J. Anat., Lond. (1965), 99, 1, pp. 161-174 161 With 3 plates and 7 text-figures Printed in Great Britain The auditory apparatus of the (Rodentia, Sciuromorpha) BY ADE PYE Institute of Laryngology and Otology, London

INTRODUCTION The family Heteromyidae possess many exceptional and interesting features for adaptation to life in the deserts. There are five living genera: Heteromys, Liomys and Perognathus, collectively known as the spiny pocket mice and rats, Dipodomys, the , and Microdipodops, the kangaroo . Despite their popular names these have no close affinity to the murid rats and mice; their closest relatives are the Geomyidae, the pocket . All except Heteromys inhabit arid or semi-arid areas of North and Central America, while Heteromys is found further south and lives in rather swampy regions. Specializations in this family include bipedal locomotion, modified excretory system for conservation of body water, ability to withstand fluctuations between extremes of temperature and a greatly enlarged middle ear cavity. There are, however, at least four other families of small whose members may inhabit deserts and have there developed similar characteristics. Three of these families belong to Rodentia: (e.g. Notomys- the Australian Jerboa rat), (e.g. Dipus the Jerboa of Asia and Africa) and (e.g. Gerbillus and Meriones, the Gerbils of Asia, North Africa and Eastern Europe). The fourth family, Dasyuridae, contains Antechinoinys, the marsupial 'Jerboa mouse' of Australia. Very little is known at present about the auditory problems facing these desert inhabitants. It is curious that this parallel evolution has occurred so many times in different families and in such widely separate geographical locations. Phylogeny of the Ileieromnyidae Webster (1960-61) discovered that the cochlea of Dipodoinys spectabilis and D. inerriamni has an unusual structure. The present author has continued and extended this study to cover all the five genera of Heteromyidae, concentrating mainly on the structure of the cochlea. These results may best be considered in relation to the phylogeny of the group. The evolutionary origin and later classification of the present-day has given rise to a great deal of controversy and the Heteromyidae provide no exception. The early classifications were inadequate in many respects and Wood (1931) re- organized Heteromyidae, including both fossil and present-day representatives, mainly on the basis of the structure of their teeth. The living genera were thus divided into three main groups: 1. Ileteronmys and Liomys 2. Perognathus 3. Microdipodops and Dipodoinys 162 ADE PYE However, after the discovery of more fossil forms, Wood (1935) concluded that Microdipodops showed more affinities to Perognathus, especially in the foot struc- ture, than to Dipodornys. The following subfamilies were then founded: 1. Heteromyinac Heteromys and Liomys 2. Perognathinae Perognathus and Microdipodops 3. Dipodomyinae Dipodomys Xohen placing Microdipodops into the Perognathinae Wood stated that the has probably been a distinct phylogenetic line for a considerable time. Later Ellerman (1940-41) agreed with the above affinities, but reorganized the animals into only two subfamilies, putting Perognathus and Microdipodops into Dipodo- myinae. Simpson (1945), however, sided with Wood and their classification is used here. The evolutionary pattern is thus suggested to be: Dipodomys Microdipodops _1___

General account of ear structure The general structure of the human auditory apparatus has been excellently described by Shambaugh (1932). Accounts of the structure of the ears in rodents are given by Wassif (1948), Fernandez (1952) and many general histology books. Keen & Grobbelaar (1941) in a comparative survey of the mammalian tympanic bulla re- marked on its large size in the gerbil Desmodillus. Later, Meriones crassus was studied by Wisner et al. (1954), Legouix etal. (1955). Howell (1932) conductedastudy on the general gross anatomy of the Heteromyidae, including the ears. Webster (1960 a, b, 1961) followed this with an intensive account of the whole ear structure in two species of Dipodotnys. Pye (1964) has continued with a survey of the auditory apparatus in the whole family, of which this paper forms the first published account.

MATE tItALS The species examined are as follows: Heteromys aorlnalus three specimens, Liornys pictus three specimens, Perogilathus californicus two specimens, Microdipodops pallidus two specimens, Dipodomys a itra- toides two specimens, Dipodomys agilis two specimens.

HISTOLOGICAL TECHNIQUE Live specimens of all genera except Heterornys were sent to London from Cali- fornia by Dr Eisenberg of Berkeley University. Heterornys was obtained from the Regional Virus Laboratory, Port of Spain, during a visit to Trinidad. It was found by experience that the histological preparation of the ear, especially of the cochlea, required careful processing and the following method was found to be most suitable Auditory apparatus of Heteromyidae 163 (Pye, 1964). All the animals were treated alike. General anaesthesia was induced by an intraperitoneal injection of Nembutal (45 mg./kg.), so that the thorax could be opened. A cannula was introduced through an incision in the wall of the left ventricle and was tied into the dorsal aorta. The right auricle was punctured and the animal was perfused with Ringer or saline solution at body temperature. When all the blood had been washed out, Wittmaack's solution (formaldehyde, potassium dichromate, glacial acetic acid and distilled water) was forced through for immediate fixation of the tissues, which then turned a yellow colour. This intra-vitam fixation method was found to be the only way to obtain standard results of the internal ear and is similar to the technique used by Wisner et al. (1954). The head was then removed, skinned, trimmed down and placed in a fresh supply of the fixative, followed by Orth's solution, each for 1 day. Further fixation and leaching of the dichromate was achieved in frequent changes of formol calcium. Decalcification occurred in a 1 % solution of hydrochloric acid; the time taken for this to be com- pleted varied between 3 and 5 weeks, depending on the size of the head. After wash- ing in running and distilled water, dehydration through the alcohols was followed by the double embedding method of Peterfi (Carleton & Leach, 1947). First embedding occurred in three changes of 1 % celloidin methyl benzoate, clearing in benzene and second embedding in three changes of wax. Vacuum embedding was considered to be essential for the dispersion of gas bubbles, especially from the middle and inner ear cavities. The sections were cut in a horizontal plane at 7-8,a thickness and the primary stains used were haematoxylin and eosin in conjunction with periodic- acid-Schiff or Van Gieson's (Carleton (1943), Claydon (1955), Lulling (1957) and Steedman (1960)). Observations of the slides, measuring and photography were accomplished under a Zeiss Ultraphot photographic microscope.

RESULTS The external ear. The external ear in all the Heteromyidae is simple in structure and small to medium in size. It appears to be largest in Heteromys. The middle ear. As the middle ear has been extensively studied by Howell (1932) and Webster (1960 a, b, 1961), this account will be kept to a minimum. The abnormal

Multiple connective - tissue sheaths

Stapes footplate

Inner 9 Outer connective connective tissue sheath C tissue sheath

Text-fig. 1. The footplate of the stapes in the oval window. Left: the inner and outer connective tissue sheaths in Heteromys. Right: the multiple connective tissue sheaths present in Dipodomys and Microdipodops. 11 Anat 99 164 ADE PYE inflation of the bulla is especially marked in Dipodomys, and Ellerman (1940-41) states that in Microdipodops this development is carried further than in any other he examined (P1. 1, fig. 1). The auditory ossicles are small, as are the intra-aural muscles associated with them. The oval window opens into the scala vestibuli much higher up the cochlear spiral than inl other mammals (PI. 1, fig. 2). Another noticeable feature is that, in the more primitive members of this family, the stapes is secured in the oval window by only two sheaths of connective tissue, one internal and one external. In Dipodomnys and Microdip)odop0s these connexions are multiple (Text-fig. 1).

Tlext-fig. 2. A schlematic drawing of the cochlea wh1ichl defines the measurements made and referred to in the text. B.Ml.W., basilar mlembrane width; B..lIT., basilar membrane thickness; (X.(:., the cells of Claudius: ARE, the hetwiht of the spiral ligament; CD, the width of the spiral ligament.

Thle innere ear. After a lprelimlinary survey of the cochleae it has been discovered that some features change considerably in structure along the cochlear spiral and especially inlterspecifically. These features have been selected for measuring and will be discussed in turn (Text-fig". 2). They are: (I) The greatest height of the cochlea. ('2) The number of half-turnls present in the cochlea. (3) Radial measurements from the centre of the spiral ganglion to the outside of each turnl. (4) Central mleasulremnelts from the centre of the spiral ganglion to the inner rod of Corti. (5) The wsidlth of the basilar membrane from the lip of the spiral ligament to the inner rod of C8orti. (6) The greatest thickness of the basilar mlembrane. Auditory apparatus of Heteromyidae 165 (7) The greatest height of the spiral ligament. (8) The greatest width of the spiral ligament. All the graphs show the mean values for the number of cochleae examined, usually from four to six. It is realized, of course, that a larger number of animals would have been desirable. General form of the cochlea In all the Heteromyidae the general form of the cochlea is a truncated cone, except that the cochlea appears to have opened out markedly at the very base. The number of half-turns seen in horizontal section varies between seven and eight (PI. 1,

_ Dipodomys agilis IrMicrodipodops Dipodomys nitratoides Liomys Heteromys Perognathus

1 2 3 mm. Text-fig. 3. The cochlear heights inIthe Heteromyidac.

Heteromys Microdipodops 1 600 H Liomys - D. nitratoides Perognathus D. agilis 1400 j

1200 I-

1000 F- ,- \ a, 800 F

Radial 600 F

400 [-

200 [- Central

I I I I I I I I I I I I I I I 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Half-turns of cochlea Text-fig. 4. The radial and central measurements in the Heteromyidae. 11-2 166 ADE PYE fig. 2). This is about the same as in the guinea-pig, but more than in many other rodents such as a mouse, hamster or rat. The height of the cochlea from the base to the apex in the modiolar plane for each animal is shown in Text-fig. 3, and indicates that in the more primitive Hetero- mvidae (Heteromys and Liomys) the cochlea is shorter than in the advanced forms. The width of the cochlea is indicated by the radial measurements (Text-figs. 2 and 4). In all these animals the radial and central measurements are alike, but the height of the cochlea is different. Another interesting feature is that the actual weight of the animal (taken as an indication of its size) does not bear any relationship with the size of the cochlea. For instance, Microdipodops has almost the same cochlear height as Dipodomsys agilis but the latter is at least four times the size of the former.

The basilar memnbrane The basilar membrane supports the organ of Corti and the associated supporting cells. The dimensions of this membrane probably play a major part in frequency analysis of sound. Generally, the basilar membrane in the primitive Heteromyidae is wide, even at the base of the cochlea, where its least measurement is 105,I in Hetero- mys and Liomys (Text-fig. 5). In the 'kangaroo' forms, however, this figure is higher still; starting at about 200/i, increasing to 300,a in the subapical turn and

350 Heteromys f%. Liomys / 300 _ Perognathus_ B M W. / _/ \.

250 ' /// 200 / ';' B. M./W. Microdipodops 150 _ D C= -D. nitratoides

100 _

50 C. ~~~~~~C.C. \.

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 ,

Perognathus Heteromys D. nitratoides (1 specimen) --- Perognathus --- D. agilis ---D. nitratoides 150 -_ Microdipodops -----D. agilis Microdipodops

100 C.H.

50 A

1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Half-turns of cochlea Text-fig. 6B.Left and centre: the thickness of the basilar membrane (.PM.T.) in Hetero.yidae. Right: the lleigllt of the cells of Hensen (C.H.) in Heteromyidae. thickening of the basilar membrane is observed in Liomys. In one specimen of Perognbathus it is large (Text-fig. 6), while in the other specimen the thickenings seem to have been present, but have collapsed and are unmeasurable. As seen from Text-fig. 6 and PI. 3, these thickenings are extremely prominent in Microdipodops and Dijpodomys, where a thickness of 50-60/,t is reached towards the apex, again with a slight decrease at the apical turn. The fine structure of these thickenings has a strange appearance, which may be seen on the photomicrographs. They are formed mainly of hyaline tissue, at least at the basal part of the cochlea. Towards the apex spaces appear in these thickenings. In some cases it looks as if the hyaline tissue has been pulled away from the other part of the membrane, which makes the measuring of the 'true' thickness inaccurate. Such thickenings of the basilar membrane are unusual among the Rodentia, so far having been described only in Meriones. In the Microchiroptera they are much more common but are especially prominent in the basal turn (Pye, 1964 and in preparation). The possible function of these features is difficult to assess until more is known of general cochlear physiology. 168 ADE PYE

The supporting cells The supporting cells also show interesting features. The cells of Hensen and Claudius are of conventional form in Heteroniys, except at the basal part of the cochlea, where the cells of Hensen are deflected towards the inner hair cells forming a large space (P1. 2, figs. 3 and 4), as in many of the Microchiroptera (Pye, 1964). In Liornys they conform to the usual pattern, but in Perognathus they are very like those in Dipodomnys described by Webster (1960). The basal turn is not unusual but from there onwards the cells of Claudius with the cells of Deiter come to form a cup on which the cells of Hensen are situated. The latter are flask-shaped in structure and send long cytoplasmic processes to the reticular membrane. The ultimate develop- ment in this line is shown by Microdipodops and Dipodomnys, where the cells of Hensen increase in size towards the apex (Text-fig. 6 and PI. 3). The cells of Claudius, however, decrease in size from the base to the apex (Text-fig. 5). This development shows a phylogenetic sequence. The arrangement and structure of the supporting cells must in some way influence cochlear physiology. It has been suggested by Legouix & Wisner (1955) that in Meriones the hypertrophied cells of Hensen may provide extra protection against very loud sounds, but as yet there does not appear to be any direct evidence for this.

Heteromys Microdipodops 800 Liomys D. nitratoides Perognathus D. agilis 700

600 -

500 -

400 -

300 - AB

200 - % AB

-- CD C

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Half-turns of cochlea Text-fig. 7. The measurements of the spiral ligament in Heteromyidae. AB, the height of the spiral ligament; CD, the width of the spiral ligament. Auditory apparatus of Heteromyidae 169

The spiral ligament The size of the spiral ligament is another important feature in cochlear function but because of its shape it is difficult to devise a single informative measurement. In the present case an indication of its approximate size has been obtained by measuring its greatest height and width (Text-fig. 2). In Heteromys both these measurements were found to be low, even at the base of the cochlea (Text-fig. 7). In other Heteromyidae the height (AB) is much greater but decreases rapidly, and the width (CD) decreases in proportion (Text-fig. 7). Generally the spiral ligament is of medium size compared with other mammals.

DISCUSSION This family is interesting in possessing so many striking modifications, not least of which is the unusual structure of its auditory apparatus. The supposed phylo- genetic line can also be followed on the basis of these structures. The hypertrophy of the middle ear increases from the primitive Heterornys and Liomys to the Dipodo- myinae and Microdipodops. The same is also true of many of the cochlear features, for example, the general size of the cochlea, the width and thickness of the basilar membrane, the bizarre structure of the supporting cells and the size of the spiral ligament. The possible functional significance of these modified cochlear structures has been discussed by Webster, but more ecological and physiological work needs to be carried out before any firm conclusions can be reached. Two interesting points emerge: (1) That Heteromys seems to show a more pronounced development of the middle and inner ear than Liomnys, although it is considered more primitive than the latter. (2) Microdiipodops often shows even more extreme modifications than Dipodoinys. Yet, as already stated, the former has probably been a separate phylogenetic line for a long period and therefore has developed independently from Dipodomyidae. If so, this is a remarkable case of parallel evolution within a single family. It would be interesting to study other desert forms and to see whether the structure of their ears is closely related to that found in the Heteromyidae. Eller- man states that: 'Great specialisations have been attained independently again and again with the of Rodentia for various modes of life'. This is certainly true, ev-en within the five living genera of the Heteromyidae.

S U M M AR Y The structure of the ears of the family Heteromyidac is discussed in relation to the phylogeny and classification of the family. A successful histological technique for the intra-vitam fixation and processing of the ears is described. The results indicate that, in the more advanced members of the family, the middle ear and especially the cochlea of the inner ear have become extremely modified. The supposed phylogenetic line can thus be followed in the structure of the ears alone. The possible functions of these special modifications cannot be assessed until more is known of the general cochlear physiology. 170 ADE PYE Thanks are due to Dr J. Eisenberg and to The Regional Virus Laboratory, Trinidad, for supplying the specimens. Histological assistance has been obtained from the Histology Section of the Department of Pathology and Bacteriology in this Institute. The canning of the preserved specimens and transportation of equipment to and from Trinidad was kindly carried out by the Trinidad Cooperative Citrus Growers Association and Messrs. Elders and Fyffes Ltd., respectively. This research has been sponsored by the Air Force Office of Scientific Research, OAR, through the European Office, Aerospace Research, United States Air Force, which also made it possible for the author to visit both Trinidad and California.

REFERENCES CARLETON, H. MI. (1943). Schafer's Essentials of Histology. London: Green and Co. CARLETON, H. M. & LEACH, E. H. (1947). Histological Technique. London: Oxford Medical Publications. CLAYDON, E. C. (1955). Practical Section Cutting and Staining. London: Churchill. CULLING, C. F. A. (1957). Handbook of Histological Technique. London: Butterworths. ELLERMAN, J. R. (1940-41). Families and Genera of Living Rodents. I, II and III. London: Trustees of British Museum. FERNANDEZ, C. (1952). Dimensions of the cochlea. J. acoust. Soc. Amer. 24, 519-523. HOWELL, A. B. (1932). The saltatorial rodent Dipodomys ; the functional and comparative anatomy of its muscular and osseous systems. Proc. Amer. Acad. Arts Sci. 67, 377-536. KEEN, J. A & GROBBELAAR, C. S. (1941). Comparative anatomy of tympanic bulla and auditory ossicles, with a note suggesting function. Trans. roy. Soc. S. Af. 28, 307-329. LEGOUIX, J. F., PETTER, F. & WISNER, A. (1954). Etude de l'audition chez mamrniferes a bulles tympaniques hypertrophiees. Mammalia, 18, 262-271. LEGOUIX, J. P. & WISNER, A. (1955). Role functionnel des bulles tympaniques geantes de pertains rongeurs (Meriones). Acustica, 5, 209-216. PYE, A. (1964). A comparative anatomical study of the auditory apparatus of selected members from the Orders Chiroptera and Rodentia. London University: Ph.D. Thesis. SHAMBAUGH, G. E. (1932). Cytology of the internal ear. In Cowdrey's Special Cytology, 3, 1111- 1333. New York: Hoeber. SIMPsoN, G. G. (1945). The principles of classification and a classification of Mammals. Bull. Amer. Mus. nat. Hist. 85, 1-350. STEEDMAN, H. F. (1960). Section Cutting in Microscopy. Oxford: Blackwell. WASSIF, K. (1948). Studies on structure of the auditory ossicles and tympanic bone in Egyptian Insectivora, Chiroptera and Rodentia. N\o. 27 Fouad 1 University, 177-213. WEBSTER, D. B. (1960 a). Auditory significance of the hypertrophied mastoid bullae in Dipodomys. Anat. Rec. 136, 299. WEBSTER, D. B. (1960 b). The morphology and functional significance of the ear of the kangaroo rat Dipodomys. Cornell University: Ph.D. Thesis and Dissertation Abs. XXI. WEBSTER, D. B. (1961). The ear apparatus of the kangaroo rat Dipodomys. Amer. J. Anat. 108, 123-248. WISNER, A., LEGOUIX, J. P. & PETTER, F. (1954). Etude histologique de l'oreille d'un rongeur a bulles tympaniques hypertrophikes: Meriones crassus. Mammalia, 18, 371-374. WOOD, A. E. (1931). The phylogeny of the heteromyid rodents. Amer. Mus. Novit. 501, 1-19. WOOD, A. E. (1935). Evolution and relationship of the heteromyid rodents with new forms from the Tertiary of Western N. America. Ann. Carneg. Mus. 24, 73-262. Auditory apparatus of Heteromyidae 171

EXPLANATION OF PLATES PLATE 1 Fig. 1. A photograph of the skull of Microdipodops from the dorsal aspect to show the enlarged middle ear cavities posterior to the braincase. Fig. 2. A horizontal section through the cochlea and the middle ear of Ileterornys, to show the general structure of the turns and the stapes in the oval window. See also PI. 2.

PLATE 2 Enlarged parts of the cochlea of Heteromys as illustrated in PI. 1, fig. 2. Fig. 3. The first half-turn, the oval window and stapes (above) and the round window (below). Fig. 4. The third half-turn, where the thickenings of the basilar membrane and the deflected cells of Hensen are apparent. Fig. 5. The fifth and seventh half-turns, where the thickenings have pulled away from the main body of the membrane. PLATE 3 Horizontal sections through the cochlea in Microdipodops. Fig. 6. The first half-turn of the cochlea. Fig. 7. The third half-turn of the cochlea, where the thickenings of the basilar membrane are apparent, also the strange supporting cells. Fig. 8. The fifth half-turn of the cochlea. 172 Plate 1

Middle ear cavity Spiral ganglion

Enlarged middle ear cavity 'N

Bralincase

Stapes footplate

First half-turn

5 mm.

i) Plate 2 173

OO#. ...t~ ~ tq

3 174 Plate 3