Craniometrical sexual dimorphism of the grey wolf ( Canis lupus, Canidae, Carnivora) in Bulgaria

: Cranial sexual dimorphism in grey wolf ( Canis lupus Linnaeus, 1758) from Bulgaria has not been studied so far. Skulls from adult individuals from across the country were studied. Thirty-six parameters (35 craniodental measurements and a volumetric one) of each skull were measured. The results showed that there was similar mean variability for all parameters in both sexes (Vc = 6.1 for males and Vc = 6.6 for females). The study presented the sexual size dimorphism in linear parameters of the skull as a male/female ratio and as a percentage (male-female)/female*100. The descriptive statistics of the obtained data revealed that the least variable parameters in both sexes were basal length, condylobasal length and cranial width. The most variable parameters of the wolf’s skull in males and females were length of crista sagittalis externa, frontal breadth, maximal occipital breadth and rostrum width of the cranium and height of the ramus of the mandible (between P 4 -M 1 ). The least sex-dependent parameters were postorbital breadth, the least diameter of bulla tympanica, the greatest diameter of bulla tympanica and brain skull volume as a non-linear parameter. The mandibular width and greatest skull length are among the parameters that exhibit the most significant sexual dimorphism. Additionally, the width of the mandibular processus articularis and the mesio-distal C length also show sexual dimorphism. The Bulgarian wolf population exhibits weak craniodental sexual dimorphism, which is male-biased, as is typical in canids with a monogamous social system. However, the sexual dimorphism is more pronounced than in the studied more northern populations.


Introduction
The grey wolf (Canis lupus Linnaeus, 1758) is a resident species in Bulgaria, with a fragmented distribution in Europe (Boitani et al., 2018).During the breeding season it mainly inhabits the mountains and partly the plains and hilly areas of Northeastern Bulgaria.It descends to the valleys during the autumn and until the middle of the winter (Popov & Sedefchev, 2003;Spassov, 2007;Spiridonov & Spassov, 2011).The species is listed "vulnerable" in the Red Data Book of Bulgaria (Spiridonov & Spassov, 2011).
Sexual dimorphism in species is primarily driven by the sexual selection (Erlinge, 1979;Gittleman & Van Valkenburgh, 1997).However, it is geographically dependent and influenced by local selective forces (Connallon, 2015).In some species, such as grey wolf, sexual dimorphism may depend on the segregation of the trophic niches of the two sexes (Ko-Historia naturalis bulgarica 46 (2024) rablev et al., 2021).On the Balkans and in Bulgaria, its sexual dimorphism has been investigated based on body parameters only (Trbojević & Ćirović, 2016;Tsingarska et al., 2019), but the cranial sexual size dimorphism has not yet been studied.The present study aimed to investigate and determine the sexual size dimorphism in some cranial features of the grey wolf in Bulgaria.

Material and methods
The collected skulls were divided into two age classes -juveniles (under 1 year of age) and adults (over 1 year of age).The juveniles were excluded from the present study because of their uncompleted growth.The age of the skulls was determined using a combination of methods, including tooth eruption and degree of incisor's wear (Fuller & Keith, 1980;Gipson et al., 2000), as well as ossification of synchondrosis intersphenoidalis and synchondrosis sphenooccipitalis (Landon et al., 1998).The method of determining age by cementum annulus counts on a canine tooth section considered the most accurate method (Goodwin & Ballard, 1985) was not applied for assessing the age of skulls due to their trophy value.
For this study, cranial material from 121 wolves (Canis lupus Linnaeus, 1758) over one year (54 males and 67 females) from the whole territory of Bulgaria (Fig. 1) was measured.The main part of the skulls (85 adult specimens) is stored in the National Museum of Natural History at the Bulgarian Academy of Sciences, Sofia.Most of these skulls are from animals hunted around the middle of the last century.Fifteen skulls, provided by hunters and forestry staff for 2 au- Thirty-six parameters (35 linear and 1 volumetric) of each skull were measured through mechanical calliper with an accuracy of 0.01mm and shown in Appendix: Table 1.Most of the measurements followed von den Driesch (1976) and are marked as such.The rest measurements were according to Onar et al. (2005), Milenković et al. (2006) and Loy et al. (2008).
Most of the parameters are shown on Fig. 2. The sexual dimorphism in linear parameters of the skull was presented as a standard male/female ratio and as a percentage (male-female)/female*100 (Hillis & Mallory, 1996;Mulders, 1997;Koroblev et al., 2020).Univariate descriptive statistics and linear regression

Results and discussion
Descriptive statistics of the data showed that the least variable parameters in both sexes were BL, CBL and CW (Appendix: Tables 2-3).The greatest skull length had a slightly higher variability in both sexes probably due to individual differences in the growth rate of the occipital crest, which was taken as a base point for this measurement.Otherwise, CBL and BL were of approximately equally low variability in both sexes.Zygomatic width is the main indicator in any craniometric study and in our case, it again had an average variability in both sexes (Vc = 7.87 for males and Vc = 7.36 for females).The most variable parameters of the wolf skull in males were CSEL, FB, maxOCB and RW of the cranium and RH of the mandible, and in females -CSEL, FB, IoB, RW, maxOCB and again RH.
In both males and females, tooth row parameters showed low variability, with males ranging from 3.14 for LTRL to 5.11 for UTRL, and females ranging from 4.06 for UCtRL to 5.95 for UTRL.Compared to other cranial parameters, the variability was weak to moderate.In both sexes UTRL had the highest variability among tooth rows.Carnassial teeth length showed similar variability in both sexes, with males having values of Vc for LP 4 -4.5 and LM 1 -5.23 and females having values of Vc for LP 4 -4.69LM 1 -4.59.These results contradict the claim of Dayan (2002) that dental features in carnivore populations are more variable than cranial ones and confirm the findings of Korablev et al. (2021).The cranial cavity volume varied significantly between males and females, with values of Vc -7.75 and Vc -9.85, respectively (Appendix: Tables 2-3).
The mean variability for all parameters was similar in both sexes (Vc = 6.1 for males and Vc = 6.6 for females).However, while Okarma & Buchalczyk (1993) reported greater variability for males feature from Bialowieża Primeval Forest (north-eastern Poland) and from Carpathian Mts (south-eastern Poland).In contrast, Hell & Paule (1982) found higher variability in females from Slovakian Carpathian Mts.The data aligns with Korablev et al. (2021), that the coefficient of variation, which expresses the variability of craniological features, is equal in both sexes.However, it is important to note that any subjective evaluations have been excluded.The variability observed is likely due to the characteristics of the local populations in different geographical areas, once the ratio of the sample size by sex in the different studies mentioned above is excluded.
Male-biased sexual dimorphism was found in all cranial parameters, ranging from 3.02% for CW to 12.08% for CSEL.Similarly, all the mandibular parameters also showed sexual dimorphism in favour of males, ranging from 4.41% for LCtRL to 10.42% for MW(M 1 ).The difference in mandibular width, was strongly determined by sex (R 2 = 0.438) (see also Fig. 3A).The parameter that showed the most significant sexual dimorphism was also the greatest skull length (Fig. 3B), the width of the mandibular processus articularis (Fig. 3C) and the mesiodistal C length (Fig. 3D).These results are consistent with previous studies, that have shown the same tendency (Gittleman & Van Valkenburgh, 1997;Larther et al., 2012;Chikachev et al., 2017;Korablev et al., 2020).
Maxillary and mandibular tooth row lengths showed minimal sexual dimorphism, ranging from 4.41% to 5.68% (Appendix: Table 4).Parameters describing the size of the carnassial teeth, along with the height of the upper canine tooth, exhibited a similar trend.The limited sex differences in carnassial teeth (P 4 and M 1 ) could be a result of the similar efforts while feeding, These teeth are used primarily for bone breaking and tissue shearing (Severtsov et al., 2016).
The cranial cavity volume showed a sexual dimorphism of 4.84% in favour of the males which value was lower than the average for all linear morphological features studied.Brain volume is considered closely linked to social behaviour, learning and behavioural plasticity (Sol et al., 2008;Benson-Amram et al., 2016).This parameter was also found to be not dependent on sex (R 2 = 0.07).
The study revealed that the average value of sexual size dimorphism in wolves from Bulgaria was 6.76%, which is approximately twice that of wolves in Central Russia -3.55% (Korablev et al., 2021).Sexual selection, particularly male competition during mating season, generally explains the nature of such dimorphism.(Erlinge, 1979;Gittleman & Van Valkenburgh, 1997;Morris & Brandt, 2014;Korablev et al., 2021).The study suggests that the difference in the diet of male and female is influenced also by their different specialisations.Males tend to specialise in killing large ungulates, while females specialise in nursing during the pup-rearing period.This is related to the segregation of the trophic niches of both sexes, as noted in previous studies (Hillis & Mallory, 1996;Korablev et al., 2021).In a related species such as the African black-backed jackal (Canis mesomelas), sexual dimorphism depends on whether it lives in allopatry or in sympatry with other related species.Macdonald et al. (2004) found that sexual dimorphism of metric cranial parameters is enhanced in allopatric populations..It is possible that the higher sexual dimorphism observed in our study is due to the lack of strong taxonomically and size-similar competitors for the wolf in Bulgaria, unlike those from the rest of the Palaearctic.
Following Larter et al. (2012) we tested the correlation between the most frequently used external parameters of the skull -CBL and ZW in male and female grey wolves.The results obtained showed the same positive linear relationship between these parameters in both, males and females(Fig.4).

Conclusion
The present study fills the gap in the knowledge on the sexual dimorphism in metric skull parameters of grey wolf from Bulgaria and the Balkans.The species exhibits weak and male-biased craniodental sexual dimorphism, like all the canids with a typical monogamous social system (Gittleman & Van Valkenburgh 1997;Bidau & Martinez 2016).However, it is more pronounced than the sexual dimorphism studied in several more northern wolf forest populations in Europe.The mandibular width and the greatest skull length are among the parameters that showed the most significant sexual dimorphism.Additionally, the width of the mandibular processus articularis and the mesiodistal C length also showed significant sexual dimorphism.The sex-separated biometric analyses from the present study will support further studies on geographic dimorphism in different grey wolf populations.

Fig. 1 .
Fig. 1.Location (red circles) and number of shot wolves, respectively the obtained skulls, measured in the present study.The map does not include some of the skulls from the National Museum of National History's collection due to unknown origin.

Fig. 2 .
Fig. 2. Most of the measured craniodental parameters of the Bulgarian grey wolf.A -left lateral view; B -dorsal view; Cventral view; D -left lateral view of the mandible.

Fig. 4 .
Fig. 4. Relationship between condylobasal length and zygomatic width in male and female grey wolves from Bulgaria.

Table 1 .
Measurements and their abbreviations.

Table 2 .
Descriptive statistics of morphometric data for male grey wolf (Canis lupus) skulls from Bulgaria.

Table 3 .
Descriptive statistics of morphometric data for female grey wolf (Canis lupus) skulls from Bulgaria.

Table 4 .
Sexual size dimorphism in craniometric features of grey wolf (Canis lupus) from Bulgaria.