Nonacosene0.54 ± 0.660.22−0.130.720.39
Unid. Branched Alkane at RT = 67.180.50 ± 0.650.520.47−0.35−0.09
Eigenvalue 11.634.804.373.18
Explained Variance (%) 29.8312.3011.198.15
Table
Table 3. Compounds in femoral gland secretions of male I. cyreni lizards. Mean (±SD) relative proportion (%) of the total ion current (TIC) area of compounds (presented in order of abundance) and factor loadings from a PCA (i.e., correlations of each compound with each PC). Correlations between the compounds and the principal components (PC) that were significant at p < 0.001 are marked in bold. Characteristic m/z fragments are shown for unidentified (Unid.) steroids.
Table 3. Compounds in femoral gland secretions of male I. cyreni lizards. Mean (±SD) relative proportion (%) of the total ion current (TIC) area of compounds (presented in order of abundance) and factor loadings from a PCA (i.e., correlations of each compound with each PC). Correlations between the compounds and the principal components (PC) that were significant at p < 0.001 are marked in bold. Characteristic m/z fragments are shown for unidentified (Unid.) steroids.
CompoundMean ± SDgPC-1gPC-2gPC-3gPC-4
Cholesterol77.92 ± 2.160.88−0.18−0.08−0.09
Campesterol5.43 ± 1.89−0.86−0.290.020.11
Hexadecanoic acid4.04 ± 0.03−0.25−0.300.400.60
9-Octadecenoic acid3.03 ± 0.02−0.710.440.27−0.25
Octadecanoic acid2.04 ± 0.030.100.110.550.75
Cholest-5-en-3-ol-, acetate1.58 ± 0.460.400.85−0.10−0.03
Sitosterol1.13 ± 0.58−0.690.21−0.180.45
Cholest-4-en-3-one0.97 ± 0.700.670.160.43−0.15
Cholest-5-en-3-ol, derivative?0.95 ± 0.430.240.800.12−0.02
4,4-Dimethyl-cholesta-5,7-dien-3-ol0.75 ± 0.27−0.79−0.210.13−0.20
Ergosta-5,8-dien-3-ol0.68 ± 0.13−0.750.05−0.32−0.12
Cholesta-3,5-diene0.51 ± 0.160.150.94−0.050.08
Cholesta-4,6-dien-3-ol0.31 ± 0.11−0.190.520.00−0.54
Ergosterol0.15 ± 0.11−0.910.140.07−0.10
Unid. Steroid (253,269,367,382)0.11 ± 0.07−0.710.170.09−0.09
Squalene0.11 ± 0.18−0.430.620.290.23
Cholesta-2,4-diene0.08 ± 0.06−0.560.66−0.030.27
Ergosta-5,22-dien-3-ol0.08 ± 0.09−0.70−0.21−0.03−0.11
Unid. Steroid (197,251,311,376)0.07 ± 0.06−0.200.20−0.760.21
Eicosanol0.02 ± 0.03−0.380.100.51−0.51
9-Hexadecenoic acid0.02 ± 0.02−0.07−0.330.73−0.12
Unid. Steroid (251,363,378)0.01 ± 0.02−0.44−0.60−0.21−0.02
Eigenvalue 7.174.462.422.05
Explained Variance (%) 32.6020.2810.999.30
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Martín, J.; Rodríguez-Ruiz, G.; López, P. Multiple Chemical Signals in Male Rock Lizards: Femoral Gland Secretions and Feces May Provide Information on Body Size but Using Different Compounds. Diversity 2023, 15, 858. https://doi.org/10.3390/d15070858

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Martín J, Rodríguez-Ruiz G, López P. Multiple Chemical Signals in Male Rock Lizards: Femoral Gland Secretions and Feces May Provide Information on Body Size but Using Different Compounds. Diversity. 2023; 15(7):858. https://doi.org/10.3390/d15070858

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Martín, José, Gonzalo Rodríguez-Ruiz, and Pilar López. 2023. "Multiple Chemical Signals in Male Rock Lizards: Femoral Gland Secretions and Feces May Provide Information on Body Size but Using Different Compounds" Diversity 15, no. 7: 858. https://doi.org/10.3390/d15070858

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