Among populations of the Miniopterus bats of western Palaearctic, intraspecific variation has not been well documented. Herein we investigate sexual and age variation of these populations using two approaches — linear and geometric morphometrics. We analysed Moroccan (M. maghrebesnis), western and eastern European (M. schreibersii), Levantine (M. schreibersii), and east-Afghanistani (M. cf. fuliginosus) specimens; variation was compared between sexes of the particular specimen sets of three above mentioned Miniopterus spp. and between four age cohorts of M. schreibersii samples. The results showed in all examined population sets males to be generally larger in size than females, the exception being the east-European animals. Significatly the most divergent sexes were those from eastern Afghanistan, the Levant and eastern Europe. The differences found between sexes in as well as between examined population sets can be attributed to different life histories and/or to food competition. Weak correlations between patterns of sexual dimorphism and the newly proposed western Palaearctic classification of the Miniopterus bats suggest only a limited contribution of sexual variation to morphological variation in general. Certain aspects of age variaton were found in all examined morphological characters except the non-metric traits, which in turn indicates the importance of these traits for identification of the particular taxon across age categories.
Bent-winged bats, the genus Miniopterus Bonaparte, 1837, represent the only genus of the family Miniopteridae. It comprises some 20 species occurring mainly in the tropics and sub-tropics of the Old World (Simmons 2005). Morphometric as well as molecular phylogenetic analyses (e.g. Tate 1941, Maeda 1982, Appleton et al. 2004, Tian et al. 2004, Miller-Butterworth et al. 2005, Benda et al. 2006, Furman et al. 2008, 2010) indicate that identification of particular taxa (species/subspecies) of the genus is often difficult according to their similar or even cryptic phenotype.
In the western Palaearctic (North Africa and Eurasia to the west of Pakistan), at least four species of the genus have been documented: Schreibers' bat, Miniopterus schreibersii Kuhl, 1817; pallid bent-winged bat, M. pallidus Thomas, 1907; Indian bent-winged bat, M. cf. fuliginosus Hodgson, 1835; and Maghrebian bent-winged bat, M. maghrebensis Puechmaille, Allegrini, Benda, Bilgin, Ibáñez & Juste, 2014. M. schreibersii occurs in southern Europe, coastal areas of North Africa and in the western areas of the Middle East (Furman et al. 2010, Šrámek et al. 2013, Puechmaille et al. 2014) while M. pallidus, morphologically almost identical with the M. schreibersii (Furman et al. 2010, Šrámek et al. 2013), occurs in the eastern portion of the Middle East (Furman et al. 2010, Šrámek et al. 2013). The Nangarhar Province of Afghanistan perhaps represents the westernmost occurrence area of another Miniopterus sp. whose taxonomic position is currently unclear; Maeda (1982) and Šrámek et al. (2013) proposed that this population is best attributed to M. fuliginosus. Recently another species, M. maghrebensis, morphologicaly very similar to M. schreibersii s. str., was described from the mountainous parts of the Maghreb (southern parts of Morocco and Tunisia). Its distribution range as well as the level of sympatry with M. schreibersii remain to be clarified.
Sexual dimorphism in morphometric characteristics was shown in bats to be relatively an important factor of species variation (e.g. Findley & Traut 1970, Schmidt 1978, Maeda 1983); however, within the genus Miniopterus, it has not been broadly studied. Maeda (1982, 1983, 1984) analysed this variation in several Australasian species (M. macrodens, M. magneter, M. australis, M. fuliginosus, and M. solomonensis). Goodman et al. (2008) studied variation in two cryptic species from Madagascar, M. gleni and M. griffithsii. Concerning the western Palaearctic populations of Miniopterus, Gaisler (1970) analysed bats from eastern Afghanistan (Jalalabad area) and found no sexual dimorhism. Crucitti (1976), Spitzenberger (1981) and Uhrin et al. (1997) examined specimens of M. schreibersii s. str. from Italy, Austria and Slovakia. In all these studies the authors found (with more or less high statistical significance) males to be larger than females in majority of their cranial or external dimensions. To our knowledge no dental as well as specific non-metrical characters have been analysed yet in western Palaearctic Miniopterus populations.
Little information is available on age variation in Miniopterus spp. Maeda (1977, 1982) studied populations from Japan and found presence of age variation in many skull characters. Van der Merwe (1978) analysed post-natal growth (body mass, hindfoot and forearm lengths) of M. natalensis (sensu Simmons 2005) from South Africa and Serra-Cobo (1987) investegated postnatal forearm growth of M. schreibersii s. str. from southern Spain.
Here, we present a detailed morphometric analysis (combination of traditional linear morphometrics, geometric morphometrics and non-metric data analyses) of cranial and dental characters of the above mentioned four western Palaearctic Miniopterus species to assess aspects of their sexual and age variation. Simultaneously, we attempt to ascertain the role of the sexual variation patterns of respective populations in their taxonomic classification (cf. Šrámek et al. 2013).
Material and Methods
To examine intrapopulation (sexual and age) variation in various cranial and dental metric or non-metric characters of western Palaearctic Miniopterus bats, we studied 342 skulls (see Appendix 1 and Material and Methods in Šrámek et al. 2013 for the list of specimens, their origin, species affiliation and determination). To examine aspects of sexual dimorphism, the specimens were divided into five population sets based on previously published results (Šrámek et al. 2013): (1) Morocco (8 ♂, 10 ♀); (2) western Europe — specimens from Spain, France, Italy, Austria (14 ♂, 20 ♀, three unsexed); (3) eastern Europe — Slovakia, Romania, Bulgaria, and Greece (including Crete) (49 ♂, 89 ♀, 14 unsexed); (4) the Levant — Turkey, Syria, Cyprus, Lebanon (54 ♂, 37 ♀)? (5) eastern Afghanistan (18 ♂, 9 22? one unsexed). Specimens lacking sex identification were not used for the sexual dimorphism analysis. According to identified rate of tooth wearing (Fig. 1) in the specimens of M. schreibersii s. str. (i.e. groups 2–4), these specimens were divided into four age groups for purpose of the age variation analysis: C0 — unweaned juveniles, no abrasion; C1 — weaned juveniles, slightly worn dentition; C2 — adults, middle worn dentition; C3 — adults, heavily worn dentition. All statistical analyses were performed using the Statistica 6.0 software.
We recorded 24 cranio-dental measurements (11 skull or mandible measurements and 13 upper or lower tooth-row dimensions) taken with a mechanical calliper (by J. Šrámek) to the nearest 0.01 mm. Further, we recorded 57 dental measurements (width, length and heigh dimensions of respective teeth) using an optical calliper (by J. Šrámek) to the nearest 0.0125 mm. For complete list of all examined measurements, see Appendix S1, Figs. S1 and S2 in Šrámek et al. (2013).
Basic descriptive statistical parameters (mean [M], minimum value [min], maximum value [max], standard deviation [SD]) were calculated separately for each measurement of each geographical group (1– 5) and sex, and of each age cohort (C0–C3). Sexual size variation was analysed via the one-way analysis of variance (ANOVA), independent t-test and Storer's index. Storer's index is a value expressing relative difference of metric (or non-metric) character(s) between sexes (Storer 1966) and is calculated according to the formula [(Mf — Mm)/Mn] × 100 (M = mean, f = female, m = male, n = all specimens). Negative values indicate the relatively larger size for males, positive values for females (cf. Bogdanowicz 1992, Benda 1994).
Mean value and Storer's index (SI) of all cranio-dental dimensions (in mm), CS and RW scores of the Miniopterus sexes in the respective populations. See methods for explanation of dimension abbreviations. * = P < 0.05, ** = P < 0.001, *** = P < 0.0001, df = degrees of freedom.
Mean value and Storer's index (SI) of dental dimensions (in mm) of the Miniopterus sexes in the respective populations. See methods for explanation of dimension abbreviations. * = P < 0.05, ** = P < 0.001, *** = P < 0.0001, df = degrees of freedom.
Cranio-dental dimensions (in mm) and CS scores of the examined M. schreibersii age cohorts. See methods for explanation of dimension abbreviations, n = number of specimens, M = mean, min = minimum value, max = maximum value, SD = standard deviation.
Selected dental dimensions (in mm) of the examined M. schreibersii age cohorts. See methods for explanation of dimension abbreviations, n = number of specimens, M = mean, min = minimum value, max = maximum value, SD = standard deviation.
Geometric morphometrics and non-metric traits
Geometric morphometrics was used to analyse skull and mandible variation between sexes of the respective population sets (groups 1–5) and also partly (see below) between age cohorts. We used the same specimens as for the linear morphometrics.
Images of skulls (lateral, ventral and dorsal views), mandibles (lateral and oclusal views) and dentition (details of the upper and lower tooth-rows) were taken with a digital camera, archived in jpg format (1360 × 1200 pixels resolution) and processed with QuickPhoto 4.1 (Promicra, Prague). The centroid size (CS) as well as the relative warp (RW) scores of all types of view for each specimen (CS1 [G1 in case of RW] — lateral view of mandible, CS2 [G2 in case of RW] — lateral view of skull, CS3 [G3 in case of RW] — ventral view of skull, CS4 [G4 in case of RW] — dorsal view of skull) were calculated using the tpsRegr 1.36 (for CS calculation; Rohlf 2009) and tpsRelw 1.46 software package (for RW calculation; Rohlf 2008). For methodology details see Šrámek et al. (2013). The RW and CS scores were analysed by the same methods as the linear metric data (basic descriptive statistics, one-way ANOVA, independent t-test; Storer's index only for CS scores). The RW analysis as well as Storer's index calculation of the data of age cohorts was not performed.
Based on images of skulls, mandibles and teeth, 49 non-metric cranial and dental characters (44 dental and five skull or mandible; see Table S1 in Šrámek et al. 2013) were investigated for each geographical group (1–5) and sex, and for each age cohort (C0–C3). Statuses of these characters were evaluated using the defined scale system 1–5 in accordance to the character state (see Fig. S3 in Šrámek et al. 2013 for details). Non-metric data were analysed in the same manner as the linear metric data.
Results of the analyses (ANOVA, t-test) as well as Storer's index values of cranio-dental and tooth dimensions (Tables 1 and 2, and Tables S1 and S2) generally showed that the most significant differences associated with sexual dimorphism were found in populations from Afghanistan, the Levant and eastern Europe while those from Morocco and western Europe diverged only in a few variables. The results indicate that males are generally larger within a given population sets, with the exception of eastern Europe. In all cranio-dental measurements the Moroccan males showed larger values than females except for dimensions associated with skull and rostral width (LaZ, LaI, LaInf, CC, P4P4, and M3M3), but the sexes just slightly diverged (P < 0.05) in the length of upper tooth-row (P4M3). In dental characters, the Moroccan females were larger in 38 of 57 variables and the sexes highly diverged (P < 0.001) in the length of lower canine (LCinf); slightly diverged in widths of the second lower incisor (WI2), second upper molar (in central part, W2M2) and third lower molar (WM3). Males of the western European bats showed values larger than those of females in almost of all cranio-dental measurements, with the exception of all upper tooth-row dimensions. Sexes moderately diverged (P < 0.01) in the condylobasal length (LCb) and only slightly diverged in the length of upper tooth-row (CM3). Teeth showed larger values more likely in males in 30 of 57 measurements; the sexes diverged (moderately) only in the first lower molar diagonal width (W3M1).
In the eastern European bats, females showed values larger than those of males in most cranio-dental measurements, with the exception of zygomatic width (LaZ), skull heights (ACr, ANc, ACo) and partly in tooth-rows length (P4M3, P4M3). Sexes highly diverged in the length characters (LCr, LCb); moderately diverged in mastoidal width (LaM), mandible and lower tooth-row length (LMd, I1Mj); and slightly diverged in length of tooth-rows (I1M3, CM3, CP4, CM3, CP4). In the majority of dental characters (40 of 57) females showed larger values than in males, with the exception of most height dimensions. The sexes were highly divergent in the widths of the second upper molar (in central part, W2M2 and the second lower incisor (LI2); moderately diverged in the second upper molar diagonal width (W1M2), length and width of the third lower incisor (LI3, WI3), length of the second lower premolar (LP2 and height of lower canine (HCinf); and slightly diverged in some dimensions of the first upper incisor (WI1, HI1), molars (W2M1, W3M2, WM3) and height of the second lower incisor (HI2).
In all cranio-dental measurements males of the Levantine bats showed larger values than those of females, with the exception of lower molar-row length (M1M3). They highly diverged in skull length (LCr, LCb), braincase dimesions (LaN, ANc); moderately diverged in mastoidal width (LaM) and length of upper tooth-row (I1M3, CM3); and slightly diverged in skull height (ACr) and partly in the tooth-rows lengths (CP4, CM3). In dental characters males showed values larger than females in 38 of 57 dimensions. Sexes highly diverged in some canine dimensions (HCsup, HCinf, LCinf); moderately in lower canine width (WCinf); and slightly in upper canine width (WCsup) and the dimensions of upper second premolar (WP4, HP4).
Males of Afghanistan Miniopterus were larger than females in all cranio-dental characters (except of lower molar-row length, M1M3). The sexes were highly divergent in condylobasal length (LCb), rostral width across the upper canines (CC) and upper tooth-row length (I1M3); moderately divergent in the skull and mandible length (LCr, LMd); and slightly in some skull widths (LaM, LaN, LaInf, P4P4, M3M3), neurocranium height (ANc) and some tooth-rows lengths (CM3, CP4). Dental measurements were larger in males in 42 of 57 dimensions, particularly in height dimensions, and sexes highly diverged in height of lower canine (HCinf, Fig. 2); moderately diverged in the length of upper canine (LCsup) and height of first upper incisor (HI1); and slightly diverged in the lengths of the first upper incisor (LI1), lower canine (LCinf) and the second premolar (LP4), in the height of the upper canine (HCsup, Fig. 2) and in the width of the first lower molar (W2M1).
Geometric morphometrics and non-metric traits
Cendroid size scores (see Table 1 and S1) showed larger values in males than in those of females in all views and examined population sets (significantly [P < 0.05] except for CS1 of the Afghanistani and western European sets, CS3 of the western European set, and for all views of the Moroccan set), except the eastern European set, where females showed larger values in all views (significantely except CS2). The significance level for respective views and groups was rather variable (see Table 1 and S1).
Twenty-two RWs were generated for the lateral skull view for both sexes of each population set, 18 for the ventral view, 14 for the dorsal view, and 14 for the lateral view of the mandible. The first four RWs, which together represented more than 50 % of total variation for each view, were used for subsequent analyses (ANOVA, t-test). These generally showed minimal shape differences between sexes of all examined population sets (Table 1 and S1).
Results of analyses (ANOVA, t-test), as well as Storer's index values, of non-metric traits showed various levels of sexual dimorphism in the respective population sets in size parameters as well as in notably variable levels of statistical significance (Table S3). The sexes of Moroccan bats only slightly diverged in P2P4inf2, CingM2sup and RmanW. The sexes of the western European bats moderately diverged in P4sup2 and CingCsup, while only slightly in Fmen and M3 sup. The sexes of eastern European bats highly diverged only in M1sup, moderately in Fmen, and slightly in P3inf2, M3sup2, M2sup, P4sup, P4sup8, CingCsup, ProcCW and Isup. The sexes of Levantine bats moderately diverged in P4inf and slightly in P3inf2, P2P4inf2, P3P4inf, FmenP2inf, M1sup5 and P4sup5. The sexes of Afghanistan bats only slightly diverged in M3sup.
The values of all cranio-dental measurements of four age cohorts and their simple comparisons (Table 3 or Fig. S1) showed certain level of age variation. In most of these dimensions, the size of the respective character was found to increase with age. This pattern was found to be inversed (the dimensions descreased in size with age) only in some dimensions concerning molariform teeth (P4M3, M1M3, P4M3 and M1M3). In the dental dimensions (Table 4 and S4 or Fig. S2), the situation was markedly less expressive than in the cranio-dental measurements and in some (mainly tooth length) dimensions (LI1, LI2, LoM1, LI3, LM2 and LM3), the above mentioned pattern was found to be inverse, i.e. with dimensions smaller in older bats.
Geometric morphometries and non-metric traits
CS scores showed considerable age variation and positive correlation between age and value of the respective centroid size, i.e. the higher cohort (older bats) the larger the centroid size value. Only the mandibular CS scores showed an inverse pattern (except for the C3 cohort whose values were markedly highest among all cohorts). For details see Table 3 or Fig. S1.
The non-metric traits data did not show a general correlation between age and value of the particular character (Table S5). Differences in character values between the respective cohorts were largely not present, with the exception of P2P4inf.
Synthesis of results of the sexual dimorphism analysis of the western Palaearctic Miniopterus bats, comprising the Moroccan, western and eastern European, Levantine and eastern Afghanistani populations, indicates that males are larger than females in all examined geographical sample sets, with the exception of eastern Europe. These results are in parallel to the majority of published studies of Miniopterus populations in different portions of the Palaearctic (Crucitti 1976, Spitzenberger 1981, Maeda 1982, 1983, 1984). Only Uhrin et al. (1997) who studied Slovakian populations, on the contrary to our results, found the males to be generally larger than females. Nevertheless, it is important to mention that the Slovakian specimens comprised a minority in our eastern European set (29 Slovakian vs. 109 Balkan samples) and individual populations thus could have different characteristics. Unfortunately, our samples were insufficient to test Slovakian bats separately as well as any other population within this sample set, with the exception of Bulgarian bats, in which males were generally larger than females. Moreover, our finding of sexual dimorphism in cranial dimension in Miniopterus bats from eastern Afghanistan is in contradiction with that by Gaisler (1970) who found no sexual differences in Miniopterus bats from the same locality. This dissimilarity may be the result of measuring specimens in different manners, as well as more precise statistical analyses of our data.
Sexual dimorphism is generally explained by several hypotheses. The most common being male-male competition for females (or selection by females; Darwin 1871, Trivers 1972) or male-female food competition (e.g. Selander 1966, Storer 1966, Earhart & Johnson 1970). Among chiropterans, there are also other proposed hypotheses, such as ‘The Big Mother’ hypothesis (Ralls 1976), which suggests that larger females give their offspring better conditions and this system subsequently lead to females of a given taxon being larger than males. Myers (1978) found positive correlation between sexual dimorphism of vespertilionid bats and number of young per litter, similarly as Brunett (1983) in Eptesicus fuscus, and proposed that female body size (particularly wing size) is positively influenced by the need to carry in flight and nourish large foetus or carry young juveniles. Williams & Findley (1979) tested this hypothesis; however, did not find this correlation. They and some other authors (Findley & Traut 1970, Findley & Wilson 1982) explained the larger size of the vespertilionid females in relation to the gravidity process and some climatic conditions (temperature, humidity) - larger females are more resistant to hypothermia and associated perturbations in embryo development and, also, the larger size provides greater energetic efficiency in maintaining homeothermy during gestation while males are hypothermic. Climatic conditions in connection with different life history traits of the respective sexes were found in some small insectivorous bat species associated with sexual dimorphism (e.g. Egsbaek & Jensen 1963, Bogdanowicz 1992). In the case of Myotis daubentonii, the precise mechanism involved in the development of sexual dimorphism was connected with different periods of time spent in hibernacula; males fly out to foraging activities in early spring while females stay and are more exposed to climatic stresses (low temperature, high humidity) (Egsbaek & Jensen 1963, Stebbings 1977, Baagøe et al. 1988). Larger size then provides greater energetic efficiency in maintaining homeothermy and thus means benefit for females.
However, our results showed males of Miniopterus bats to be generally larger than females (with an exception of the eastern Europan populations). Hence, sexual dimorphism differences are presumably related to different factors or, to the same factors but affected by other mechanisms. We speculate that these factors involved may be in parallel to the case of Myotis daubentonii, specifically associated with different climatic conditions affecting differentially aspects between the sexes in life history traits at the population level, such as the use of seasonally different shelters (hibernacula) or different periods of their usage, forming of sex-specific colonies. These factors may also be directly related to differences in patterns of sexual dimorphism at the population level (i.e. females larger than males in eastern Europe vs. males larger than females in Morocco, western Europe, the Levant, and Afghanistan). Nevertheless, to elucidate the machanisms leading to development of sexual dimorphism of the respective Miniopterus populations concerning their life histories of their sexes remain to be studied, since they were generally not much explored yet (Spitzenberger 1981, Boye 2004).
Another factor that might contribute to sexual dimorphism in Miniopterus may be associated with the feeding strategies (food competition). We documented in some sample sets (mainly in Levantine and Afghanistani sets) very marked dimorphism in dentition (positively correlating with the level of dimorphism in skull dimensions), particularly in canines and this finding may thus indicate intraspecific food competition that consequently led to male adaptations to different prey types of size than in females, and consequently the generally larger size in the former. However, this was not documented in other bat species (Krzanowski 1971) and this hypothesis is not corroborated with some of our other results, specifically weak dimorphism and larger dentition in females of eastern European and Moroccan populations. To conclude, the differences in patterns of sexual dimorphism between studied population sets might be best explained by different effects or combination of effects of two different factors — distinct life histories and feeding strategies.
The level of sexual dimorphism found among the examined population sets does not fully correlate with the classification of the western Palaearctic bats of the genus Miniopterus as presented in recent revision (Šrámek et al. 2013). Most particularly, the different patterns of dimorphism found in the western (larger males) and eastern (larger females) European populations are surprising. In other areas, these patterns were quite similar to each other; however, the level of significance diverged. The most significant dimorphism was found in bats from eastern Afghanistan (classified as M. cf. fuliginosus), the Levant (M. schreibersii) and eastern Europe (M. schreibersii). In Moroccan (recently defined as a new species, M. maghrebensis) and western European (M. schreibersii) populations, the rate of dimorphism was less pronounced and similar to each other. However, it may be important to mention that the rate of dimorphism found in the respective population sets could be affected by different sample sizes ('n' affects P value calculation). To conclude, results rather suggest that in this genus, sexual dimorphism probably has had a minor contribution to measureable aspects of morphological variation with little signal associated with recent phylogenetic evolution (cf. Šrámek et al. 2013).
Age variation was demonstrated to occur in all linear-metric characteristics and CS scores, while almost no age variation was found in non-metric traits. This finding clearly indicates importance of the non-metric traits for species identification of taxa across age categories. The negative correlation found between age and size in several linear-metric dimensions (e.g. P4M3, M1M3. LI1, LI2) and one non-metric trait (P2P4inf) can be attributed to abrasion or to the relative difference generated by mandible development, i.e. tooth size does not change with mandible development.
We thank Rainer Hutterer (Zoological Museum and Institute Alexander Koenig, Bonn, Germany) and Riyad Sadek (American University Beirut, Lebanon), for allowing access to specimens under their care, and Steve Goodman for invaluable help with the style and language revision of the early version of the manuscript. The study was supported by the Ministry of Culture of the Czech Republic (No. DKRVO 2014/14, 00023272).
Supplementary online materials
Fig. S1. Graphs of mean values of cranio-dental measurements and of mean CS values for respective cohorts. See methods for explanation of dimension abbreviations.
Fig. S2. Graphs of mean values of dental measurements for respective cohorts. See methods for explanation of dimension abbreviations.
Table S1. Cranio-dental dimensions (in mm), CS and RW scores of the Miniopterus sexes in the respective populations. See methods for explanation of dimension abbreviations, n = number of specimens, M = mean, min = minimum value, max = maximum value, SD = standard deviation, * = P< 0.05, ** = P< 0.001, *** = p< 0.0001, F = F-values from ANOVA and T-test, SI = Storer's index.
Table S2. Dental dimensions (in mm) of the Miniopterus sexes in the respective populations. See methods for explanation of dimension abbreviations, n = number of specimens, M = mean, min = minimum value, max = maximum value, SD = standard deviation, * = P < 0.05, ** = P< 0.001, *** = P< 0.0001, F = F-values from ANOVA and T-test, SI = Storer's index.
Table S3. Non-metric traits of the Miniopterus sexes in the respective populations. See methods for explanation of dimension abbreviations, n = number of specimens, M = mean, min = minimum value, max = maximum value, SD = standard deviation, * = P < 0.05, ** = P< 0.001, *** = p< 0.0001, F = F-values from ANOVA and T-test, SI = Storer's index.
Table S4. Dental dimensions (in mm) of the examined M. schreibersii age cohorts. See methods for explanation of dimension abbreviations. M = mean, min = minimum value, max = maximum value, and SD = standard deviation.
Table S5. Non-metric traits of the examined M. schreibersii age cohorts. See methods for explanation of dimension abbreviations. M = mean, min = minimum value, max = maximum value, and SD = standard deviation.