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Geometric Morphometry of Skulls Characteristics of Nine Species of Eothenomys.

Byline: Xiao-Ying Ren, Di Zhang and Wan-Long Zhu

Key words

Geometric morphometric, Cranium, Mandible, Eothenomys, Ecological adaptation.


Morphological characteristics of small mammals were variable and likely to be affected by age or geographic factors, such as altitudinal and latitudinal (Renaud et al., 2010). Previously studies showed that there had geographical gradients of cranial (Fadda and Corti, 2001), mandibular (Duarte et al., 2000) and dental shape (Renaud, 1999) in mammals. Taxonomic differentiations were commonly associated with morphological divergences (Renaud and Michaux, 2003); now more and more geometric morphometry variations of skulls were reported (Auffray et al., 2009; Renaud and Auffray, 2010).

Morphometrics is a method for the quantitative description, variation, analysis and interpretation of shape, which is a fundamental area of research in biology (Rohlf, 1990; Meng et al., 2018). Unlike the traditional approaches, the geometric method is aimed at comparison of the shapes themselves (Pavlinov, 2001). Traditional morphometrics were restricted to univariate comparisons of individuals' linear measurements to bivariate plots (Janzekovic and Krystufek, 2004). The geometric morphometric techniques have several advantages over the traditional approaches (Hautier et al., 2009; Auffray et al., 2009), which used all the information available about the land mark location, while adhering to the geometric definition of shape rigorously (Loy et al., 1996). Land marks of homologous points allow the visualization of shape changes among samples (Capanna et al., 1996).

Eothenomys were the inherent species in Hengduan Mountains region (Zheng, 1993), which had special status in Microtus, Arvicolinae. Eothenomys were useful for palaeoecological, palaeoclimatical, palaeogeographical and evolutionary indicators because they had abundant in fossil and archaeological records (Andrews, 1990). Still now, it is not possible to accurately identify all species in Eothenomys by using traditional methods. There were still several unsolved problems in the taxonomy of Eothenomys and this leads to search for different methods such as DNA studies (Luo et al., 2004) or karyotypes (Ma and Jiang, 1996). In order to investigate the geometric morphometrics of the craniums and mandible in nine species of Eothenomys (E. fidelis, E. melanogaster, E. chinensis, E. proditor, E. custos, E. cachinus, E. eleusis, E. miletus and E.olitor), ANOVA analyses, principal components analysis, thin plate spline, UPGMA and Multidimensional Scaling were used in the present study.

We hypothesized that there may had differences of skulls characteristics of nine species of Eothenomys, and these difference of geometric morphometry of skulls characteristics were mainly for adaptation to their habitats, respectively.



All specimens used in the present study were from the Kunming Institute of Zoology. 179 skulls were photographed with a Nikon camera, the crania and mandibles were analyzed separately, 469 photographs were taken and subsequently analyzed (Table I). Different landmark numbers were used to describe the configurations of the skulls (Table II).

Measurement of skulls characteristics

Landmarks were digitized on standardized pictures of the dorsal, lateral and ventral views of the cranium, and latera view of the mandibles (Fig. 1). Landmarks were marked on the digital picture of skull using TPSDIG2 (Rohlf, 1990; Rohlf and Slice, 1990). In order to avoid bias due to measurement errors, each sample was measured sixth independently. All the landmarks showed good repeatability.

Statistical analysis

Group differences of the morphometric variables were performed by ANOVA analyses with SPSS (13.0). Principal component analysis (PCA) was applied to investigate phenetic relationships. Shape differences were visualized with transformation grids using thin plate spline (TPS) algorithm (Bookstein, 1991). Pair-wise procrustes distances were calculated between group averages using Morphologika2 v2.5 program. Procrustes distance matrix was served as the basis to calculate a similarity tree using the UPGMA method in the PHYLIP program package (Felsenstein, 2009). Multidimensional Scaling, weight matrix and Hierarchical Cluster analysis were used whole skull shapes of nine species with four faces.

Table I.- Numbers of samples examined from different species.


###Dorsal###Lateral Ventral

E. miletus###15###11###14###9

E. olitor###1###1###1###1

E. proditor###5###5###6###4

E. custos###4###5###5###5

E. chinensis###4###4###3###2

E. cachinus###21###6###6###2

E. melanogaster###29###26###63###32

E. eleusis###35###31###59###37

E. fidelis###5###4###5###3

Table II.- Numbers of landmark.





ANOVA analyses showed that there had significant differences in skull shape among species in centroid size (Table III). Principal component analysis also showed that significant differences were found among nine species. On the dorsal view of the cranium, the first and second PC cumulative variance explained 86%, E. melanogastor is clearly separated from all other species. On the lateral view of the cranium, the first and second PC cumulative variance explained 86%, which was classified into two parts, one part included E. eleusis, E. cachinus and E.miletus, another part included E. melanogastor, E. custor, E. fidelis and E. proditor. On the ventral view of the cranium, the first and second PC cumulative variance explained 87%; E. melanogastor is clearly separated from all other species.

On the lateral view of the mandibles, the first and second PC cumulative variance explained 82%, E.miletus, E. melanogastor and E. chinensis were clearly identifiable. Thin plate spline (TPS) analysis showed that the shape differences associated with the particular canonical variety in different species, which were mostly in orbital cavity and alveolar region (Fig. 2).

Table III.- ANOVA for the cranial size of Eothenomys (significant values are in italics).

###Sum of squares###df###F###P





The similarity three trees showed that E. miletus and E. eleusis were more closely (Fig. 3A, B, C), E. melanogaster and E. fidelis were gathered together (Fig. 3A, C), E. melanogaster and E. custos were gathered together, E. fidelis and E. olitor were gathered together (Fig. 3B, D). In Multidimensional Scaling analysis, E. fidelis, E.proditor, E. olitor, E. custos and E. chinensis were above the diagonal (between Dimension 1 and 2); E. miletus, E. eleusis, E. cachinus and E. melanogaster were below the diagonal (Fig. 4). Dimension weights plot caudated from weight matrix analyze was revealed that varieties of ventral of the cranium and mandible had effects on Dimension 2, and varieties of lateral and dorsal of the cranium had effects on Dimension 1 (Fig. 5).


The relationship between taxonomy and geometric morphological characteristics

The classification and identification of the genus Eothenomys remained confusion, which was especially typical in the classification studies on this genus. It had been identified as Clethrionomys rufocanus: Clethrionomys and the Eothenomys/Caryomys complex (Hinton, 1926; Allen, 1940; Jones and Johnson, 1965). Allen (1940) further classified the genus Eothenomys into three subgenera: Eothenomys, Anteliomys and Caryomys. Musser and Carleton (1993) pointed that Eothenomys were reassigned to a genus independently. Wang and Li (2000) classified only two subgenera in Eothenomys, Eothenomys and Antelionomys. In the present study, UPGMA trees showed that the genus Eothenomys may classified into two subgenera.

In Multidimensional Scaling analysis, E. fidelis, E. proditor, E. olitor, E. custos and E. chinensis were above the diagonal; E. miletus, E. eleusis, E. cachinus and E. melanogaster were below the diagonal, suggesting that the genus Eothenomys could classified into two subgenera, which supported the results for Wang and Li (2000).

Historically, genus Eothenomys included 14 species, as described by Milne-Edwards (1872), Thomas (1911a, b, 1914, 1921), Allen (1912), Cabrera (1922), Hinton (1923), Tokuda and Kano (1937) and Wang and Li (2000). Furthermore, it was debated about 9 species: E. shaneius, E. regulus, E. lemminus, E. andersoni, E. smithii, E. wardi, E. fidelis, E. Eva and E. inez (Liu and Liu, 2005). Hinton (1923) regarded Eothenomys as a distinct genus and described E. fidelis. Allen (1924) renamed Microtus fidelis. Wang and Li (2000) used E. fidelis as a synonym of E. miletus. Wang (2003) recognized E. fidelis was a distinct species. In the present study, the results showed that only E. melanogastor is clearly separated from all other species on cranium and mandible using by principal component analysis.

Overlapping is visible between E. fidelis and E. chinensis, E. proditor and E. miletus on the dorsal view of the cranium, but E. miletus and E. fidelis separated clearly from each other as on the lateral view of the cranium. According to UPGMA trees, E. miletus appeared to separate from E. fidelis, which supported the results those of Wang (2003).

Based on UPGMA trees, E. miletus and E. eleusis appeared to be more closely related to others (Fig. 3). Although E. eleusis and E. miletus were proposed as separate subspecies or species (Allen, 1940; Hinton, 1923; Musser and Carleton, 1993; Thomas, 1912a, b; Wang and Li, 2000), the cyt b sequences from these two speceies were nearly identical (Luo et al., 2004). But Multidimensional Scaling analyzes revealed that they had some distance. Anterior ventral points of the upper incisor alveolus of E. miletus was different from E. eleusis. ANOVA analyses showed that significant differences in skull shape were found among species in centroid size. As noted earlier, the radiation of Oriental voles were probably recent, and most likely began about 2 Mya according to the fossil record (Zheng, 1993). The environment can interact to various degrees in mandible morphology (Renaud and Millien, 2001). Based on the above researches, we inferred that the radiation of E. miletus and E. eleusis began to radiation recently.

The relationship between environment and geometric morphological characteristics

The random differentiation associated with environment were observed in cranial (Fadda and Corti, 2001), mandibular (Duarte et al., 2000; Renaud and Auffray, 2010) and dental shape differentiation (Renaud, 1999; Hautier et al., 2007), geographical gradients (Renaud and Millien, 2001). Shapes in European wood mouse had a latitudinal gradient changing (Renaud and Michaux, 2003; Renaud et al., 2005). Eothenomys has been recorded near Trans-Himalayan Tange includes various north-south extending ranges and adjacent mountainous areas on the east skirts of the Qinghai-Tibetan Plateau. The geological configuration of this area was complicated (Li and Wang, 1986). In the present study, skull shapes in E. melanogaster was rather smaller, and E. chinensis was larger, which was supported for Bergmann's rule (Bergmann, 1847; Mayr, 1942). Moreover, variations of environment could influence the centroid size of Eothenomys.

On the mandible, E. proditor had a higher centroid size, E. proditor has been recorded from woodland and meadow around 2800-4200m (Luo et al., 2004), which was inhabited the highest altitude among Eothenomys, and the food quality was lower. Other study showed that the intergeneric comparisons between mandible shapes suggested that the latitudinal change in morphology observed within the wood mice may be related to change in diet (Renaud and Michaux, 2003). So, E. proditor should diversify their shape of mandible in order to maintain its high-energy demand.

Using thin plate spline analysis, significant morphological differentiations were shown for both orbital cavity and alveolar region in intraspecific skulls. And some differences were interspecific character, such as the point between the anterior tip of suture nasal and premaxilla, anterior tip of suture between nasal and premaxilla, anterior and posterior most ventral points of the upper incisor alveolus, and coronoid process, angular process ascending ramus and condylar process. The main function of the nose is helping animal to breathe. When animal inhale through their nose, air passes through nostrils into a short and narrow area known as the nasal passage that leads to the back of the pharynx, and down into the windpipe and lungs. The external air that we breathe is also warmed and moistened as it passes through the nose. Therefore, the main difference of interspecies was nasal bone, which may reflect adaptations to temperature and humidity.

However, the different of nose could view on lateral and dorsal of cranium, which have a main effect on Dimension 1 in Multidimensional Scaling analysis, and E. miletus, E. eleusis, E. cachinus and E. melanogaster were below the diagonal, so it may imply that speciation of four species were main affected by temperature and humidity. Change of premaxilla and mandible were interacted to the quality diet (Renaud and Michaux, 2003, 2007; Duarte et al., 2000; Renaud and Millien, 2001), varieties of ventral of the cranium and mandible had effects on Dimension 2, which may imply that E. olitor E. custos and E. chinensis were affected by diet quality.


In conclusion, there had significant differences in skull shapes of centroid size in Eothenomys. The genus Eothenomys could be classified into two subgenera. Moreover, the environmental factors (temperature, humidity and diet) may lead to the difference of geometric morphometry of skulls characteristics in Eothenomys.


This research was financially supported by National Science Foundation of China (No. 31760118; 31560126). We wish to thank Prof. Burkart Engesser at Historisches Museum Basel, Switzerland for correcting the English usage in the draft. Thank you for the anonymous reviewers and the editor of the journal for their valuable comments.

Statement of conflict of interests

The authors declare that they have no competing interests.


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Author:Ren, Xiao-Ying; Zhang, Di; Zhu, Wan-Long
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9CHIN
Date:Apr 30, 2019
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