Morphologies of Murinae species

The features of the glans penis and baculum are summarized in Table 1. All examined Murinae species (A. speciosus, A. argenteus, M. musculus, M. caroli, M. minutus, and R. norvegicus) had a medial bacular mound and lateral bacular mounds that together formed a trident structure (bacular mounds) (Fig. 2A–F). In the Murinae species, the stalk was stained by Alizarin Red S, while the medial and lateral bacular mounds were not stained.

Table 1.

Measurements (mm) and features of the glans penis and baculum in six Murinae species and five Arvicolinae species

Apodemus speciosus: The medial and lateral bacular mounds were developed and protruded slightly outside of the outer skin of the glans penis (Fig. 2A). A dorsal papilla was present, and the tip of the urethral process was divided into three forks (Table 1); the medial fork was larger than the outer two forks. Two grooves were present on the dorsal part of the outer skin (Fig. 1A). The medial distal segment of the cartilage was rod-like and tapered slightly toward the distal part.

Apodemus argenteus: The medial and lateral bacular mounds were developed but protruded only slightly from the outer skin of the glans penis (Fig. 2B). A dorsal papilla was present, and the tip of the urethral process was divided into three forks (Table 1); the medial fork was as large as the outer two forks. No groove was present on the dorsal part of the outer skin. The stalk of the baculum was wider than in A. speciosus of a similar body size. The medial distal segment was thin and short, and its junction with the stalk was rounded.

Mus musculus: The medial bacular mounds were developed, whereas the lateral bacular mounds were buried in the surrounding tissue and only the tips were exposed (Fig. 2C). The tip of the medial bacular mound was divided into two small parts and the tip of the urethral process was divided into two parts (Table 1). One shallow groove was present on the ventral surface of the outer skin. The stalk was thin at the tip and widened gradually as it approached the base, and the medial distal segment was short, even though the medial bacular mound protruded to the outside.

Mus caroli: The medial bacular mounds were developed, whereas the lateral bacular mounds were buried in the surrounding tissue and only the tips were exposed (Fig. 2D). The tip of the medial bacular mound was divided into two small parts and the tip of the urethral process was divided into two parts (Table 1). There were two pairs large rim papillae and some small rim papillae on the outer crater. One shallow groove was present on the ventral surface of the outer skin. The stalk was thin at the tip and widened gradually as it approached the base.

Micromys minutus: The medial bacular mounds were well developed, whereas the lateral bacular mounds were buried in the surrounding tissue and only the tips were exposed (Fig. 2E). The tip of the medial bacular mound was not divided and the tip of the urethral process was divided into two parts (Table 1). The ventral side of the outer skin had one deep groove. The base of the stalk was broken, so we do not know the details of its morphology, but it exhibited little widening at the base. The medial distal segment was exceptionally long, in contrast to those of M. musculus and M. caroli.

Fig. 2.

Views of the internal features of the distal end of the glans penis (upper row) and ventral views of the stalk and bacular mound (lower row) of Murinae (A–F) and Arvicolinae (G–K) species. (A) Apodemus speciosus (TOY-214), (B) A. argenteus (TOY-215), (C) Mus musculus (TOY-224), (D) M. caroli (M2272), (E) Micromys minutus (M12843), (F) Rattus norvegicus (TOY-309), (G) Alexandromys montebelli (M12810), (H) Myodes rutilus (TOY-292), (I) Craseomys rufocanus (TOY-121), (J) C. smithii (M13298), and (K) C. andersoni (M11486). Blue and green arrows represent lateral and medial bacular mounds, respectively. The base of the M. minutus stalk was broken (E). Scale bars represent 1 mm.

Rattus norvegicus (juvenile): The tip of the urethral process was divided into two parts and the presence of a dorsal papilla was not confirmed (Table 1). Most of the trident structure was encased inside the outer skin of the glans penis and was not prominent (Fig. 2F). There was no groove in the center of the ventral surface, but a pair of shallow grooves was present on the lateral side. The base of the stalk was not very wide; the medial distal segment was deep and thick.

Morphologies of Arvicolinae

All Arvicolinae species (A. montebelli, M. rutilus, C. rufocanus, C. andersoni, and C. smithii) had a developed medial bacular mound and lateral bacular mounds that formed a trident structure (Fig. 2G–K). In the Arvicolinae species, the stalk, as well as the medial and lateral distal segments of the bacular mounds, were stained by Alizarin Red S. The medial bacular mound and stalk were not connected by bone, but by cartilage. Only the proximal part of the medial and lateral bacular mounds was associated with the surrounding tissue; the other parts were independent and outward protrusion was confirmed. The urethral process was divided into three parts consisting of a small medial part and two lateral parts, and each part had smaller projections (Table 1). There was a groove on the ventral surface of the outer skin that was identified as common to all five species. Stalks also had similar shapes in the five Arvicolinae species, and the width of the base of the stalk was wider than in the Murinae species (ranges of SBW/STL: 0.24–0.48 in Murinae and 0.45–0.59 in Arvicolinae). Myodes rutilus had a specific morphology of the lateral distal segments in their angle and shallower tip as seen from the lateral view, distinct from other Arvicolinae species (Fig. 2H).

Fig. 3.

Penis sections of Apodemus speciosus. Photographs of whole penis sections (A-a, B-a, C-a, and D-a), and enlarged views of the regions in the rectangles in A-a, B-a, C-a, and D-a (A-b, B-b, C-b, and D-b). mb, medial bacular mounds consisting of fibrocartilage; lb, lateral bacular mounds; up, urethral process; dp, dorsal papilla; cc, corpus cavernosum; st, stalk. Blue and green arrowheads indicate cavernous spaces and veins, respectively. Scale bars represent 0.5 mm. Diagram of the locations of four A. speciosus penis sections (lower panel). A, distal part of the lateral bacular mounds; B, central part of the lateral bacular mounds; C, region connecting the medial and lateral bacular mounds and the stalk; and D, central part of the stalk.

Analysis of A. speciosus tissue sections

Medial and lateral bacular mounds were typically found in sexually mature adults as well as in juveniles. The medial distal segment of the medial bacular mound (mb) was composed of fibrocartilage (Fig. 3A-a, B-a). Lateral bacular mounds (lb) were composed of corpus spongiosum, with cavernous spaces in the middle of the distal part of the lateral bacular mounds (Fig. 3A-b). The more proximal part of the lateral bacular mounds was occupied by cavernous spaces throughout (Fig. 3B-b). The development of cavernous spaces in the lateral bacular mounds was also observed in juveniles. The lateral bacular mounds had multiple vessels along the cavernous spaces (Fig. 3C-b). The lateral bacular mounds did not have skeletal muscle tissue (Fig. 3A-b, B-b). The corpus cavernosum (cc) along the stalk (st) was found to have blood vessels extending from the lateral bacular mounds (Fig. 3D-b). The urethral process (up) and part of the dorsal papilla (dp) which composed of corpus spongiosum also had cavernous spaces (Fig. 3A-a, B-a). By contrast, the medial bacular mound did not have cavernous spaces (Fig. 3A-a, B-a). The medial and lateral bacular mounds were connected to each other on the dorsal plane, and the back was also connected to other surrounding tissues (Fig. 3A-a, B-a). There were epidermis continuous with the outer skin of the glans penis and distal tip outside of the medial and lateral bacular mounds. The lateral bacular mounds, the urethral process, and the dorsal papilla, were mostly composed of corpus spongiosum, and were surrounded with other tissues; thus, those corpora spongiosa were not connected to each other and existed independently. The stalk was mostly composed of lamellar bone and proximal end of stalk was composed of hyaline cartilage.

Fig. 4.

Penis sections of Craseomys rufocanus. Photographs of whole penis sections (A-a, B-a, C-a, and D-a), and enlarged views of the regions in the rectangles in A-a, B-a, C-a, and D-a (A-b, B-b, C-b, and D-b). mb, medial bacular mounds consisting of fibrocartilage; lb, lateral bacular mounds; up, urethral process; dp, dorsal papilla; cc, corpus cavernosum; st, stalk. Blue and green arrowheads indicate cavernous spaces and veins, respectively. Scale bars represent 0.5 mm. Diagram of the locations of four C. rufocanus penis sections (lower panel). A, distal part of the lateral bacular mounds; B, central part of the lateral bacular mounds; C, region connecting the medial and lateral bacular mounds and the stalk; and D, central part of the stalk.

Analysis of C. rufocanus tissue sections

Medial and lateral bacular mounds were typically found in the sexually mature adults as well as the juveniles. The medial and lateral distal segments of the medial bacular mound (mb) and lateral bacular mounds (lb) were composed of bones (Fig. 4A-a, B-a). The lateral bacular mounds consisted of bone on the outside and soft tissue consisting of cavernous space in both the distal region (Fig. 4A-b) and inside (Fig. 4B-b). Blood vessels were observed in soft tissue (Fig. 4B-b, C-b) and skeletal muscle tissue was not present. The corpus cavernosum (cc) along the stalk (st) contained blood vessels extending from the lateral bacular mounds (Fig. 4D-b). The urethral process (up) and the dorsal papilla (dp) also contained cavernous spaces (Fig. 4B-a, C-a). The dorsal papilla was large and similar in size to cross sections of the medial or lateral bacular mounds (Fig. 4B-a). By contrast, the medial bacular mound did not contain cavernous spaces (Fig. 4A-a, B-a). The lateral bacular mounds, the urethral process, and the dorsal papilla, were mostly composed of corpus spongiosum, and were surrounded with other tissues; thus, those corpora spongiosa were not connected to each other and existed independently.

Differences in the baculum and glans penis among species

This is the first study that compares both the external and internal morphologies of the male genitalia of Japanese Murinae and Arvicolinae. Medial and lateral bacular mounds were identified in all of the species examined in seven genera of both subfamilies, indicating the common presence of a trident structure (Fig. 2). In M. caroli, medial bacular mounds were present as a protrusion from the foreskin (Tokuda 1934), while the presence of lateral bacular mounds was confirmed for the first time in this study. The medial bacular mound protruded from the glans penis and was highly developed while the lateral bacular mounds were degenerate in M. musculus, M. caroli, and M. minutus (Fig. 2). Yang and Fang (1988) reported the presence of lateral bacular mounds in M. musculus and M. minutus, but it is not clear from their figures whether the medial bacular mound is projecting. Rodriguez et al. (2011) reported the presence of MCC (MUMP corpus cavernosum) in the glans penis in laboratory mice, and this was thought to correspond to the lateral bacular mounds in the present study.

In this study, all Murinae species had medial distal segments that consisted of cartilage and lateral bacular mounds that consisted of soft tissue occupied by the corpus spongiosum (Fig. 2A–F). In the Murinae, two types of medial distal segments have been reported: those were ossified and cartilaginous segments (Yang and Fang 1988). The medial distal segment has been reported to ossify in some rat species including R. norvegicus and two Niviventer species (Yang and Fang 1988; Yonezawa et al. 2011). In laboratory rats, the medial distal segment does not ossify in young individuals up to day 40, but ossifies later as they grow (Yonezawa et al. 2011). The specimen of R. norvegicus used in this study was still juvenile (BW, 25.5 g), and the medial distal segment may not have ossified yet and was still present as cartilage. Therefore, it is suggested that the medial distal segment does not ossify in small Murinae, but is thought to ossify concurrently with growth in large Murinae such as Rattus and Niviventer. Yang and Fang (1988) described cartilage inside the lateral bacular mounds, but histological studies of rats (Murakami 1987; Kelly 2000) and the present study suggest that the lateral bacular mounds do not consist of cartilage, but instead are soft tissue consisting of corpus spongiosum.

Large interspecific differences were found in the medial and lateral bacular mounds of the glans penis in Murinae, but differences were also found in other areas. The glans penis of the genus Apodemus is distinguished by the presence of a trigeminal urethral process in contrast with bifurcated processes in other genera; the tip of the medial distal segment of genus Mus was bifurcated and the tips of other genera were not bifurcated; the glans penis of genus Micromys differed in that the medial distal segment was exceptionally long; and the glans penis of genus Rattus also resembled Apodemus in its juveniles but was distinguishable by bifurcation of the urethral process and the absence of dorsal papilla (Fig. 2 and Table 1). Consistent with Tokuda (1934), who advocated the importance of penile morphology as a diagnostic taxonomic character, the penile morphology of each species was distinct and diverse, possibly related to and dependent on genus-specific reproductive strategies.

Fig. 5.

Muroidea subfamilies with confirmed trident structures indicated on the molecular phylogenetic tree of Steppan and Schenk (2017). Black circles indicate subfamilies in which the trident structure was confirmed. White circle indicates the one subfamily in which the trident structure was not confirmed. Black triangle indicates a subfamily where dorsal lobe and lateral lobes that are suspected to be homologous to the medial bacular mound and the lateral bacular mounds of the trident structure were confirmed. There have been no studies on the internal penis structures of the remaining taxa.

On the other hand, both the medial and lateral distal segments were ossified in all five species of Arvicolinae examined in this study (Fig. 2G–K). In Arivicolinae, various types of ossification have been reported: distal segments entirely ossified and others entirely cartilaginous, an ossified medial distal segment and lateral bacular mounds that are soft tissue, an ossified medial distal segment and lateral distal segments that are cartilaginous, and a cartilaginous medial distal segment and lateral bacular mounds that are soft tissue (Hooper and Hart 1962). In addition, the taper of lateral distal segments of M. rutilus differed from that of the other species so that the distal part of the lateral distal segments appeared wider in this study (Fig. 2H).

Medial and lateral bacular mounds that form a trident structure have been identified in many subfamilies of Muridae and Cricetidae including Deomynae (Kivanc et al. 1997), Gerbilinae (Yiğit et al. 1997), and Murinae (Yang and Fang 1988; this study) in the Muridae; and Sigmodontinae (Hooper 1962; Hooper and Musser 1964; Bezerra 2005), Arvicolinae (Hooper and Hart 1962; this study), Tylomyinae (Hooper 1960; Hooper and Musser 1964), and Cricetinae (Callery 1951) in the Cricetidae (Fig. 5). By contrast, no trident structure has been reported in Neotominae (Hooper 1958; Hooper 1960) in the Cricetidae. The presence or absence of a trident structure in Lophiomyinae of the Muridae is unknown due to a lack of study. In reference to phylogenetic relationship of Muroidea (Steppan and Schenk 2017), trident structure is suggested to have evolved before the split between Muridae and Cricetidae with secondary loss in Neotominae of Cricetidae (Fig. 5). Dorsal lobe and lateral lobes have been identified in Spalacinae of the Spalacidae (Kankiliç et al. 2009). If these structures in Spalacinae are homologous to medial and lateral bacular mounds in Muridae and Cricetidae, acquisition of the trident structure is suggested to have occurred in earlier stage of Muroidea diversification. Presence or absence of the trident structures in species of Nesomyidae, Calomyscidae, Platacanthomyidae, and Spalacidae should be examined to reconstruct the evolution of the trident structure beyond the stalk in the Muroidea. Further study using bone staining and tissue sections is required to cover Muroidea species.

Model of the movement mechanism of the lateral bacular mounds

Kelly (2000) proposed that the baculum increases the overall flexural stiffness of the penis during copulation by transferring bending and compressive forces from the distal end of the glans to the tensile wall of the corpus cavernosum. In this study, the presence of cavernous spaces and blood vessels were confirmed in the lateral bacular mounds in both A. speciosus and C. rufocanus. Blood flowing through the vessels of the corpus cavernosum is suggested to enter the vessels of the lateral bacular mounds and accumulate in the cavernous space of the corpus spongiosum, and induce erection of the glans penis. Since no muscle tissue was found in the corpus spongiosum of lateral bacular mounds or corpus cavernosum, muscle tissue may not be involved in the mobility of lateral bacular mounds. In A. speciosus, the lateral bacular mounds have no ossified lateral distal segment, and therefore, almost all the internal space was occupied by the corpus spongiosum, and proximal parts of the lateral bacular mounds were attached to the distal part of the stalk (Fig. 3). In C. rufocanus, lateral bacular mounds had lateral distal segments and therefore less cavernous space (Fig. 4). Inflow of blood into the lateral bacular mounds is considered to increase the volume of the lateral bacular mounds in A. speciosus and C. rufocanus, thereby moving the lateral bacular mounds laterally using the junction of the proximal parts of the lateral bacular mounds and the stalk as a fulcrum (Fig. 6). This blood flow mechanism is thought to contribute to an efficient increase in the cross-sectional area of the glans penis. Kelly (2000) and Rodriguez et al. (2011) have suggested that corpus spongiosum of lateral bacular mounds were involved in penis expansion and hardening during copulation. Additionally, the present study clarified the specific movement mechanism for the first time. In laboratory rats, it has been reported that contraction of ischiocavernosus muscles attached to the proximal part of the pelvis and the corpus spongiosum causes blood flow into the corpus spongiosum, resulting in expansion of the cross-sectional area of the glans penis (Hart and Melese-D'Hospital 1983). Ischiocavernosus muscles contacting the pelvis may be involved in initiating blood flow in the corpus cavernosum connected to the cavernous space of corpus spongiosum.

Fig. 6.

Model of the movement mechanism of the lateral bacular mounds in non-erection (A) and erection (B) states. The cavernous space serves as a pool of blood in the corpus cavernosum in the lateral bacular mounds. Lateral bacular mounds expand driven with blood flow in the cavernous space and move laterally using the junction of the proximal parts of the lateral bacular mounds and the stalk as a fulcrum.

The size of the dorsal papilla differed significantly between A. speciosus and C. rufocanus (Figs. 3B-a, 4B-a). Although the dorsal papilla of A. speciosus was very small, the dorsal papilla of C. rufocanus was as large as the lateral bacular mounds and was almost fully occupied by cavernous space. Therefore, in C. rufocanus, it is thought that blood flowing into the dorsal papilla also contributes to the increase in the cross-sectional area of the glans penis, in addition to the lateral bacular mounds. These findings suggest the importance of blood flow into the corpus spongiosum for expansion and erection of the glans penis in Muroidea species and the differences between families in the structures involved in blood flow: lateral bacular mounds in Muridae (A. speciosus in this study), and lateral bacular mounds together with the dorsal papilla in Cricetidae (C. rufocanus in this study). Differences in the way the glans penis expands are suggested to be related to the pattern of ossification of the trident structure.