Biomechanical adaptations for burrowing in the incisor enamel microstructure of Geomyidae and Heteromyidae (Rodentia: Geomyoidea)

Abstract The enamel microstructure of fossil and extant Geomyoidea (Geomyidae, Heteromyidae) lower incisors incorporates three‐ or two‐layered schmelzmusters with uniserial, transverse Hunter‐Schreger bands having parallel and perpendicular or exclusively perpendicular oriented interprismatic matrix. Phylogenetically, these schmelzmusters are regarded as moderately (enamel type 2) to highly derived (enamel type 3). Our analysis detected a zone of modified radial enamel close to the enamel–dentine junction. Modified radial enamel shows a strong phylogenetic signal within the clade Geomorpha as it is restricted to fossil and extant Geomyoidea and absent in Heliscomyidae, Florentiamyidae, and Eomyidae. This character dates back to at least the early Oligocene (early Arikareean, 29 Ma), where it occurs in entoptychine gophers. We contend that this specialized incisor enamel architecture developed as a biomechanical adaptation to regular burrowing activities including chisel‐tooth digging and a fiber‐rich diet and was probably present in the common ancestor of the clade. We regard the occurrence of modified radial enamel in lower incisors of scratch‐digging Geomyidae and Heteromyidae as the retention of a plesiomorphic character that is selectively neutral. The shared occurrence of modified radial enamel is a strong, genetically anchored argument for the close phylogenetic relationship of Geomyidae and Heteromyidae on the dental microstructure level.

Modern geomyoids are endemic to North, Central, and northern South America, and their fossil record dates back to the early Oligocene (Orellan, 33 Ma) (Korth, 1993).Geomyoids and related geomorphs are well represented in Cenozoic faunas across North America, and in the Oligocene and Miocene epochs, they were diverse and radiated into several extinct lineages, such as Heliscomyidae, Florentiamyidae, entoptychine Geomyidae, and Mioheteromyinae, particularly in the Great Plains and adjacent mountain regions (Asher et al., 2019;Flynn et al., 2008).Most authors suggest Geomyidae and Heteromyidae as sister group to the extinct basal geomorphs Heliscomyidae and Florentiamyidae and phylogenetically related to the extinct Eomyidae (Asher et al., 2019;Fahlbusch, 1985;Flynn, 2008;Flynn et al., 2008;Jiménez-Hidalgo et al., 2018;Korth, 1994;McKenna & Bell, 1997).
Geomyidae (pocket gophers) are small-to medium-bodied rodents with anatomies highly adapted to a subterranean-burrowing (fossorial) lifestyle in open habitats.They possess a wedge-shaped, massive skull with a broad, forward-sloping occipital surface, heavy muscle attachments, and protruding lower incisors that are used as chisels for tunneling by representatives of Thomomyini ( MS Hafner, 2016;Wahlert, 1985) (Figure 1).The lips enclose the posterior side of the incisors, preventing the ingestion of sediment while tooth-digging.
Strong and long claws on the front legs are used for shoveling, especially in the scratch-digging Geomyini ( MS Hafner, 2016).The earliest fossil record of Geomyidae is entoptychine gophers from the early Oligocene (Arikareean) (Flynn et al., 2008).Geomyid findings from the early Arikareean (early Oligocene) in the Pacific Northwest and Northern Rocky Mountains (Calede & Rasmussen, 2020;Samuels & Hopkins, 2017) point to a broad distribution of geomyids early in their history.Full exploitation of the subterranean niche in geomyids dates back to at least the early Oligocene (early Arikareean, Ar1), when the entoptychine gopher Gregorymys veloxikua created toothexcavated foraging burrows in southern Mexico (Jiménez-Hidalgo et al., 2018;Ortiz-Caballero et al., 2020).Nevertheless, there likely was more paleoecological diversity in early members of the clade (other than Gregorymys) with some taxa possibly semi-fossorial (not subterranean), others more fossorial or, in contrast, adapted to a terrestrial lifestyle (Calede et al., 2019).It should be mentioned that Asher et al. (2019) placed Gregorymys outside Geomyoidea, a view that our study does not support.
Lower incisor schmelzmuster and incisor morphology of North American geomorph rodents have not been studied in detail previously.Only a few random fossil and extant taxa were analyzed by Wahlert (1968) and Kalthoff (2000).

| MATERIAL S AND ME THODS
Enamel microstructure analysis is a powerful tool for answering systematic-phylogenetic questions at the genus and higher taxonomic levels.Enamel formation is controlled by genetic and epigenetic factors; as a consequence, few samples of each taxonomic subgroup are sufficient to characterize its main schmelzmuster features.
Here, we describe the schmelzmuster of lower incisors of ten species of Geomyidae, ten species of Heteromyidae, and one or two species of Heliscomyidae.The sample from geomyoids covers taxa from the late Oligocene/Early Miocene (Arikareean) to Recent, the two heliscomyid samples come from the late Eocene (Chadronian) and early Oligocene (Orellan) (Appendix S1).
In geomyines, the enamel microstructure is similar in upper and lower incisors.However, as in many rodent clades, upper and lower incisors in Dipodomyinae and Perognathinae have different schmelzmuster with the lower incisor being more apomorphic (Kalthoff, 2000).Only lower incisors were available for sectioning for Entoptychinae, Mioheteromyinae, Heteromyinae, and Heliscomyidae.For reasons of comparability, we chose lower incisors for this study.Schmelzmuster type denotations follow Kalthoff (2000), and character polarity for microstructure characters follows Martin (1999).Enamel thickness categories are as follows: less than 70 µm relate to thin; between 71 and 90 µm relate to moderate; greater than 90 µm relate to thick; and greater than 140 µm relate to very thick.
Preparation for enamel microstructure analysis follows Kalthoff (2000) and Koenigswald and Mörs (2001).Transverse and longitudinal sections were studied and documented using a scanning electron microscope (SEM) (Quanta FEG 650,located

| RE SULTS
All taxa feature uniserial Hunter-Schreger bands (HSB).Table 1 summarizes the results and measurements; Appendix S2 gives the raw result data per specimen.

Heteromyidae: Mioheteromyinae
Schizodontomys sulcidens (Figures 3q and 4b ordii, California).The PE consists of radial enamel that in its outermost portion merges into primitive radial enamel.The incisor cross section is oval-shaped and the middle labial part of the enamel is flattened.

Heteromyidae: Heteromyinae
Heteromys anomalus (Figure 3p), extant, KOE 4231.Heteromys anomalus has a two-layered schmelzmuster with mostly transversely oriented HSB and moderate inclination; mesially HSB become diagonal (schmelzmuster type 3a).The thick PI shows IPM at right angles but the angle is markedly reduced over 2 to 3 prisms at the junction PI/PE.At the EDJ, the IPM is thickened and plate-like in a zone measuring 3 to 4 prisms.The PE is made up of radial enamel.
The incisor cross section is pear-shaped with rounded enamel.

Heteromyidae: Perognathinae
Perognathus mclaughlini (Figures 3r and 4a), Perognathus rexroadensis (Figure 3s The PE is made up of radial enamel and a thin PLEX at the OES.The incisor cross section is oval-shaped and the middle labial part of the enamel is flattened.

| D ISCUSS I ON
The enamel microstructure is comparatively homogeneous in Geomyidae with respect to schmelzmuster and enamel thickness (Table 1).Entoptychinae (Pleurolicus, Entoptychus, Gregorymys) and Geomyinae (Pliogeomys, Geomys, Cratogeomys, Thomomys) have a three-layered schmelzmuster with transversely and, in part, diagonally oriented HSB.The PI is twofold with IPM at different, mostly high angles to the prism long axes in a thick IPI and prism-parallel IPM in a thin OPI; the PE consists of radial enamel.Enamel is moderate to greatly thick in both Entoptychinae and Geomyinae.From an evolutionary perspective, this schmelzmuster is moderately derived and represents types 2 and 2a of Kalthoff (2000).The incisor cross section is triangular or subtriangular with characteristically flattened enamel in all analyzed taxa (Figure 3a-k).
Compared to Geomyidae, the enamel microstructure in Heteromyidae is almost as homogeneous but is more de- Nesomyinae, Otomyinae, Sigmodontinae, and Trilophomys (Kalthoff, 2000).Schmelzmuster types 2 and 3 evolved recurrently in rodent incisors and, therefore, are homoplastic structures conveying limited phylogenetic value.
A remarkable structure occurs at the EDJ in both Geomyidae and Heteromyidae: thickened IPM fibers form plates between prism rows in a zone of 3-4 prism thickness; the plate-like IPM is perpendicular to the prism long axes and does not anastomose.The IPM plates relate to the inter-row sheets of Boyde (1969); however, the sheets are at most half as thick as a prism, that is, 1-1.5 µm.The inter-row sheets induce a strong reduction of the HSB decussation angle, forcing them to arrange in nearly or fully parallel orientations.
The inter-row sheets were incorrectly identified as a "starting zone" by Kalthoff (2000).
Combined, these characters (IPM developed as inter-row sheets, direction of IPM inter-row sheet fibers perpendicular to prism direction, subparallel prisms in radial rows) define the modified radial enamel of Pfretzschner (1993Pfretzschner ( , 1994)).In high-crowned teeth of large mammals (and in rodent incisors as variety of hypsodont teeth) prisms steeply ascend toward the occlusal surface as a reaction against increased abrasion (Rensberger & Koenigswald, 1980).In addition, special microstructures may occur adjacent to the EDJ as a reaction to tension forces (Pfretzschner, 1993(Pfretzschner, , 1994)), all showing decussation of linear elements in a radial-vertical direction.In modified radial enamel, these elements are the (sub)parallel prisms and the interjacent inter-row sheets.
We detected modified radial enamel close to the EDJ exclusively within Geomyoidea but not in Geomorpha outgroups, such as Heliscomyidae; nor was modified radial enamel reported to occur in the lower incisors of Eomyidae (Wahlert & Koenigswald, 1985) or Florentiamyidae (Wahlert, 1983).As this enamel type was found to be biomechanically beneficial for reducing tension and bending forces in high-crowned teeth, we assume a similar form-function association in Geomyidae and Heteromyidae (Pfretzschner, 1993(Pfretzschner, , 1994;;Vassallo et al., 2021).Consequently, we explain the presence of modified radial enamel as an adaptation to prevent structural failure triggered by increased mechanical stress acting on this tooth position due to regular burrowing activities (including chisel-tooth digging) and feeding on abrasive, fiber-rich plants and plant parts that grow underground (e.g., forbs, roots, stems, bulbs, tubers).
In general, the lower incisors are the more active gnawing teeth in rodents compared to the upper pair, a fact that has been quantified by several studies: A recent experimental analysis on the kinematics of chisel-tooth digging by African mole rats showed that the upper incisors are used as an anchor while the lower incisor excavates the soil (Van Wassenbergh et al., 2017).Judging from the displacement of the respective incisors during excavation, the work-and with that, concomitant compressive stress-of the lower incisors is three times greater than that of the upper pair (Van Wassenbergh et al., 2017).Large differences in yearly growth rates between upper and lower incisors in the chisel-tooth digging pocket gophers  Flynn et al., 2008;Joeckel & Tucker, 2013), rely on their strong and long claws for digging and they use incisors to remove rocks and cut roots (Lessa & Thaeler, 1989).In contrast, chisel-tooth diggers, such as the markedly proodont Thomomys, use their broad, triangular incisors to loosen soil to build their extended burrow systems (Jones & Baxter, 2004).Interestingly, fossoriality was established early in geomyid history as remains of early Oligocene (early Arikareean, Ar1) Gregorymys were found inside a burrow system (Jiménez-Hidalgo et al., 2018;Ortiz-Caballero et al., 2020).The burrows show incisor marks but lack claw marks, contrary to early Miocene (Arikareean) presumed Gregorymys burrows that show a combination of incisor and claw marks (Gobetz & Martin, 2006).However, there is contradictory evidence regarding chisel-tooth digging in entoptychine gophers: the skull of Gregorymys does not display the necessary adaptations for this burrowing mode (Calede et al., 2019) whereas skulls and postcranial material of Pleurolicus and Entoptychus show specializations for fossoriality and are in that respect most similar to extant tooth-digging species (Calede et al., 2019;Rensberger, 1971Rensberger, , 1973)).
Geomyids and heteromyids are sister taxa, sharing a common, yet unknown, ancestor.The presence of modified radial enamel close to the EDJ in both clades throughout all taxa suggests acquisition of this character in the ancestor, which we assume had a fossorial lifestyle and employed tooth-digging as the primary excavation mode.We interpret the presence of modified radial enamel as biomechanically advantageous for geomyids engaged in tooth-digging and/or using their large, triangular and flattened lower incisors for dirt and root removal during scratch-digging.Although heteromyids self-construct burrows, use of their incisors as digging tools or aids has never been reported; moreover their skull architecture and incisor shape argue against such behavior (Calede et al., 2019;McIntosh & Cox, 2016).We regard lower incisor modified radial enamel in Heteromyidae as a retained plesiomorphic character that, apparently, is selectively neutral.
Modified radial enamel might still be biomechanically advantageous for dietary specialists living in desert conditions, such as the heteromyid Dipodomys microps.This species has flat, chisel-shaped lower incisors, with which it peels the salt-coated outer leaf layers of halophytic plants (Kenagy, 1972) to reach the inner, soft tissues; a behavior that might impose certain stresses on incisors.
On the other hand, modified radial enamel also occurs in the lower incisors of a few other muroid taxa with uniserial Hunter-Schreger bands (Kalthoff, 2000).These are (a) the Congo Forest Rat Deomys ferrugineus (Deomyinae), a terrestrial species with no burrowing activities (Ray & Malcolm, 2013); (b) Milne-Edwards's Tufted-tail Rat Eliurus myoxinus (Nesomyinae), which is a scansorial species dependent on forest environments (Goodman, 2016); (c) Bastard's Big-footed Mouse Macrotarsomys bastardi (Nesomyinae), which has terrestrial adaptations but uses self-constructed burrows (Carleton & Goodman, 2003); and (d) the Southern African Pouched Mouse Saccostomus campestris (Cricetomyinae), showing terrestrial and scansorial adaptations but also short legs and strong toes, well-adapted to digging (Perrin, 2013).At present, we cannot explain the occurrence and obviously parallel evolution of modified radial enamel in these species.No underlying phylogenetic signal seems to be present and, regarding biomechanics, it remains to be tested whether modified radial enamel can be linked to specialized foraging behavior like, for example, insectivory in Deomys ferrugineus.
In the light of the above reported evidence of modified radial enamel in some taxa unrelated to Geomyoidea and among each other, a reviewer suggested that this enamel trait might as well be homoplastic in Geomyidae and Heteromyidae.However, we disagree because modified radial enamel is a character consistently present in both families.This is a clear difference to the above examples, where this character occurs isolated.
Our motivation for this study is a serendipitous discovery of a conspicuous microstructure (i.e., modified radial enamel) in the lower incisors of Thomomys, Chaetodipus, and Dipodomys.Here, we intend to (a) describe the schmelzmuster and lower incisor morphology in a representative sample of fossil and extant Geomyoidea (Geomyidae, Heteromyidae), (b) document the occurrence of modified radial enamel, (c) discuss its assumed biomechanical and higher F I G U R E 1 The chisel-tooth digging Botta's Pocket Gopher Thomomys bottae (Eydoux and P. Gervais, 1836) emerging from its tunnel.Photo credit: Chuck Abbe, Nine Sisters Photography, Wikimedia Commons (CC BY 2.0) level phylogenetic implications, and (d) compare these enamel microstructure results with examples in basal geomorphs (Heliscomyidae).

Heliscomyidae
Heliscomys sp.(Figures 3w and 5a), Early Oligocene: Orellan, KOE 3466; Heliscomys vetus (Figures3x and 5b), Late Eocene: Chadronian, KOE 3528.Heliscomys sp. and Heliscomys vetus have a three-layered schmelzmuster (schmelzmuster type 2a), in which HSB are oriented transversely to diagonally with a high inclination.The IPM in the IPI is perpendicular to the prisms, this angle decreases markedly in the thin OPI, which measures only 1-2 prisms.The PE consists of radial enamel.The incisor cross section is slender and oval-shaped and the middle labial part of the enamel is flattened.
rived.Perognathinae (Perognathus, Chaetodipus), and the extant Dipodomyinae (Dipodomys) and Heteromyinae (Heteromys) have schmelzmuster type 3 with transversely oriented HSB and angled IPM in an only onefold PI and radial enamel in the PE.The Miocene (Barstovian) dipodomyine Cupidinimus and the Miocene (Arikareean) mioheteromyine Schizodontomys feature a more plesiomorphic, three-layered schmelzmuster of type 2, similar to that described for geomyids.The thickness of the enamel varies somewhat from very thick in Schizodontomys and Heteromys to moderate/thick in perognathines, and from moderate/thick in Miocene dipodomyines to thick in extant dipodomyines.The incisor cross section is oval with a flattened middle labial part of the enamel with the exception of Schizodontomys, Heteromys, Perognathus rexroadensis, and P. mclaughlini, which all have rounded enamel (Figure 3p-s).The schmelzmuster types 2 and 3 occurring in Geomyidae and Heteromyidae, respectively, are represented in the lower incisors of various other fossil and extant rodent clades with uniserial HSB: the more uncommon type 2 in members of Deomyinae, Murinae, and Neotominae; the very common type 3 in Arvicolinae, Cricetinae, Dendromurinae, Gerbillinae, Murinae, Myocricetodontinae,

at the Swedish Museum of Natural History in Stockholm), at acceleration voltages of 15- 20 kV and magnifications of x 30 to × 5,000. All measurements are given
in µm and were carried out on transverse sections.

IPI, inner portio interna; IPM, inter- prismatic matrix; MRE, modified radial enamel; OPI, outer portio TA B L E 1
Summary table of measurement ranges, schmelzmuster type, stratigraphic age, and taxonomic information for the analyzed

taxa Taxa No of species Schmelzmuster type Thickness of E (µm) PI or IPI (µm) % OPI (µm) if present % PE (µm) % Modified radial enamel
At the bend to mesial and lateral, the enamel thickens somewhat.In the thick IPI, the IPM is perpendicular to the HSB, the latter decussating at a high angle; the OPI with prism-parallel IPM is 4 to 5 prisms thick.At the EDJ, a zone measuring about 3 prisms shows thick plate-like IPM forcing the HSB to decussate at a very low angle.The thin OPI shows prism-parallel IPM over a thickness of 2 to 3 prisms.The PE has radial enamel.The entire incisor cross section could not be evaluated; the labial part of the enamel is less flat than in Gregorymys and Entoptychus.Scanning electron micrographs of lower incisor enamel microstructure in Geomyidae.(a) Thomomys talpoides, transverse section, KOE 650.Arrows point to thickened IPM.(b) Geomys quinni, longitudinal section, KOE 3248.Arrows point to plate-like IPM.(c) Geomys sp., transverse section at bend toward lateral, KOE 3511.Modified radial enamel extends over two to three prism rows.
Geomyidae: EntoptychinaeGregorymys cf.curtus (Figures2f and 3h), Early Miocene: latest Arikareean, KOE 3256.This taxon has a three-layered schmelzmuster (schmelzmuster type 2) with continuous transversely oriented HSB showing a steep inclination.In the IPI, HSB decussate at high acute angles and the IPM is perpendicular to the prism long axes.In the thin OPI, the IPM turns into a very low angled to parallel direction in respect to the prisms.At the EDJ is a 2-3 prism thick zone, in which the HSB decussate at a low angle forced by the plate-like IPM in between them.The PE is made up of radial enamel.The incisor cross section is triangular-shaped with labially flat enamel.Entoptychus sp.(Figures 2g and 3g), Early Miocene: late Arikareean, KOE 3244.Entoptychus sp. has a three-layered schmelzmuster with transversely oriented HSB showing moderate to steep inclination; HSB are diagonally oriented at the mesial and lateral ends of the enamel (schmelzmuster type 2a).The enamel thickens somewhat at the bend to mesial and lateral.The PI is made up of a thick IPI, in which HSB decussate at a generally high angle except for a 1-2 prism thin zone near the EDJ where the decussation angle is low because of thick plate-like IPM.In the equally thin OPI (1-2 prisms thick), IPM runs parallel to the prism long axes.The PE has radial enamel.The incisor cross section is subtriangular in shape with a rounded dentine body and labially flat enamel.Pleurolicus sp.(Figures 2h and 3i), Late Oligocene/Early Miocene: Arikareean, KOE 3257.Pleurolicus sp.shows a three-layered schmelzmuster (schmelzmuster type 2) with transversal HSB having a moderate to steep inclination.haveaverylowdecussationanglebecause of thick, plate-like IPM.The plate-like character of the IPM gets pronounced at the turn of the enamel toward lateral.The IPM is at right angles in the IPI and prism-parallel in the thin, 1-2 prism thick OPI.The PE shows radial enamel.The incisor cross section is triangular-shaped with labially flat enamel.cf.Geomys quinni (Figures 2b and 3b), Early Pliocene: early Blancan, KOE 3248.cf.Geomys quinni shows a three-layered schmelzmuster (schmelzmuster type 2) with transversely oriented HSB throughout the entire enamel; the HSB are steeply inclined.The IPI shows HSB with perpendicular IPM, and the OPI measures only 3-4 prisms and has prism-parallel IPM.There is a conspicuous, 3-4 prism thick zone at the EDJ consisting of thick, plate-like IPM allowing HSB to decussate only at a very low angle.The HSB decussation angle gets larger toward the PI/PE junction but seems not to be fully perpendicular.The PE consists of radial enamel.The incisor cross section is triangular-shaped with labially flat enamel.Geomys bursarius (Figure3d), Holocene, KOE 3275; Geomys bursarius (d) Thomomys bottae, detail of longitudinal section, KOE 3283.Modified radial enamel with plate-like IPM is well expressed.(e)Cratogeomyscastanops,transversesection,KOE3284.Obvious modified radial enamel with plate-like IPM, extending over about five prism rows.(f)Gregorymyscf.curtus,longitudinalsection,KOE3256.Modified radial enamel is already present in this entoptychine gopher genus, which is the first to appear in the early Oligocene in North America.(g)Entoptychussp., transverse section, KOE 3244.Arrows point to thickened IPM.(h) Pleurolicus sp., longitudinal section, KOE 3257.Modified radial enamel is rather thin in this genus.Abbreviations: EDJ, enameldentine junction; IPI, inner portio interna; IPM, interprismatic matrix; MRE, modified radial enamel; OPI, outer portio interna; OES, outer enamel surface; PE, portio externa section.Also, HSB layers decussate at varying angles: angles closer to the EDJ are about 45 degrees and rise closer to 90 degrees, never being fully perpendicular.There is a conspicuous zone at the EDJ, where the IPM is thick and plate-like forcing the first four HSB layers to decussate at a very low angle.Toward the lateral parts of enamel; the HSB are steeply inclined.The PI is twofold having an IPI, in which IPM is perpendicular to the prisms and a 1-3 prism thick OPI, in which IPM is at low angle or parallel to the prisms.Right at the EDJ is a four prism thick zone with thick, plate-like IPM, forcing the decussation angle of the HSB to about zero.Toward the junction PI/PE, the HSB decussation angle rises reaching 90 degrees.The PE consists of radial enamel.The OES is wrinkled causing a wavy OES as seen in transverse sections.The incisor cross section is subtriangular in shape with a rounded dentine body and labially flat enamel.Pliogeomys buisi (Figure3a), Late Miocene/Early Pliocene: Late Thomomys bottae (Figures2d and 3j), Late Pleistocene (ca 20,000 BP), KOE 3283; Thomomys talpoides (Figures2a and 3k), extant, KOE 650 (figured inKalthoff, 2000: fig.40: a1).Both species of the genus Thomomys feature a three-layered schmelzmuster (schmelzmuster type 2a) with transversely to slightly diagonally oriented HSB with steep inclination.The PI is twofold with a thick IPI with perpendicular