Evolution of bone compactness in extant and extinct moles (Talpidae): exploring humeral microstructure in small fossorial mammals
© Meier et al; licensee BioMed Central Ltd. 2013
Received: 8 November 2012
Accepted: 19 February 2013
Published: 26 February 2013
Talpids include forms with different degree of fossoriality, with major specializations in the humerus in the case of the fully fossorial moles. We studied the humeral microanatomy of eleven extant and eight extinct talpid taxa of different lifestyles and of two non-fossorial outgroups and examined the effects of size and phylogeny. We tested the hypothesis that bone microanatomy is different in highly derived humeri of fossorial taxa than in terrestrial and semi-aquatic ones, likely due to special mechanical strains to which they are exposed to during digging. This study is the first comprehensive examination of histological parameters in an ecologically diverse and small-sized mammalian clade.
No pattern of global bone compactness was found in the humeri of talpids that could be related to biomechanical specialization, phylogeny or size. The transition zone from the medullary cavity to the cortical compacta was larger and the ellipse ratio smaller in fossorial talpids than in non-fossorial talpids. No differences were detected between the two distantly related fossorial clades, Talpini and Scalopini.
At this small size, the overall morphology of the humerus plays a predominant role in absorbing the load, and microanatomical features such as an increase in bone compactness are less important, perhaps due to insufficient gravitational effects. The ellipse ratio of bone compactness shows relatively high intraspecific variation, and therefore predictions from this ratio based on single specimens are invalid.
KeywordsWolff’s law Paleohistology Size Phylogeny Placentalia
Talpidae is a diverse clade of small-sized lipotyphlan mammals which occupy different habitats, ranging from terrestrial to semi-aquatic to fossorial . They are widely distributed throughout the largely temperate regions of the northern continents and their rich fossil record since the Eocene includes many genera from many sites and ages . The phylogenetic relationships within the clade are not yet fully resolved; a comprehensive morphological study  and ongoing molecular work [4–6] serve as a framework in which to understand complex biogeographic and ecomorphological patterns of evolution.
The relation between microanatomical structure and mechanical adaptations of long bones, such as the humerus, has been studied in many amniote taxa (e.g., [15–22]). Variations in the histological proportions of cortex and medulla can be biomechanical indicators of lifestyles . In general, terrestrial taxa have a moderately thick, compact cortex with little or no spongiosa in the mid-shaft region, long bones of flying animals show hollow medullas, and terrestrial or swimming taxa have a spongiosa inside the medulla . However, most of these studies focus on adaptations to an aquatic lifestyle [16, 19, 20, 23, 24]; in contrast studies of microanatomy in fossorial taxa are lacking.
There is evidence that cortical bone primarily responds to strain only prior to sexual maturity [25, 26]. However, Wolff’s ‘Law’, which postulates that bone increases in density and/or cortical thickness in response to the loads it is placed under during an individual’s life, does not always hold, although rules for ‘bone functional adaptation’ to mechanical loading do exist (, p. 484; [28, 29]). It has been shown that bone thickness is influenced also by other variables such as temperature, and that its development is mediated by genetic mutations and/or modified transcript levels [30–32].
In addition, body size has been suggested to restrict Wolff’s ‘Law’, with bone not responding to biomechanical strains in femora of small animals such as shrews and bats . A high intraspecific variation in cortical thickness in each tested species suggested that Wolff’s Law is not applicable below a certain body size in mammals . In addition, bone density was not significantly different between terrestrial and semi-aquatic rodents within the size range of moles . Phylogeny may be the most important factor coupled with the organisation of bone compactness in mammalian long bones [16, 33].
Moles form an ideal subject of research on the evolution of microanatomical structure due to their biomechanical diversity. A relationship between an aquatic and terrestrial lifestyle and humeral microanatomy has been postulated, but this is based on taxonomically broad studies of amniotes . Here, we investigate bone cortical thickness in fossil and living talpid taxa, representing thus one of the first comprehensive examination of bone compactness in any mammalian clade. We test the hypothesis that compactness is higher in humeri in the most fossorial species due to the severe mechanical strains to which they are exposed to during digging. In testing it, we examine several issues around Wolff’s ‘Law’ and others detailed above.
Materials and methods
Humeri of 11 extant and 8 fossil talpid species representing terrestrial, semi-aquatic and fossorial forms were studied (Figure 1). Two non-talpid species, the Asian house shrew (Suncus murinus, Soricidae), and the mouse-eared bat (Myotis myotis, Chiroptera), served as outgroups (Additional file 1).
The phylogenetic framework is a composite that best integrates the current knowledge on extant and fossil taxa, a subject not fully resolved . Relationships among extant species follows [3, 6], the latter supported the basal position of Condylura (see also ). The position of Geotrypus is based on the recent and comprehensive analysis based on new fossil data by Schwermann & Martin, 2012 . The position of the following fossil taxa is based on the listed references: Asthenoscapter[35, 36], Desmanella, Mygatalpa[35, 36], Paratalpa[35, 36] and Proscapanus.
Histomorphometric analyses were performed using the image-processing software BONE PROFILER , which has been used to determine bone compactness in amniote long bones [20, 24]; for examples of applications see also Houssaye  and Hayashi et al. . For whole-cross-section-profiling the program sets a section centre and places a grid over the section dividing the bone tissue into 60 radial sectors and 51 concentric shapes. It then measures the degree of solid bone in each of the 51 subdivisions of each sector. The measurements of all sectors are then integrated into a global compactness of the section. The parameters S (reciprocal of the slope at the inflexion point), P (distance to transition point), Min (lower asymptote of sigmoid curve), Max (upper asymptote), Cc (compactness in the bone centre), CDI (cortico-diaphyseal index; ), and Cg (bone global compactness) are calculated by the program. Parameters S, P, and Min have been shown to evince biomechanical information .
Student’s paired t-tests were performed for five bone profile variables (i.e. Cg, CDI, P, S). The groups tested for differences in these values were fully fossorial versus other moles, i.e. Scalopini versus Talpini, and outgroups versus Talpidae. In addition, phylogenetic ANOVA was performed to make phylogenetically corrected between-groups comparisons . We adopted equal lengths for all branches, since estimates of branch lengths for talpids are still very tentative (see  for a similar procedure). Phylogenetically corrected contrasts were computed in PDAP module of Mesquite program  and assessed by ANOVA in PAST program .
Talpid humeri are hollow in the center, with few showing coarse trabeculae (Figure 1). In general, the humeri of the semi-aquatic Desmanini and terrestrial/semi-fossorial Urotrichini, except for the Russian Desman (Desmana moschata), are much smaller than those of the two fossorial clades: Talpini and Scalopini. The inner cross-sections of non-fossorial taxa (Desmanini, Condylura and Urotrichus) are, overall, more circular compared to those of Talpini and Scalopini. These latter show a rather buckled outline and a slightly elliptic medullary cavity, reflecting the torsion of the humerus and the deep reaching distal end of the deltopectoral crest (Figure 2). The cross-section of Condylura is unique in that it displays a typical fossorial outline combined with a very round medullary cavity as present in semi-aquatic and terrestrial species.
Analysis using phylogenetic ANOVA to test for differences on different variables and groups
fossorial - non fossorial
Scalopini - Talpini
Ourgroups - Talpidae
Global compactness (Cg)
Reciprocal of the slope at the inflexion point (S)
Significance of difference between mean values of the functional groups with Two-sided Student’s t-test, same variance
The Talpini and Scalopini show no differences in any of the tested variables; all values are within normal range of variance. However, although the results indicate a slightly lower ellipse ratio in Scalopini (0.516 ± 0.41) compared to Talpini (0.560 ± 0.36), these are not statistically significant.
No difference in global bone compactness (Cg) was detected between the clades of moles or their functional groupings (Figure 4, Tables 1 and 2). It might be expected that Cg would be more elevated in cross-sections of highly derived humeri of fossorial taxa than in terrestrial and semi-aquatic ones in relation to the severe mechanical strains to which they are exposed during digging. The results here reject this hypothesis.
It has been suggested that bone structure in small species is much simpler than in larger ones; for instance, small terrestrial mammals generally have a thin cortex and little or no spongiosa . This implies that the overall morphology of the humerus, at small size, may be all that is required to cope with the strains of digging, and that microanatomical specializations are less likely to occur. In small mammals, the cortical dimensions are probably already mechanically efficient without further adaptation. Dawson  calculated the bone tissue strength for the shrew (Blarina brevicauda) and two bat species (Myotis lucifugus and Pipistrellus subflavus), which are comparable in size with moles, using the formula of Koch ; the inherent tissue strength (estimated by Ascenzi & Bonucci ) exceeded the predicted loading by a factor 100. Dawson  thus suggested that Wolff’s Law does not apply in these diminutive mammals. The results from global bone compactness analysis in this study confirm this statement. However, another parameter calculated by BONE PROFILER, the S value, is significantly larger in fossorial talpids than in non-fossorial ones. In amniote long bones in general, S also exhibited an adaptive relationship . In addition, the ellipse ratio showed a highly significant relationship between fossorial and nonfossorial talpids.
No difference was seen in the cross-sections of the two fossorial clades Talpini and Scalopini, which is in congruence with earlier reported results of close convergence in them . Based on stress performance modelling with finite element analysis, Piras et al. (, p.13) stated that once the taxa ‘reached the optimal phenotypic status, their humerus did not undergo further morphological changes’. Piras et al.  found in the two fully fossorial clades Talpini and Scalopini a slowing of the evolutionary rate of humeri which are better adapted to mechanical stress and the similar path of development of characteristics that lead to a decrease of stress; moreover a lower variance of fossorial humerus shapes when compared to those of non fossorial ones was found.
It has been reported earlier that analysing only one specimen per species can be sufficient as interspecific variation is much higher than intraspecific variation . While this might be true for studies on a higher taxonomic level (e.g. for the Lissamphibia, ), intraspecific variation in Talpidae is relatively high, and therefore predictions based on single specimens should be avoided. For example, differences in age, size, sex and nutrition of the specimens can contribute to variation in bone- density [16, 49]. An influence on microanatomy by these factors cannot be ruled out. Future studies on bone microstructure variation therefore need not only include additional taxa but also use several specimens per species.
Fully fossorial talpids are distinguishable from other talpids by the S-value, the reciprocal of slope of the sigmoid curve (Figure 5, Table 2) as well as by the ellipse ratio of the medullary cavity of the humeral cross section (Figure 7), although a phylogenetic corrected statistical analysis did not offer significant results. How these variables behave in other fossorial versus nonfossorial species of mammalian clades of similar size, remains yet to be investigated. Furthermore, the absence of significant differences in the two fully fossorial clades, Talpini and Scalopini, and the low variance compared to nonfossorial taxa, indicate that fossorial adaptation is further evidence of the high degree of evolutionary parallelism in these clades.
We thank G. Rössner (Munich), D. P. Lunde (Washington DC), R. J. Asher (Cambridge), A. Averianov (St. Petersburg), L. Costeur (Basel), R. Ziegler (Stuttgart), B. Oberholzer (Zürich) and O. Hampe (Berlin) for kind access to specimens and two anonymous reviewers for useful suggestions to improve the this study. This work was supported by the Swiss National Fund Grant 3100A0133032/1.
- Gorman ML, Stone DR: The natural history of moles. 1990, New York: Cornell University Press, 138-Google Scholar
- Mc Kenna MC, Bell SK: Classification of mammals above the species level. 1997, New York: Columbia University PressGoogle Scholar
- Sánchez-Villagra MR, Horovitz I, Motokawa M: A comprehensive morphological analysis of talpid moles (Mammalia) phylogenetic relationships. Cladistics. 2006, 22: 59-88. 10.1111/j.1096-0031.2006.00087.x.View ArticleGoogle Scholar
- Shinohara A, Campbell KL, Suzuki H: Molecular phylogenetic relationships of moles, shrew moles, and desmans from the New and Old worlds. Mol Phylogenet Evol. 2003, 27: 247-258. 10.1016/S1055-7903(02)00416-5.PubMedView ArticleGoogle Scholar
- Shinohara A, Suzuki H, Tsuchiya K, Zhang Y-P, Luo J, Jiang X-L, Wang Y-X, Campbell KL: Evolution and biogeography of talpid moles from continental East Asia and the Japanese Islands inferred from mitochondrial and nuclear gene sequences. Zoolog Sci. 2004, 21: 1177-1185. 10.2108/zsj.21.1177.PubMedView ArticleGoogle Scholar
- Crumpton N, Thompson R: The holes of moles: Osteological correlates of the trigeminal nerve in Talpidae. J Mamm Evol. 2012, 10.1007/s10914-012-9213-2.Google Scholar
- Koyabu D, Endo H, Mitgutsch C, Suwa G, Catania KC, Zollikofer CP, Oda S-I, Koyasu K, Ando M, Sánchez-Villagra MR: Heterochrony and developmental modularity of cranial osteogenesis in lipotyphlan mammals. EvoDevo. 2011, 2: 21-10.1186/2041-9139-2-21.PubMed CentralPubMedView ArticleGoogle Scholar
- Piras P, Sansalone G, Teresi L, Kotsakis T, Colangelo P, Loy A: Testing convergent and parallel adaptations in talpids humeral mechanical performance by means of Geometric Morphometrics and Finite Element Analysis. J Morphol. 2012, 273: 696-711. 10.1002/jmor.20015.PubMedView ArticleGoogle Scholar
- Sánchez-Villagra MR, Menke PR, Geisler JH: Patterns of evolutionary transformation in the humerus of moles (Talpidae, Mammalia): a character analysis. Nat Hist. 2004, 170: 163-170.Google Scholar
- Whidden HP: Comparative myology of moles and the phylogeny of the Talpidae (Mammalia, Lipotyphla). Am Mus Novit. 2000, 3294: 1-53.View ArticleGoogle Scholar
- Freeman R: The anatomy of the shoulder and upper arm of the mole (Talpa europaea). Journal of Anatomy and Physiology. 1886, 20: 201-219.Google Scholar
- Reed CA: Locomotion and appendicular anatomy in three soricoid insectivores. Am Midl Nat. 1951, 45: 513-671. 10.2307/2421996.View ArticleGoogle Scholar
- Yalden DW: The anatomy of mole locomotion. J Zool. 1966, 149: 55-64.View ArticleGoogle Scholar
- Gambaryan PP, Gasc J-P, Renous S: Cinefluorographical study of the burrowing movements in the common mole, Talpa europaea (Lipotyphla, Talpidae). Russian J Theriol. 2002, 1: 91-109.Google Scholar
- Dawson DL: Functional interpretations of the radiographic anatomy of the femora of Myotis lucifugus, Pipistrellus subflavus, and Blarina brevicauda. Am J Anat. 1980, 157: 1-15. 10.1002/aja.1001570102.PubMedView ArticleGoogle Scholar
- Stein BR: Bone density and adaptation in semiaquatic mammals. J Mammal. 1989, 70: 467-476. 10.2307/1381418.View ArticleGoogle Scholar
- Francillon-Vieillot H, De Buffrénil V, Castanet J, Géraudie J, Meunier FJ, Sire J-Y, Zylberberg L, De Ricqlès AJ: Microstructure and mineralization of vertebrate skeletal tissues. Skeletal biomineralization: Patterns, processes and evolutionary trends. Edited by: Carter JG. 1990, New York: Reinhold, Van Nostrand, 471-530.Google Scholar
- Girondot M, Laurin M: Bone Profiler: A tool to quantify, model and statistically compare bone-section compactness profiles. Bone. 2003, 23: 458-461.Google Scholar
- Germain D, Laurin M: Microanatomy of the radius and lifestyle in amniotes (Vertebrata, Tetrapoda). Zoologica Scripta. 2005, 34: 335-350. 10.1111/j.1463-6409.2005.00198.x.View ArticleGoogle Scholar
- Canoville A, Laurin M: Evolution of humeral microanatomy and lifestyle in amniotes, and some comments on palaeobiological inferences. Biol J Linn Soc. 2010, 100: 384-406. 10.1111/j.1095-8312.2010.01431.x.View ArticleGoogle Scholar
- Laurin M, Canoville A, Germain D: Bone microanatomy and lifestyle: A descriptive approach. Comptes Rendus Palevol. 2011, 10: 381-402. 10.1016/j.crpv.2011.02.003.View ArticleGoogle Scholar
- Hugi J, Sánchez-Villagra MR: Life history and skeletal adaptations in the Galapagos marine iguana (Amblyrhynchus cristatus) as reconstructed with bone histological data—A comparative study of iguanines. J Herpetol. 2012, 46: 312-324. 10.1670/11-071.View ArticleGoogle Scholar
- Wall WP: The correlation between high limb-bone density and aquatic habits in recent mammals. Sediment Geol. 1983, 57: 197-207.Google Scholar
- Houssaye A: Bone histology of aquatic reptiles: what does it tell us about secondary adaptation to an aquatic life?. Biol J Linn Soc. 2012, 108: 3-21.View ArticleGoogle Scholar
- Pearson OM, Lieberman DE: The aging of Wolff’s “Law”: Ontogeny and responses to mechanical loading in cortical bone. Am J Phys Anthropol. 2004, 125: 63-99. 10.1002/ajpa.20155.View ArticleGoogle Scholar
- Young MT, Brusatte SL, Ruta M, De Andrade MB: The evolution of metriorhynchoidea (Mesoeucrocodylia, Thalattosuchia): An integrated approach using Geometric Morphometrics, Analysis of Disparity, and Biomechanics. Zoological Journal of the Linnean Society. 2010, 158: 801-859. 10.1111/j.1096-3642.2009.00571.x.View ArticleGoogle Scholar
- Ruff C, Holt B, Trinkaus E: Who’s afraid of the big bad Wolff?: “Wolff’s law” and bone functional adaptation. Am J Phys Anthropol. 2006, 129: 484-498. 10.1002/ajpa.20371.PubMedView ArticleGoogle Scholar
- Frost HM: Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolff’s Law: The bone modeling problem. Anat Rec. 1990, 226: 403-413. 10.1002/ar.1092260402.PubMedView ArticleGoogle Scholar
- Frost HM: Skeletal structural adaptations to mechanical usage (SATMU): 2. Redefining Wolff’s Law: The remodeling problem. Anat Rec. 1990, 226: 414-422. 10.1002/ar.1092260403.PubMedView ArticleGoogle Scholar
- Cretekos CJ, Wang Y, Green ED, Program NCS, Martin JF, Rasweiler JJ, Behringer RR: Regulatory divergence modifies limb length between mammals. Genes Dev. 2008, 22: 141-151. 10.1101/gad.1620408.PubMed CentralPubMedView ArticleGoogle Scholar
- Serrat MA, King D, Lovejoy CO: Temperature regulates limb length in homeotherms by directly modulating cartilage growth. Proc Natl Acad Sci. 2008, 105: 19348-19353. 10.1073/pnas.0803319105.PubMed CentralPubMedView ArticleGoogle Scholar
- Morimoto RI: The heat shock response: Systems biology of proteotoxic stress in aging and disease. Cold Spring Harb Symp Quant Biol. 2011, 76: 91-99. 10.1101/sqb.2012.76.010637.PubMedView ArticleGoogle Scholar
- Legendre L, Le Roy N, Martinez-Maza C, Montes L, Laurin M, Cubo J: Phylogenetic signal in bone histology of amniotes revisited. Zoologica Scripta. 2012, 42: 44-53.View ArticleGoogle Scholar
- Schwermann A, Martin T: A partial skeleton of Geotrypus antiquus (Talpidae, Mammalia) from the Late Oligocene of the Enspel Fossillagerstätte in Germany. Paläontol Z. 2012, 86: 409-439.View ArticleGoogle Scholar
- Hutchison JH: Notes on type specimens of European Miocene talpidae and a tentative classification of Old World Tertiary Talpidae (Insectivora: Mammalia). Geobios. 1974, 7: 211-256. 10.1016/S0016-6995(74)80009-4.View ArticleGoogle Scholar
- Ziegler R: Order Insectivora: Talpids. The Miocene land mammals of Europe. Edited by: Rössner GE, Heissig K. 1999, München: Dr. Friedrich Pfeil, 53-74.Google Scholar
- Canoville A, Laurin M: Microanatomical diversity of the humerus and lifestyle in lissamphibians. Acta Zoologica. 2009, 90: 110-122. 10.1111/j.1463-6395.2008.00328.x.View ArticleGoogle Scholar
- Kolb C, Sánchez-Villagra MR, Scheyer TM: The palaeohistology of the basal ichthyosaur Mixosaurus, Baur, 1887 (Ichthyopterygia, Mixosauridae) from the Middle Triassic: palaeobiological implications. Comptes Rendus Palevol. 2011, 10: 403-411. 10.1016/j.crpv.2010.10.008.View ArticleGoogle Scholar
- Chinsamy A, Raath MA: Preparation of fossil bone for histological examination. Palaeontologia africana. 1992, 29: 39-44.Google Scholar
- Enlow DH, Brown SO: A comparative histological study of recent fossil and recent bone tissues. Part III. Tex J Sci. 1958, 10: 187-230.Google Scholar
- Hayashi S, Houssaye A, Nakajima Y, Chiba K, Ando T, Sawamura H, Inuzuka N, Naotomo Kaneko N, Tomohiro O: Bone histology suggests increasing aquatic adaptations in Desmostylia (Mammalia, Afrotheria). PLoS One. 2012, in pressGoogle Scholar
- Castanet J, Curry Rogers K, Cubo J, Jacques-Boisard J: Periosteal bone growth rates in extant ratites (Ostriche and Emu). Implications for assessing growth in dinosaurs. Comptes Rendus de l’Académie des Sciences - Series III - Sciences de la Vie. 2000, 323: 543-550. 10.1016/S0764-4469(00)00181-5.Google Scholar
- Garland T, Midford PE, Ives AR: An introduction to phylogenetically based statistical methods, with a new method for confidence intervals on ancestral values. Am Zool. 1999, 39: 374-388.View ArticleGoogle Scholar
- Maximino C: Evolutionary changes in the complexity of the tectum of nontetrapods: A cladistic approach. PLoS One. 2008, 3: e3582-10.1371/journal.pone.0003582.PubMed CentralPubMedView ArticleGoogle Scholar
- Hammer Ø, Harper DAT, Ryan PD: PAST: Paleontological statistics software package for education and data analysis. Palaeontol Electron. 2001, 4: 9-Google Scholar
- Koch JC: The laws of bone architecture. Am J Anat. 1917, 21: 298-View ArticleGoogle Scholar
- Ascenzi A, Bonucci E: The compressive properties of single osteons. Anat Rec. 1968, 161: 377-391. 10.1002/ar.1091610309.PubMedView ArticleGoogle Scholar
- Laurin M, Girondot M, Loth M-M: The evolution of long bone microstructure and lifestyle in lissamphibians. Paleobiology. 2004, 30: 589-613. 10.1666/0094-8373(2004)030<0589:TEOLBM>2.0.CO;2.View ArticleGoogle Scholar
- Hall BK: Bones and cartilage: Developmental and evolutionary skeletal biology. 2005, Oxford: Elsevier Ltd, 83-90.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.