Acta Medica Mediterranea, 2018, 34: 583

GEOMETRIC MORPHOMETRIC ANALYSIS OF HEAD SHAPE IN A SPOTTED WHIP ( RAVERGIERI) IN DIFFERENT HABITATS OF

MINOO MOSHTAGHIE1, MOHAMMAD KABOLI2*, MANSOUR SALEHI3 1Department of Environment and Energy, Science and Research Branch, Islamic Azad University, Tehran, Iran - 2Department of Environment, Faculty of Natural Resources, University of Tehran, Iran - 3Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences

ABSTRACT

Morphological patterns of existing variation and investigative concept related to diversity can be very important in understan- ding the evolution of morphology. It is known that phylogenetic history, size and ecology can all promote the evolution of morpholo- gical variation; however, some methods have been recently developed to analyze these aspects for particular body structures. Spotted whip snake appears to have the widest distribution of the Hemorrhois snake , especially in Iran. Morphological difference resulting from habitat can occur in several populations of H. ravergieri. Some studies have studied the relation between morphology and habitat use, revealing that motion is the main facilitator of this evolutionary relationship. Now, we consider intraspecific ecomorphological in three habitat types. Geometric morphometric analyses can provide a more detailed image of head shape in three directions (dorsal, lateral and ventral) on three types of habitat: north-west of Iran, north-east of Iran and central Zagros of Iran. Adult specimens were selected and characteristics of head were analyzed, along with body and head size for understanding the proximate mechanisms, which might be involved. The morphological variation between H. ravergieri indicated that variation in size and head dimensions between and sexes are highly significant in habitat. Males and females differed in head characters and body size; especially, males were found to exhibit a disproportionate increase in head size and dimensions. H. ravergieri from north-east and central Zagros are smaller, than those living in north-west are. The results revealed that considering the previous findings on the populations, the morphological differences were probably not caused by competition between the popu- lations; instead, these traits could be attributed to the natural selection.

Keywords: geometric morphometric, Hemorrhois ravergieri, head shape, habitat.

DOI: 10.19193/0393-6384_2018_2s_92

Received November 30, 2017; Accepted January 20, 2018

Introduction Spotted whip belonging to family (genus Hemorrhois), have four species, While species identification and their delin- namely Hemorrhois ravergieri, H. nummifer, H. eation should be regarded as one of the most impor- hippocrepis and H. algirus. Although they are non- tant problems that should be taken in to account in venomous snake species, their behaviors are similar the study of and evolution, they can be to those of venomous snakes(33,34,39). This genus has a quite necessary in the discovery of life’s diversi- wide species dispersal in different habitats of Asia, ty(38). To find the patterns of variation within and/or southern Europe and northern Africa(35,39). H. between the species, the evolutionary biology ravergieri has the widest distribution range among should be addressed. Some species (especially the genus; smallest size of which is about (50 cm) snake) have a wide variety of morphs in different and the longest one was reported about 159 cm(14). habitats due to the adaptation phenomenon(4). 584 Minoo Moshtaghie, Mohammad Kaboli et Al

It is known that each species displays a wide cal characteristics. It should be noted that the main range of shapes in different habitants; consequently, goal in geometric morphometrics is to measure morphological patterns can be employed to deter- morphological similarities and differences; the mine apparent characteristics, as well as other main concern has been solving patterns of biogeo- behavior changes that occur in different ecosys- graphical variation in different phenotypes in the tems. So more complex measurement techniques case of Hemorrhois species in Iran. such as geometrical shape can be employed for the Finally, geometric morphometrics can be purpose of better identification(41). employed in order to conduct a fine-scale analysis It has been shown that various ecological roles of dorsal, lateral and ventral head shape develop- of a structure, in addition to its size, can play a cru- ment; this approach can facilitate the study of dif- cial role in determining the evolution of morpholo- ferent head regions, thereby finding those that are gy. In the case of squamates (including snakes), it particularly modified during ontogeny. has been revealed that the head and cranium shapes can predict such things as the foraging behaviour, Material and methods prey capture, refuge use, defense and aggressive behaviours(4,19,36). For the vast majority of snake Locality and field study species, it has been shown that the head size limits Field samplings were performed during 2015- the maximum prey size. There are also few studies 2016, under the permission, with the reference that have already addressed the form-function rela- number: 29304/95, from the Department of tionship between squamate head morphology and Environment, Iran. Forty nine specimens were col- bite performance(21,32). lected from extensive fields of Iran including (i) In recent years, there have a relatively large north-east of Iran, (ii) north-west of Iran, and (iii): number of functional studies considering geometric central Zagros. Only 34 matures (22 male and 12 morphometric analyses, e.g.,(30). It has been revealed female) specimens, which did not suffer from dam- in the the last decades that the morphometrics could age or head scales deformations, were selected to be improved by methods in which the geometry of be used for photography. shape has been preserved in the analysis of biologi- Statistical analysis cal forms to make use of new image technologies Sex and state of sexual maturity were verified in order to get data and visualize the obtained after dissection(22). After recording the sex and results(1). Some methods have also been developed snout-to-vent length (SVL), the graph paper was set recenetly to illustrate multivariate variations in underneath the heads in order to record scales, and shape that could be simply understood within a photos of the dorsal, lateral and ventral views of the geographic framework(8). head (Figure 1) were taken using a Cannon The pioneering traditional morphometric study Professional camera with 18-megapixel resolution. on the H. ravergieri (in Iran) reported a significant difference in ventral scales between males and females. From another point of view, for the clarifi- cation of species biosystematics, geometric mor- phometric, phylogenetic and molecular studies are recommended, Due to different head shape and col- ors observed between different populations of H. ravergieri in Iran, morphometric studies are unable to distinguish them by morphometric characters(15). Although a large number of the previous stud- ies have been concerned the genetic and phyloge- netic properties of Colubridae family all over the (27) Figure 1: Landmarks positioned on A: dorsal view, B: world , geometric morphometric and genetic ventral view and C: lateral view of head in the H. raver- information is meager regarding the populations of gieri. H. ravergieri, especially those inhabiting in Iran. In this regard, a comparison between H. ravergieri Geometric morphometrics is a well-estab- species in different habitats can be helpful in find- lished technique(10,24,2) that effectively allows non- ing whether or not there are significant morphologi- shape information to remove from geometric struc- Geometric morphometric analysis of head shape in a spotted whip snake... 585 tures(5,13) and has been proved to be more powerful two categories (male and female). In this study, sex- than traditional morphometrics(29). ual categories were statistically different in popula- A geometric morphometric analysis was per- tions. formed using high quality pictures from the dorsal, Specimens were grouped in three geographic lateral and ventral head of 38 specimens. 34 land- samples based on geographic units: (i) north-east of marks in dorsal view, 17 landmarks in lateral view Iran, (ii) north-west of Iran, and (iii): central and 16 landmarks in ventral view (Figures 1a,1b, Zagros. Some biometric variables were tested for and 1c) were digitized for each specimen using homoscedasticity (Levene’s test, P > 0.05). The TpsDig2(30). Since landmark positions can convey SVL and head dimensions seem to be the most important phylogenetic, developmental and func- important variables in the analyses (p= 0.011) tional information, it is very necessary to digitize (Table 1). and analyze landmarks correctly before commenc- Morphometric category mean Standard deviation ing any morphometric study(41). These configura- characters tions were superimposed using a General SVL male 79.50 b 14.08

Procrustes Analysis (GPA) as previously suggested female 84.18 a 6.03 by(31). The measurement errors present in the dataset TL male 22.27 b 4.02 obtained with the above-mentioned experimental design were quantified using a Procrustes ANOVA. female 23.73 a 2.44 After that, a file in tps format was created for all HL male 3.4 0.24 groups studied with tpsUtil(30). female 3.56 0.25

Landmarks were applied for all specimens. A HW male 1.84 0.4 Generalized Least-Squares (GLS) fit was conducted female 1.96 0.28 in order for minimizing Procrustes shape distances between landmark configurations; this can lead to SL male 0.99 0.22 standardizing orientation through minimization of female 0.98 0.15 the summed squared distances between the corre- SW male 1.3 1.68 sponding landmarks. Residuals resulting from the female 0.98 0.07 superimposition could be modeled by employing the thin-plate spline interpolating function(5,26), this can Table 1: Descriptive statistics of biometric variables. Morphometric characters between male and female just in SVL lead to the creation of the bending energy matrix, (p=0.011) & TL (p=0.039) have significant. thereby showing how the visualization of how each specimens’ configuration can differ from that of the Therefore, scales of head characters in males consensus on a deformation grid. In H. ravergieri, and females populations were examined. The mor- landmarks were employed based on the intersection phological variation between H. ravergieri deter- of dorsal, lateral and ventral head scales (Fig. 1). mined by Procrustes ANOVA indicated that varia- Visualization of shape changes as explained tion in size and head dimensions between species by the canonical variates (CV) was done using and sexes is highly significant (Table 2). MorphoJ software, by generating deformed outline drawings with the average shape as a reference(25). Canonical scores were computed for males and females of each population. Deformation grids were produced by regression of shape variables against canonical scores using MorphoJ(29). Table 2: regression of head characters against SVL. Results The examination of GPD between different Ranges and mean for studied characters in groups provided significant results in most cases, each group have been provided in Table 1. The indicating shape differences between sexes and measurement error of the head was estimated from groups. Dorsal and lateral head shape differed sig- the Procrustes ANOVA for each analysis. The first nificantly between ontogenetic classes within each category related to sex (male and female), the sig- sex of the species. For both the dorsal and the later- nificant value was examined in all locations and in al configuration of landmarks, GPD between males 586 Minoo Moshtaghie, Mohammad Kaboli et Al was lower than those observed for the respective 4), but in ventral side, no significant differences females. Dorsal, lateral, and ventral head shapes were distinguished between genders in all popula- differed significantly between sexes. Differences in tion. Ontogenetic shape change, analyzed through shape between populations were analyzed by means examination of deformation grids, was little differ- of canonical variates analysis (CVA) applying ent in all groups It is reported that for both dorsal 10,000 permutations, north-west samples have sig- and the lateral configurations of landmarks, defor- nificant different with other groups (Figure 3). mation patterns are similar between sexes, but shape change is more intense in males (Figure 4).

Table 4: P-Value between males and females of each location in three direction (dorsal, lateral and ventral). P value with bold-faced type represents significant diffe- rence. Discussion Figure 3: Means of each group’s scores on the first and second canonical root of the canonical variants analysis Monitoring has shown that they are to separations locality. well known for their ecological diversity and body Analyses of head dimensions with SVL size disparity(9,20). It has been hypothesized that geo- showed that males and females of species differ sig- graphic variation in their pattern could be strongly nificantly in their metric characters ontogenetic associated with their habitat(16). The scales of rep- characteristics, analyzed through examination of tiles could be employed as a tool in the study of deformation grids, and were similar in both sexes. their intra- and inter-species morphological varia- Examination of all landmark sides’ configurations tions(7,23) On the other hand, a significant positive revealed that shape differences between male and relationship between the head size and shape in female are primarily related to the posterior region monitoring has been well substantiated, showing of the head (Figure 4). that a large proportion of initial head shape dispari- ty could be explained by allometric scaling. Previous studies may also confirm an allometric relationship between body size and body shape(37,11). Geometric morphometrics could be assumed to have a higher sensitivity and detect finer differences since a wide variety of variables should be taken in to account. However, many snakes have been reported to be monomorphic in sex(3,12,40). Linear morphometric parameters can be helpful in highlighting divergent head growth trajectories between the species living in all locations(23). In addition, the high discrimina- Figure 3: Means of each group’s scores on the first and tive power of GM vs. multivariate methods could second canonical root of the canonical variants analysis be more perceptible in cases where only head to separations locality. dimensions for multivariate analyses would be Dorsally, the posterior part of the pileus, employed. While the identified shape differences including the front parietal, parietal, interparietal, may be difficult to describe, they show agreement and occipital scales, is the region that appears with the available knowledge regarding the head deformed (Table 4). Laterally, shape differences characteristics of the H. ravergieri. between the two ontogenetic stages are due to It has been shown that males exhibit signifi- deformation of the tympanic area of the head (Table cant variations in the head characters, relative to Geometric morphometric analysis of head shape in a spotted whip snake... 587 their SVL (p=0.011) and tail length (TL) (p=0.039), (Figures 2(a)-(d) and 4). The results showed a posi- compared to females (Table 1). Furthermore, it tive relationship regarding the male-female differ- should be noted that HL and HW are hypermetric entiation in the geometric body size. The pattern of with regard to body size in species (Figure 2c). sexual differences on the dorsal configuration of landmarks by deformation grids was in agreement with the empirical notion of specialists, showing that males have a more robust habitus, while females are more likely to have a more rounded head; this was consistent with a previous study addressing head shape dimorphism in the Lacertidae(7). H. ravergieri from north-east and cen- tral Zagros are smaller, than those living in north- west are. Geometric morphometric give a detailed image of head shape modifications, consistent with the hypothesis of mechanical constraints related to microhabitat and refuge use. For example, Eye regions in north-west snakes are larger than other groups or nostril in central Zagros is the smallest (Figures 4). Finally, habitat-related differentiation is more striking in males than in females, pointing Table 2: (a)Relationship of head width (HW) with snout- towards interactions between sex and natural selec- vent length (SVL). (b) Relationship of head lenght (HL) with snout-vent length (SVL). (c) Relationship of head tion. lenght (HL) with head width (HW). (d) Relationship of Snout length (SL) with Snout width (SW). Conclusion

Generally, there is a direct relation between It is thought that geometric morphometrics width, height and length of head, and type of could act as a powerful tool for the detection of preys(18). These selective pressures could be attrib- small-scale morphological differences, thereby uted to the antagonistic behavior of males for terri- leading to the good discrimination of very closely tory defense and/or mate acquisition, as those with related populations; what is obtained is consider- bigger heads could be assumed to have lead and ably better than that achieved by multivariate meth- exercise superiority. In addition, a bigger head ods. The differences identified in the case of head could be beneficial for males during competition shape scales, and differences in head dimensions, and defense(7,18). The hypermetric features of head were in agreement with the previous studies, show- length in relation to SVL can be seen in (Figure 2b). ing the structural and functional differences Despite this, head width could be inevitably mod- between members of each population in a given ified along with the increase in the head size (r= locality. It could also be concluded that the simulta- 0.824); also, the similarity of head dimensions neous application of geometric morphometrics could be related to anatomical and structural restric- could contribute to the analysis of different mor- tions, which could limit cranium growth or genetic phological aspects and determination of the evolu- correlations between sexes(28). tionary processes. The results of regression analyses considering Nevertheless, it should be noted that applica- head size and shape variables were found to con- tion of geometric morphometrics in the study of form to those obtained for the modification of head head shape of H.ravergieri is still unexplored; as an dimensions with the increase in the head width and illustration, it is necessary to determine which part length (Figures 2b, 2c, and 2d). This clearly shows of the information obtained by landmark methods that shape could be directly related to size; this is could be regarded as a phylogenetic signal. While not surprising since organismal size transformations this study revealed the great discriminatory capabil- cannot emerge without consequent shape transfor- ity of geometric morphometrics in the case of mations(6,41). 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