Talpa fossilis or Talpa europaea? Using geometric morphometrics and allometric trajectories of humeral moles remains from Hungary to answer a taxonomic debate
Article number: 18.2.42A
Copyright Society for Vertebrate Paleontology, August 2015
Submission: 21 April 2015. Acceptance: 22 July 2015
The taxonomic validity of Talpa fossilis has been subject to a longstanding debate. Talpa fossilis has been considered as a chronospecies, stratigraphic species, and finally as junior synonym of Talpa europaea. In this study, the large humeral sample of T. fossilis and T. europaea from the Plio-Pleistocene of Hungary is re-investigated using a geometric morphometrics analysis. Furthermore, the differences in the static allometric slopes under the allometric constraint hypothesis were tested. The results indicate that T. fossilis and T. europaea have significant differences in both size and shape. The allometric slopes of T. fossilis and T. europaea were found to be different, revealing that the two taxa follow different patterns of shape modification according to size. In light of this evidence, T. fossilis and T. europaea are supported as two distinct species.
Gabriele Sansalone. Università di Roma Tre, Dipartimento di Scienze, L.S. Murialdo, 1 - 00146 Roma, Italy and Center of evolutionary ecology, Pesche, Italy.
Tassos Kotsakis. Università di Roma Tre, Dipartimento di Scienze, L.S. Murialdo, 1 - 00146 Roma, Italy and Center of evolutionary ecology, Pesche, Italy.
Paolo Piras. Università di Roma Tre, Dipartimento di Scienze, L.S. Murialdo, 1 - 00146 Roma, Italy and Center of evolutionary ecology, Pesche, Italy. and Dipartimento di Scienze Cardiovascolari,Respiratorie, Nefrologiche, Anestesiologiche e Geriatriche, Sapienza-Universita` di Roma, Roma, Italy and Dipartimento di Ingegneria Strutturale e Geotecnica Sapienza, Università di Roma.
Keywords: Talpidae; geometric morphometrics; humerus; allometry; systematics; Plio-Pleistocene
Final citation: Sansalone, Gabriele, Kotsakis, Tassos, and Piras, Paolo. 2015. Talpa fossilis or Talpa europaea? Using geometric morphometrics and allometric trajectories of humeral moles remains from Hungary to answer a taxonomic debate. Palaeontologia Electronica 18.2.42A: 1-17. https://doi.org/10.26879/560
Copyright: © 2015 Society of Vertebrate Paleontology. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Ontogenetic, static, and evolutionary allometries are recognized depending on whether the relationship between shape and size is taken over the development of an individual, across individuals at a similar developmental stage within a population, or across separate evolutionary lineages (Cock, 1966; Gould, 1966; Cheverud, 1982). The allometric-constraint hypothesis states that the allometric slope remains stable at the above-species level and is able to constrain the evolutionary divergence in the morphospace along its specific trajectories (Firmat et al., 2014; Pélabon et al., 2014; Voje et al., 2014). Many recent comparative studies have provided evidence of the constraining role of static allometry, revealing that intraspecific static allometric trajectories remain constant through time (Voje and Hansen, 2013; Firmat et al., 2014; Voje et al., 2014). On the basis of the allometric-constraint hypothesis, statistically significant differences in static allometric slopes are not expected within the same species. Firmat et al. (2014), analysing a time-ordered sample of Mimomys savini, showed how the static allometric slopes remained constant through time. With the allometric-constraint as a background hypothesis, divergence in allometric slopes, in populations distributed across a time-ordered series, could indicate the presence of separate species. The humerus of highly fossorial moles is well suited for this kind of investigation, as this skeletal element is often abundant and well preserved in fossil assemblages.
Diverse lifestyles have evolved among Talpidae, which includes species that are ambulatory (the shrew-like moles), semi-aquatic (desmans), semi-fossorial (shrew moles), and fully fossorial (Hutchison, 1976; Yates and Moore, 1990). The fully fossorial taxa evolved a unique humeral morphology (Gambaryan et al., 2003; Sánchez-Villagra et al., 2004; Piras et al., 2012). As a result of the adaptation to burrowing, the humerus is widened and flattened, and presents an elliptically shaped, ventrally directed head of humerus, a heavily expanded proximal end, an enlarged teres major tubercle, a deep brachialis fossa, a large, hemicylindrical clavicular facet, and an enlarged medial epicondyle bearing a deep fossa for the attachment of the Flexor digitorum profundus tendon-muscle (Edwards, 1937; Hutchinson, 1968). The complexity of the humerus makes this bone a potentially rich source of evolutionary characters (Sánchez-Villagra et al., 2004); this bone experienced transformations that are of taxonomic and systematic interest (van den Hoek Ostende, 1997; Ziegler, 2003; Klietmann et al., 2014; Sansalone et al., in press).
Systematics and taxonomy of fossil moles have often been based on humeri, in particular when teeth materials are morphologically indistinguishable or very scarce (Ziegler, 2003). Moreover, many fossil species have been described on humeral remains alone (Ziegler, 2006). In the present contribution the humeral fossil material of Talpa fossilis and T. europaea from several Hungarian localities and from the German locality of Petersbuch 1 were re-investigated (Figure 1; Table 1). Hungarian localities provided a large amount of fossil material belonging to the genus Talpa. Classification of this material was based not only upon humeral remains, but also upon other skeletal elements, such as mandibles, teeth, and skull rostra (Petényi, 1864; Kormos, 1930; Kretzoi, 1938; Sulimski, 1959; Jánossy, 1979). The Late Pliocene-Middle Pleistocene Talpa material has been attributed to T. fossilis, whereas the Late-Pleistocene specimens to T. europaea (Jánossy, 1979). A geometric morphometric (GM) analysis of the humerus was used to quantitatively evaluate the differences (if any) in their shape and size as well as in their relationship. The static allometric trajectories of the two taxa were also investigated.
Pomel (1848), describing Talpa fossilis, suggested that the new species was somewhat larger and more robust and differed from other species in the shape of the carpals. However, the description made by Pomel (1848) has gone unnoticed since T. fossilis is attributed to Petényi (1864), who described T. vulgaris fossilis from the Hungarian fossil bearing locality of Beremend. The original description translated by van Cleef-Roders and van den Hoek Ostende (2001) states: “ Talpa vulgaris fossilis Petényi. The fossil bone material of this animal totally agrees with the corresponding bones of the recent common mole, both in morphology and size; thus this fossil mole does not differ from the recent mole on the specific level, if one does not take into account the only difference noted by me, viz. that in the modern mole the labial side of the mandible has only two foramina mentale, one under the second premolar, the other under the first molar, whereas in the fossil mole three of such foramina are found, one under the first premolar, but two-be it one of them very shallow- under the first molar.”
Following the original description of Petényi (1864), Talpa fossilis could not be distinguished from T. europaea Linnaeus, 1758, and justified a distinction only at subspecific level. Despite this, the middle-sized Talpa specimens from Late Pliocene to Middle Pleistocene deposits are often attributed to T. fossilis , whereas in more recent deposits they are often classified as T. europaea (Jánossy, 1979). Kormos (1930) described T. praeglacialis from the Early Pleistocene locality Püspokfürdo (Betfia 2), accounting for a higher number of foramina mentale than in T. europaea. Kretzoi (1938) then placed T. praeglacialis in synonymy with T. fossilis. Von Koenigswald (1970), in his discussion about the Talpa specimens from the German locality Petersbuch 1, suggested that there were no differences in size and morphology between T. fossilis and T. europaea . Rabeder (1972), in his discussion about the Talpa material from Hundsheim, suggested the presence of some slight morphological differences between T. fossilis and T. europaea such as the width of the M3 talonid and the number of the mental foramina. However, such differences were thought to be distinctive at the subspecific level only. Robert (1983), investigating the moles remains from the Middle Pleistocene of Saint Saveur, France, proposed to retain the name T. fossilis as a chronospecies since she observed a gradual size increase during the Pleistocene. Niethammer (1990), following von Koenigswald (1970), considered T. fossilis as a stratigraphic species. However, van Cleef-Roders and van den Hoek Ostende (2001) pointed out that the material from Saint Saveur could be referred to T. caeca sensu latu because of the division of the mesostyle and the lack of humeri in the sample in addition to questioning the evolutionary trend proposed by Robert (1983). Van Cleef-Roders and van den Hoek Ostende (2001), reviewing the literature concerning T. fossilis, suggested considering T. fossilis as a junior synonym of T. europaea. They pointed out, however, that including all the material belonging to T. fossilis into T. europaea would probably be misleading. Thus, they noted the need for a more thorough review of the fossil assemblages bearing both taxa.
From this brief summary of the major contributions to the debate, it is evident that most researchers tend to consider Talpa fossilis as a subspecies or as a synonym of T. europaea. By contrast, many authors (Martín-Suárez and Mein, 2004; Sánchez-Villagra et al., 2004; Crochet et al., 2009; Colangelo et al., 2010; Rzebik-Kowalska, 2014) still consider the specific distinction as valid. Moreover, many of the measurements and morphological differences reported in the literature remain statistically untested. This study contributes to the debate by introducing the study of static allometric trajectories via geometric morphometrics (GM) analysis in order to reveal (if any) the potential different shape-size relationship between T. fossilis and T. europaea. Statistically significant differences in static allometric trajectories could suggest, on the basis of the allometric-constraint hypothesis (Firmat et al., 2014; Voje et al., 2014), that a specific distinction could be justified upon an inherent underlying biological process.
MATERIAL AND METHODS
A total of 113 left humeri belonging to Talpa europaea Linnaeus, 1758 (n = 67) and Talpa fossilis Petényi, 1864 (n = 46) were analyzed. See Table 1 and Figure 1 for specimen localities and corresponding ages. See Appendix for specimen accession numbers and housing institutions.
The humeri have been photographed in caudal view at a fixed distance of 50 cm with a Nikon D100 camera with a Micro-Nikkor 105 mm lens. We digitized 21 landmarks and 15 semi-landmarks (Figure 2.1) using the tpsDig2 software (Rohlf, 2006). Semi-landmarks are used to optimally translate, scale, and rotate landmarks, as well as to slide along the outline curve until they match as closely as possible the positions of the corresponding points along an outline in a reference configuration (Adams et al., 2004; Perez et al., 2006). Thus, semi-landmarks are useful to depict the shape of curved lines where landmarks cannot be detected. Successively a generalized procrustes analysis (GPA) (Bookstein, 1991; Goodall, 1991) implemented in the procSym() function from R-package “Morpho” (Schlager, 2014) was used to rotate, translate, and scale landmark configurations to the unit centroid size (CS = the square root of the sum of squared distances of a set of landmarks from their centroid) (Bookstein, 1986). Rotation of the scaled and translated landmark sets starts by comparison with a reference configuration, usually the first specimen in the dataset. Once the first rotation is completed, a mean shape is calculated and the rotation process is repeated using the mean shape as the reference configuration for the sample (including the reference-specimen configuration). The mean shape/rotation procedure is iterated to minimize rotation differences between subsequent iterations through a least-square procedure (Rohlf and Slice, 1990). The residual differences correspond to real shape differences plus measurement error. A principal components analysis (PCA) was used to visualize the ordination of the aligned specimens. The significance of the observed shape differences among species was evaluated by performing a permutational multivariate analysis of variance (perMANOVA) on procrustes coordinates using the adonis() function included in the “vegan” R package (Oksanen et al., 2013). Deformations associated to the extreme of PC axes have been visualized through deformation grids. The grids have been built using the thin-plate spline methodology (Bookstein, 1989).
The significance of size differences has been evaluated by performing a permutational univariate analysis of variance (perANOVA) on CS using the function adonis(). Size variation was visualized using a boxplot.
Measurement error associated with landmarks digitization was determined by first calculating the mean Procrustes distances between all combinations of two specimens in the dataset using TPSsmall software. The same values were then calculated for each cluster of replicas (three replicas were generated). The mean Procrustes distances were calculated for each set of replica’s per specimen, including the same values for the total dataset. As an overall measure for digitisation error, the minimum and maximum values observed in all separate specimens were extracted, and the average of all mean values for those specimens was calculated. The amount of digitisation error with respect to the total variation in shape can be expressed as a percentage. Finally, the ratio of the mean value for total digitisation and the mean of the total dataset was calculated.
The relationship between size (independent variable) and shape (dependent variable) was tested performing a multivariate regression of shape on size values averaged by species. All individuals analyzed are adult or subadult based on the ossification status of humeral epiphysis and diaphysis. Thus the allometric trajectories belong to the category of static allometry. A permutational multivariate analysis of covariance (perMANCOVA), using species as groups and size as covariate, was used to test for differences in slopes among species (Zelditch et al., 2004, 2012). This analysis was performed using the function adonis(). If slopes do not differ significantly (in this case the species and size interaction of the MANCOVA is not statistically significant), it is possible to control for the allometric effect and compute size-corrected shape variables (Viscosi and Cardini, 2011; Viscosi et al., 2012; Zelditch et al., 2012). For the sake of visualization a canonical correlation analysis (CCA) was performed, which determines a Y axis that represents the amount of Y (shape variables) that is best explained by the independent variable X (CS). In order to study interspecific shape differences, the intraspecific variation was removed by performing separate per-species multivariate regressions between shape and size. Then, for each species, the residuals were added to species specific shapes predicted at maximum and minimum, species-specific size values. This procedure ensures elimination of intraspecific allometry while maintaining the interspecific size-shape differences due to evolutionary allometry (Piras et al., 2011, 2014). The differences between the predicted shape variables have been evaluated performing a perMANOVA using the function adonis(). This strategy, common in GM studies, allows the standardization of shape variables at determined size values (Zelditch et al., 2004, 2012). Finally, the Euclidean distances between shapes predicted at 10 equal CS values for Talpa fossilis and T. europaea were plotted in order to visualize the course of interspecific morphological distances along the static allometry.
Variation Partitioning Analysis
A variation partitioning analysis (VARPART) (Legendre and Legendre, 2012; Legendre et al., 2012) was performed to take into account the influence of space and climate on shape variables. The technique of variation partitioning is used when two or more complementary sets of hypotheses can be invoked to explain the variation of a response variable (Legendre, 2008). For this purpose, the latitude and longitude coordinates of each locality were transformed using the principal coordinates of neighbour matrices method (PCNM; Borcard and Legendre, 2002; Borcard et al., 2004). PCNM produces orthogonal (linearly independent) spatial variables over a much wider range of spatial scales (Borcard and Legendre, 2002). The residuals of multivariate regression of the shape variables on the transformed geographical coordinates were calculated in order to remove the effect (if any) of spatial influence. Then, the residual scores to the mean shape value were summed. Once the spatial effect has been removed it is possible to run a perMANOVA test to ascertain if significant shape differences still occur among populations." It was hypothesized that a spatial influence would only occur for T. euroapea specimens.
The VARPART analysis was performed using the varpart() function in “vegan” R package (Oksanen et al., 2013). The significance of the model was tested with a redundancy analysis (RDA). Climatic data were taken from Zachos et al. (2001). The oxygen isotopic data have been already used as a proxy for climatic changes in several previous works (Raia et al., 2005; Meloro et al., 2008; Sansalone et al., 2015) and was included as the climatic data in the VARPART model. The VARPART analysis was performed on the whole sample as well as on the separate samples of T. europaea and T. fossilis.
The measurement error analysis revealed that 2.9% of the total variation was due to digitisation error. Since the measurement error was smaller than 5%, it was judged as not significantly influencing further analysis.
Variation Partitioning Analysis
The results of the VARPART analysis are summarized in Table 2. A significant geographic and temporal interaction in the whole sample was found, whereas there was no correlation with climate. However, temporal and climate interactions were found to be negligible in both the separate species. A significant geographic influence was found only for Talpa europaea.
Shape and Size Analyses
The PCA performed on the aligned procrustes coordinates (Figure 3.1-2) show a good degree of separation between Talpa fossilis and T. europaea in particular along the PC1 (17.18% of the total variance). At positive values of the PC1 the humeral morphology shows an enlargement of the pectoral ridge and of the teres tubercle, an enlargement of the greater tuberosity and a medial shift of the humeral head. At negative values of the PC1 it presents a contraction of the pectoral ridge and a reduction of the teres tubercle, a reduction of the greater tuberosity and a lateral shift of the humeral head. At positive values of the PC2 (13.6% of the total variance) the humeral morphology shows an elongation of the teres tubercle and a shortening of the pectoral ridge. At negative values of the PC2 it shows a shortening of the teres tubercle and an elongation of the pectoral ridge. At positive values of the PC3 (8.17% of the total variance) the humeral shape shows a contraction of the lateral epicondyle and an enlargement of the minor sulcus. At negative values of the PC3 it shows an enlargement of the lateral epicondyle and a contraction of the minor sulcus.
The perMANOVA test returned a statistically significant difference (p < 0.001; F 1,111 = 12.98) between the two species. Even when using the space corrected values for Talpa europaea the perMANOVA test returned a statistically significant result (p < 0.001; F 1,111 = 13.48). The boxplot computed for the CS (Figure 4) showed a statistically significant size variation (perANOVA, p < 0.001; F 1,111 = 46.041) between T. europaea and T. fossilis, with the former being larger than the latter.
According to the significant interaction (p = 0.0199; F 1 = 1.92) between species and size effects in the perMANCOVA, specific allometric trajectories were proved to be non-parallel between the two species. Multivariate regression of shape data on size returned a statistically significant result (p < 0.001; F 1,109 = 7.86; R2 = 0.061), with size accounting for 6% of the total shape variance (Figure 5.1). Separate multivariate regressions on Talpa fossilis and T. europaea returned statistically significant results (p = 0.003, R2 = 0.048; p < 0.001, R2 = 0.034; respectively). PerMANOVA returned statistically significant values for the shape variables standardized at maximum and minimum CS values (p < 0.001, F 1,111 = 26.51 and p < 0.001, F 1,111 = 12.09; respectively). Euclidean distances show a decrement toward the CS value of 3.3 (though not becoming zero) from that value they tend to augment (Figure 5.2).
DISCUSSION AND CONCLUDING REMARKS
The separation between Talpa fossilis and T. europaea, observed along the PC1 (Figure 3.1-2), is due to changes in the teres tubercle and pectoral ridge. In particular, T. europaea has a larger teres tubercle and pectoral ridge than T. fossilis. In these humeral regions two of the main muscles associated with the burrowing motion are inserted (Figure 2.2) (Gambaryan et al., 2003; Piras et al., 2012, 2015). On the teres tubercle the Teres major and Latissimus dorsi muscles are inserted, while on the pectoral ridge the Pectoralis Pars Sternalis muscle inserts (Dobson, 1882; Freeman, 1886; Gambaryan et al., 2003). These muscles account for the 42.5% of the total digging muscle weight (Gambaryan et al., 2003). Piras et al. (2012) showed that the enlargement of the teres tubercle and of the pectoral ridge was a key factor in the evolution of fossoriality, and that the development of these anatomical regions significantly reduced the mechanic stress on the humerus across Talpidae phylogeny. Sansalone et al. (in press) re-assessed the taxonomy of the Plio-Pleistocene Neurotrichine moles and applied GM techniques on humeral remains. Their analysis showed the primitive morphology of the teres tubercle, pectoral ridge, and bicipital tunnel in the extinct Polish Rzebikia polonica and R. skoczeni, whereas the extant North American Neurotrichus gibbsii was in a more derived state (Sansalone et al., in press). Within this framework, we suggest that T. europaea was better adapted to burrowing, as this taxon had a larger area of insertion for the main digging muscles and is more specialized than T. fossilis.
This study showed that the allometric trajectories of Talpa europaea and T. fossilis were significantly different (they cross each other) by having different starting and ending points. This evidence excludes the possibility that T. fossilis should be considered as a stratigraphic species (Rabeder, 1972). In this case we would expect to find very similar static allometric trajectories, as predicted by the allometric-constraint theory (Voje and Hansen, 2013; Firmat et al., 2014; Voje et al., 2014). The humerus of highly fossorial moles experienced a strong phenotypical channeling in order to adapt to the underground environment (Nevo, 1979; Sánchez-Villagra et al., 2006; Piras et al., 2012, 2015). The absence of a climatic influence on humeral shape, related to the climatic stability of the subterranean ecotope (Nevo, 1979), supports the evidence that the different allometric slopes likely indicate the presence of two separate species. Furthermore, the hypothesis that T. fossilis be considered a chronospecies of T. europaea (Robert, 1983) has been proven to be false. The significant shape differences found between T. europaea and T. fossilis, even when the shape variables are predicted at the same CS values, indicate that the two taxa have different allometric patterns and humeral morphologies even under the same adaptive pressure.
Given these results Talpa fossilis is considered to be a distinct species from T. europaea. Separating these two taxa would also fit with molecular data. According to Colangelo et al. (2010) and Feuda et al. (2015) the T. europaea basal split has a mean divergence time estimate of 0.7 m.y. The time estimate would be in agreement with the fossil record and the species distinction usually presented in literature (Sulimski, 1959; Jánossy, 1979). From the results of this study, T. fossilis, which has a more primitive humeral morphology, is hypothesized to have originated from a different lineage than T. europaea. The fossil record (Ziegler, 1999; van den Hoek Ostende and Fejfar, 2006; Ziegler, 2006; Engesser, 2009) support the presence of four Talpa species since the early Miocene: T. tenuidentata Ziegler, 1990 (MN2-3; 22-18 Ma), T. minuta de Blainville, 1840 (MN2-9; 22-10 Ma), T. vallesensis de Villalta Comella and Crusafont Pairó, 1944 (MN7-11; 12-8.5 Ma) and T. gilothi Storch, 1978 (MN9-11; 11.5-8.5 Ma). It is possible that the T. fossilis lineage originated from an offshoot of the Miocene mole lineages, which had a different allometric pattern and primitive humeral features, and spread across Europe during the Pliocene, surviving until the end of the Middle Pleistocene. In another possible scenario, T. fossilis could be derived from an eastern branch and may have colonized Europe during the Early Pliocene. According to Colangelo et al. (2010), T. europaea was derived from the eastern lineage of the genus Talpa. Therefore, it is hypothesized that T. europaea , during its colonization routes, could has come in contact with T. fossilis and out-competed it due to its larger size and superior digging capability. Competition in moles is a well-known phenomenon and is described in detail for the genera Talpa and Mogera (Abe, 1996; Loy et al., 1996; Loy and Capanna, 1998; van Cleef-Roders and van den Hoek Ostende, 2001; Yokohata, 2005; Bego et al., 2008; Loy, 2008). Furthermore, highly fossorial moles have been extensively reported to be solitary, territorial, and actively patrolling their tunnels, which they defend against intruders (Gorman and Stone, 1990; Loy et al., 1994).
The results of this study suggest that the landmark-based shape analysis is useful in supporting systematics in palaeontological investigations, in particular when morphological differences are elusive. In order to extend these findings on humeral static allometric trajectories to larger portions of the occurrence areas of these two species, the systematics of every assemblage bearing Talpa fossilis and T. europaea should be reviewed, based not only on humeral morphologies, but also on other skeletal elements following the methodologies used in this study.
We are grateful to Dr. M. Gasparik from Natural History Museum, Budapest, Hungary and Prof. L. Kordos from Hungarian Geological Institute, Budapest, Hungary for allowing the visit of the Talpidae collection. We want to thank Dr. T. Matthews and an anonymous referee for their useful comments during the manuscript revision.
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