J. Anat. (1990), 168, 143-166 143 With 17 figures Printed in Great Britain

The megachiropteran pineal organ: a comparative morphological and volumetric investigation with special emphasis on the remarkably large pineal of praedatrix

KUNWAR P. BHATNAGAR, HEIKO D. FRAHM*t AND H. STEPHAN* Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Health Sciences Center, Louisville, Kentucky 40292, USA and * Max-Planck-Institut fur Hirnforschung, Vergleichende Neurobiologie, Deutschordenstrasse 46, D-6000 Frankfurt a.M. 71, FRG (Accepted 4 July 1989)

INTRODUCTION In a recent comparative study, morphological and volumetric data were presented on the pineal organs of 19 species of megachiropteran fruit of the family Pteropodidae (Bhatnagar, Frahm & Stephan, 1986). Since that time brains of several other species of fruit bats have become available in our laboratory. The relatively and absolutely large pineal of Dobsonia species, especially that of Dobsonia praedatrix, forms part of this report. While absolutely large pineal organs have been reported among birds in the emu Dromaeus novaehollandiae (Cobb & Edinger, 1962) and among in neonates of the antarctic seals Mirounga leonina and Leptonychotes weddelli (Bryden, Griffiths, Kennaway & Ledingham, 1986), the shape, size, and topography of the Dobsonia pineal is unlike any that has been described so far, at least among bats. Furthermore, instead of remaining deeply situated either under or between the cerebral hemispheres, as in other pteropodids, the pineal organ in Dobsonia extends to the brain surface and covers the cerebellum anteriorly. This report deals not only with the morphology of the large pineal organ of the New Guinean naked-backed Dobsoniapraedatrix Andersen, but also documents further the comparative pineal morphology of 16 additional species of megachiropteran bats and compares this with the available data on other megachiropteran pineals.

MATERIALS AND METHODS This investigation is based on 45 pineal organs from adult of both sexes belonging to 17 species of the family Pteropodidae (Table 1). The specimens were collected in Africa, Papua New Guinea, Australia and South-East Asia by Heinz Stephan. Under Nembutal anaesthesia, the animals were weighed, measured, photographed, and then perfused through the left ventricle with normal saline followed by freshly made Bouin's fluid. Brains were carefully removed from the cranial vault, weighed, measured (see Table 3) and immersed in Bouin's fluid for four days, then transferred to 70 % alcohol which was changed several times during the first few weeks. Detailed notes on preparation in the field are given in Stephan, Frahm & Baron (1981). (These can be obtained from the authors upon request.) Morphological t Present address: Anatomisches Institut der Universitat K6ln, Joseph-Stelzmann-Str. 9, D-5000 Koin 41, FRG. 144 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN Table 1. Selected data on the pineal organ of 17 species of megachiropteran bats (Family Pteropodidae) Body Brain Mean Pineal + Size Pineal Code Sex weight weight dimensions volume CV index type no. Spec:-s ,y (g) (mg) (1) (2) (3) (4) (5) (6) Subfamily Tribe Pteropini Subtribe Rousettina 1319 torquata 2,1 36-4 1160 037 055 0-0818 508 72 A Subtribe Pteropodina 1439 tonganus 2,1 435 0 6025 1 70 1-04 1-4883 13-3 212 AB Subtribe Dobsoniina 1485 Dobsonia inermis 3,0 150-0 2700 3 65 1 45 0 7596 8-0 237 a,fC* 1489 Dobsonia moluccensis 1,0 465 0 5300 4 58 1-92 2 3000 313 1490 Dobsonia spec. 1,1 230-0 3775 4-22 2 35 4-9226 9-8 1123 a,8C*a,flC* 1495 Dobsonia praedatrix 1,2 184-0 3030 4-22 4-51 16-3447 19-6 4393 a,dC* Tribe Epomophorini 1503 Hypsignathus monstrosus 1,0 310-0 3480 0-62 091 0-4100 75 A 1509 franqueti 1,2 120-0 2215 0-83 0 93 0 5360 13-1 197 A 1539 Scotonycteris zenkeri 1,1 214 710 038 0.44 0-0361 3-1 47 A 1541 argynnis 2,2 27-0 840 0 35 0-62 0-0768 245 85 A Tribe Cynopterini Subtribe Cynopterina 1611 raptor 2,1 26-1 731 0-42 0 50 0-0778 6-8 88 A Subfamily Macroglossinae Tribe Macroglossini 1621 woermanni 0,3 181 680 0-42 059 0-0927 33.5 137 A 1625 minimus 1,2 14-5 560 0-28 0-37 0 0190 39.3 33 A 1631 crassa 1,2 17-6 623 0 33 0-38 0-0421 44.3 64 A Tribe Notopterini 1635 melanops 1,1 47-6 1285 0-42 047 0-0820 21 6 60 A 1639 Nesonycteris woodfordi 0,3 356 1020 0-35 055 0-0671 27-0 60 A 1641 macdonaldi 1,2 704 1456 050 0-62 0-1280 21 7 70 A * See Discussion. The standard values of body and brain weights were taken by H. Stephan in the field. (1) The anteroposterior extent (in mm) of pineal was calculated from serial coronal sections. The expression of this dimension, therefore, should not be taken as the pineal 'length'. (2) The greatest width (in mm) of pineal was calculated at its widest point. Both expressions of size (1,2) are from serial sections and therefore they include a shrinkage artefact of approximately 20%. (3) Pineal volume (in mm3), corrected to fresh volume (see Materials and Methods). (4) CV, the coefficient of variation, i.e. the standard deviation in percentage of the mean (see Results), (5) The formula for the calculation of pineal size indices (SI) is: actual pineal size SI, expressed in percent = . x 100, computed pineal size where the actual pineal size is calculated from serial sections, and the computed pineal size is derived from the allometric formula, antilog [-2094 + (0 735 x log body weight)]. The value, -2-094 is the y-intercept of the reference baseline running through the family Vespertilionidae and the value, 0 735 is the mean ascent of regression lines of bat families and/or subfamilies. Pineal size here refers to pineal volume. For further explanation see Materials and Methods. (6) Classification ofpineal types is based on Vollrath (1979). For further explanation see Results and Table 3 in Bhatnagar et al. (1986). observations were made on 20 ,um serial coronal brain sections stained with cresyl violet, gallocyanin, or by the Heidenhain-Woelcke procedure. Selected sections were stained with the one-step Gomori trichrome or Masson trichrome. One brain of Dobsonia praedatrix was cut sagittally. The anteroposterior extent and greatest width of the pineal organ (Table 1) were obtained in each species from enlarged photomicrographs. Megachiropteran pineal organ 145 The volume of the pineal organ was calculated in all 45 specimens. Even though detailed description for this determination is provided in Bhatnagar et al. (1986), a brief explanation of the procedure is given again. Equidistant serial sections through the epiphysis were photographed directly from the slides, the borders delineated and the pineal cut out. Volumes were calculated according to the formula: V = APx WSx D/MS where V = epiphyseal volume in mm3; AP = average area of the photographic paper in mm2/mg; WS = total weight of the cut-out pineal photographs in mg; D = distance between measured sections in mm x section thickness; M2 = square of linear magnification of the photographs. A conversion factor to account for the shrinkage due to fixation was applied to correct the pineal volumes using the formula: Corrected epiphyseal volume = serial section epiphyseal volume (V) x conversion factor (CF), volume of fresh brain where CF = (VFB) serial section brain volume (VSB)' where VFB = weight of fresh brain obtained in the field specific gravity of the brain (= 1 036) and VSB = volume determined through the photographic method (see above and Stephan et al. 1981 for details). The standard values of body and brain weights were derived by H. Stephan in the field from several specimens of each species. Regression line analyses of megachiropteran pineal volumes against body weight result in similar slopes, as reported by Bhatnagar et al. (1986) (a = 0735). The average ascent for the five tribes Pteropini, Epomophorini, Cynopterini, Macroglossini and Notopterini was a = 0767; the average slope for the two subfamilies Pteropodinae and Macroglossinae was a = 0 730. To make a direct comparison possible between the chiropteran pineal indices in Bhatnagar et al. (1986) and the additional species reported in the present paper, and taking into consideration the quite similar slopes mentioned above, it was considered unnecessary to change from the one used in Bhatnagar et al. (1986). This slope is now confirmed and stabilised by the additional data on 17 species. As reported in Bhatnagar et al. (1986) the reference line is drawn through the Vespertilionidae. Any value on the reference line is considered to represent the average volume of the pineal for a typical vespertilionid and has an index of 100 (%). The distances from the reference line are expressed by deviations from 100 and are a measure of the degree of deviation of the pineal volumes from those of typical Vespertilionids. The indices are listed in Tables 1 and 2 and scaled in Figures 14-17. The allometric formula used for determining the pineal size indices was: log pineal volume =-2 094 + 0735 x log body weight. Data previously reported on 19 species of megachiropteran pineal organs (Bhatnagar et al. 1986) were combined with that on the 17 new species of megachiropterans of this study (Tables 1 and 2). Thus, the conclusions encompass a total of 36 pteropodid species belonging to the two subfamilies and five of the six tribes of the family Pteropodidae, excluding the tribe Harpyionycterini. 146 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN Table 2. Revised data on the pineal organ of 19 species of megachiropteran bats* Body Brain Mean Pineal + Size Pineal Code Sex weight weight dimensions volume CV index type no. Species S,Y (g) (mg) (1) (2) (3) (4) (5) (6) Subfamily Pteropodinae Tribe Pteropini Subtribe Rousettina 1297 helvwn 1,1 263 4290 680 780 0-2763 49.7 57 A 1299 aegyptiacus 1,1 134 2250 800 1260 0-8134 23-0 276 A Subtribe Pteropodina 1325 Pteropus alecto 0,2 595 7040 1840 1120 1-7070 205 194 AB 1343 Pteropus conspicillatus 1,0 750 8350 1800 1100 1-5085 144 AB 1375 Pteropus lylei 2,2 322 6130 1945 1400 2-9184 253 520 AB 1411 Pteropus poliocephalus 1,1 695 7230 2800 1920 7-8000 0-8 789 AB 1425 Pteropus scapulatus 2,0 375 5360 980 1030 0 6385 340 102 AB 1445 Pteropus vampyrus 1,1 1015 9400 2020 1250 1-8268 0-1 140 AB Tribe Epomophorini 1519 labiatus 1,2 63-4 1580 605 770 0-2408 21-0 142 A 1533 pusillus 2,1 25 5 814 400 540 0 0605 60 5 69 A Tribe Cynopterini Subtribe Cynopterina 1545 brachyotis 2,1 33-1 980 610 830 0-2762 13-5 262 A 1547 Cynopterus horsfieldi 1,2 58 8 1370 675 1010 0 4853 40-1 302 A 1553 ecaudatus 1,1 24-0 800 780 980 0-3725 6-7 447 A 1567 Chironax melanocephalus 1,1 17-6 610 440 530 0-0920 32-7 139 A 1579 Balionycteris maculata 0,2 14-3 510 560 770 0 2000 13-1 351 A Subtribe Nyctimenina 1607 robinsoni 1,1 47-0 1230 570 790 0-2303 9.5 169 A Subfamily Macroglossinae Tribe Macroglossini 1619 spelaea 1,2 550 1300 405 570 00655 36-4 43 A 1627 Macroglossus sobrinus 3,1 21-6 690 485 430 0-0862 37.5 112 A 1629 Syconycteris australis 0,1 14-7 570 420 510 0 0574 99 A * The above data have been extracted from Table 1 in Bhatnagar et al. (1986) and are partially revised. Additional data on body weight and/or brain weight of certain species resulted in minor changes which influenced the pineal volume and/or size index and necessitated repetition. These data were used in computing Figures 14-17. For explanations and abbreviations refer to Table 1.

RESULTS Morphological observations Classification of the pineal organ A general scheme of classification of vertebrate pineal organs as proposed by Vollrath (1979) has been applied to bat pineals also (for details, see Table 3, Bhatnagar et al. 1986). Type A pineals are small, oval or conical and are located adjacent to the third ventricle; Type AB pineals are longer than their greatest width by a factor of at least two; Type ABC pineals are rod-like, reach the brain surface and are close to the skull. For parts of the pineal organ (deep, middle and superficial) greatly reduced in size, the respective capital letter is substituted by the corresponding lower case letter of the Greek alphabet (e.g. a4#C); for a missing pineal segment, the letter representing the respective segment is omitted (e.g. aC). The remarkably large pineal organ of Dobsonia praedatrix Extending from the roof of the third ventricle to the brain surface and covering the cerebellum anteriorly, the af/C-type, mushroom-shaped pineal organ of Dobsonia Megachiropteran pineal organ 147 Table 3. Data on individual specimens ofDobsonia praedatrix Code Body Brain Pineal Length (mm) Brain dimensions (mm) no. weight weight 1495 Sex (g) (mg) Dimensions Vol (mm3) HB FA E T BL HemL BW BH 32431 ,3 237 2878 4-22 4 58 18-391 170 120 25 5 26-0 29 5 17-8 16-3 13-3 32451 9 156 2927 4-82 493 12-382 150 108 23 5 26-0 28-8 18-2 16-9 13-5 32481 9 177 3014 3-62 4 03 16-723 155 115 24-0 27-0 29-8 18-7 16-8 13-6 Mean 4-22 4-51 16-3447 CV+ 19-6 SI 4393 324622 172 2741 - 153 110 23-0 24-5 27-3 16-2 16-4 13-5 3244 Y 157 3079 - 148 110 26-0 24-0 28-3 18-2 17-0 14-1 3247* s 159 3339 - 160 107 24-5 26-5 28-3 18-4 17-3 14-5 3249 Y 186 2976 - 160 112-5 26-0 23-0 28-6 17-6 16-6 14-0 Collection date: between 29 December 1981 and 6 January 1982. Place and Biotope: New Guinean caves. 3243-3245: St Peter, Chanel Coll, Kokopo, 30 km SE of Rabaul. 3246-3249: Keravat. Abbreviations: BH, brain height; BL, brain length; BW, brain width; E, ear; FA, forearm; HB, head and body; HemL, hemisphere length; SI, size index; T, tail. For other abbreviations refer to Tables I and 2. Brains serially sectioned: 1 coronally; 2sagittally. * Subadult.

praedatrix was approximately 5-33 mm long, subtending an angle of about 30° with respect to the third ventricle and was wedged between the cerebral hemispheres and the cerebellum (Figs. 1, 2). Anteroposteriorly it extended 4-22 mm with the greatest width of 4-51 mm measured at the umbrella-like cap (Figs. 3a, 4; Table 1). It should be emphasised that these measurements were taken from serial sections and include an approximately 20 % shrinkage artefact (a volume shrinkage of 50% is equivalent to 20-6 % of linear shrinkage). The 'stalk' was approximately 11 mm in diameter and consisted of a two-pronged base enclosing the third ventricle (Figs. 2-4) which extended dorsal to the pineal in a very small suprapineal recess complete with choroid plexus. Through this pineal base passed the most posterior fibres of the habenular commissure. Furthermore, in the region between the prongs, the ependymal lining of the third ventricle appeared deficient (Figs. 5, 9b) so as to allow unrestricted contact between the circulating cerebrospinal fluid and the pinealocytes and other paren- chymatous elements. Progressing posteriorly, the pineal gradually achieved a position dorsal to the cerebellum (Fig. 4c-f) enlarging into an umbrella-like cap. Large calibre blood vessels, particularly the superior sagittal sinus and its tributaries, were juxtaposed with the pineal on its dorsal and lateral aspects, but not in the 'stalk' region. Vessels penetrated the pineal dividing it into 'lobes'. Numerous groups of neurons, about 10,um in nuclear diameter, were observed in the connective tissue along these blood vessels (Fig. 6). The entire pineal was encapsulated by a pineal sheath. Individual pinealocytes were clustered together in cell cords surrounded by variable amounts of connective tissue (Fig. 7). In general, the pineal organ appeared to be very compact and solid. In the basal, 'stalk' region, the dorsal and ventral aspects of the pineal were grossly different, being much more compact ventrally than in the dorsal region. The expanded superficial part of the pineal consisted of a distinct cortex and medulla (Fig. 7). The cortex was much more compact than the medulla and contained closely packed pinealocytes surrounded by fewer connective tissue elements. The 148 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN

Fig. 1 (a-b). Dorsal (a) and lateral (b) views of the brain of Dobsonia praedatrix (d, 1495/3243). P, pineal organ. c. x 3-4. Megachiropteran pineal organ 149

Fig. 2. Sagittal section through the brain of Dobsonia praedatrix (S9 1495/3246). AC, anterior commissure; C, cerebellum; CC, corpus callosum; CP, choroid plexus; HC, habenular commissure; NH, neurohypophysis; OB, olfactory bulb; OC, optic chiasma; P, pineal organ; PC, posterior commissure; SC, superior colliculus; T, telencephalon; TH, thalamus; III, third ventricle; IV, fourth ventricle. c. x 5 75. Cresyl violet, 20 ,um. medullary portion showed abundant connective tissue in which fewer pinealocytes were interspersed. The morphology of the pineal organ in the other three species of Dobsonia, i.e. D. inermis, D. moluccensis and D. species, was very similar to that of D. praedatrix. The differences were in the overall size (Figs. 3, 8) of the pineal and the apparent lack of intrapineal neurons. The pineal in D. moluccensis and D. species was posterior to the habenular structures, thus remaining anatomically unrelated to them. In D. inermis, the most posterior fibres of the habenular commissure passed through the pineal base as in D. praedatrix. In all Dobsonia species the ependymal lining of the third ventricle was deficient in the basal pineal region, thereby allowing pinealocytes to come in contact with the cerebrospinal fluid. The pineal organ in the other pteropodids For a general morphological description of the megachiropteran pineal organ reference is made to Bhatnagar et al. (1986). Only those observations which are typical for these additional thirteen species will be recorded briefly here and discussed fully later in this report. Except in Pteropus all pineal organs were small, deeply situated (Figs. 9-13) and were of Type A (Table 1). Pteropus tonganus is an excellent species in which to show the presence of a vascular cuff around the pineal (Fig. 9a). Blood vessels of varying calibres form this cuff. In no other bat species was such a profuse pineal vasculature observed. The pineal remained unrelated to the habenular nucleus and the commissure, both of which were small and more anterior. Clusters of pinealocytes, uncovered by ependyma, were seen to project into the third ventricle. A ganglion containing large neurons was seen within the highly vascular connective tissue surrounding the pineal. The pineal of the large bat Hypsignathus monstrosus was very small (Fig. 10 a, b; Table 1) and deeply situated in between the habenular eminences. The habenular commissure passed through much ofthe anterior half ofthe pineal. The subcommissural organ and the pineal were situated very close to each other. A large suprapineal recess extended over and posterior to the pineal. Connective tissue prevailed in the parenchyma; cords 150 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN

af

1 mm b ~~~d' . Fig. 3. For legend see opposite. Megachiropteran pineal organ 151

Fig. 4(a-J). Pineal organ of Dobsonia praedatrix (,, 3243) in relation to brain regions as seen in tracings made from serial cross-sections. Of especial note are the juxtaposed superior sagittal sinus and tributaries which are dorsal (c, d), and the cerebellum (c-f) which is ventral to the pineal. Refer also to Figure 2. BV, blood vessels; C, cerebellum; LV, lateral ventricles; M, mesencephalon; P, pineal organ; SCO, subcommissural organ; SPR, suprapineal recess; SSS, superior sagittal sinus; T, telencephalon; III, third ventricle with choroid plexus. Cresyl violet, 20 ,um. of pinealocytes were lacking. In Casinycteris argynnis, Myonycteris torquata and Scotonycteris zenkeri a suprapineal recess was lacking; in the other species though, such a recess, however small, was observed. In several other species, such as Epomops franqueti, S. zenkeri and C. argynnis, the pinealocytes were seen in open

Fig. 3 (a-d). Schematic reconstruction of the acfC-type pineal organ in (a) Dobsonia praedatrix (3243); (b) D. inermis (3225); (c) D. moluccensis (3232) and (d) D. moluccensis species (3237). The distance between successive sections in (a-d) is 300, 200, 240 and 300 ,um respectively. Pineal cut-outs at equal intervals obtained from enlarged photomicrographs were serially arranged in an overlapping manner for these reconstructions. The scale refers only to the individual coronal outlines of pineal and not to the overall reconstruction. 152 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN l |_|lsf7;f||.9i|1liF |

7 Megachiropteran pineal organ 153 communication with the third ventricle and consequently with the circulating cerebrospinal fluid. Like P. tonganus, the pineal organ in S. zenkeri and Megaloglossus woermanni (Fig. 12) was posterior to the habenular structures and was thus unrelated to them. Intrapineal cavities were observed in all three specimens of M. woermanni (Fig. 12). Such cavities have been reported in the pineals of several microchiropteran bats (Bhatnagar et al. 1986). Morphologically distinct dorsal and ventral subdivisions of the pineal were evident in M. torquata, Syconycteris crassa and Notopteris macdonaldi. The pineal in S. crassa had a ring-like configuration surrounding the third ventricle anteriorly; the ventral aspect of this ring was formed by the subcommissural organ (Fig. 13). No sex-related difference in the pineal morphology of megachiropteran bats was observed. Volumetric observations The data on 19 megachiropteran pineals reported by Bhatnagar et al. (1986) have been revised (Table 2) because of minor changes in body weight and/or brain weight of certain species which influenced the pineal volume and consequently the pineal size indices. These data were pooled with the 17 species of this study (Table 1). In the double logarithmic plot of pineal volume versus body weight (Fig. 14), all 36 species appeared to fit quite well with the exception of Dobsonia praedatrix, the species with an exceptionally large pineal. Also, the correlation between body size and the size of the pineal, expressed as volume, was found to be positive in all 36 species of Megachiroptera. For the overall regression, a correlation factor (r) of 0-827 was determined; this value rose to 0-864 after excluding the aberrant Dobsonia praedatrix (Fig. 14). The best correlation between body size and pineal size was obtained in the tribe Notopterini (r = 0 992) and in the six species belonging to the tribe of Epomophorini (r = 0-908). Glaring exceptions were seen in the large bodied bat Hypsignathus monstrosus (body weight 310 g; SI, 75), and the small-bodied bat Balionycteris maculata (body weight 14-3 g; SI, 351). When the two subfamilies, Pteropodinae and Macroglossinae (family Pteropodidae), were compared, it became evident that despite a complete overlap of Macroglossinae with some species of Pteropodinae, the latter subfamily included a large number of species whose pineal size indices exceeded the range covered by the Macroglossinae (Fig. 15). The extraordinarily high position occupied by the pineal in D. praedatrix becomes especially obvious in this comparison; its size index (4393; volume 16-3447 mm3, 0 56 % of the brain) was more than 50 times as large as would be expected in an average Macroglossine bat of equivalent body weight. Extending such a comparison further to five of the six tribes of the family Pteropodidae (Harpyionycterini are not represented in our material) it was seen that

Fig. 5 (a-b). Coronal section through the base of the pineal organ of Dobsonia praedatrix. Note the discontinuous ependymal lining (arrows) through which an open communication exists between the pineal parenchyma and the third ventricle with its circulating CSF. The enclosed area is enlarged in (b). c. x 85, x 200 respectively. Cresyl violet, 20 rnm. Fig. 6(a-b). Intrapineal neurons (N) interspersed amongst the pinealocytes (P) of Dobsonia praedatrix. They are few in number and closely associated with blood vessels (BV). c. x 125, x 480 respectively. Cresyl violet, Masson trichrome, 20 /am. Fig. 7. Coronal section through the mushroom-like expansion of the Dobsonia pineal. A distinct cortex (C) composed ofdensely-packed pinealocytes, and abundance ofconnective tissue (C7) in the medullary region (inset) are clearly seen. c. x 50, x 250 respectively. Gomori and Masson trichrome respectively, 20 /tm.

6 ANA 168 154 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN

Fig. 8 (a-c). Dorsal views of brains of the three species ofDobsonia. (a) Dobsonia moluccensis (3232); (b) Dobsonia spec. (3237); (c) Dobsonia inermis (3225). P, pineal organ. c. x 2-80.

Fig. 9(a-b). Coronal sections through the pineal organ of Pteropus tonganus. (a) Note the vascular cuffcomposed of numerous blood vessels (BV) of varying calibres and surrounding the pineal organ (P). (b) At the pineal base, the ependymal lining is discontinuous and deficient (arrows). Pinealocyte clusters (PC) are seen projecting into the third ventricle (III). BV, blood vessels; SCO, subcommissural organ. c. x 37, x 57 respectively. Cresyl violet, 20,m. Fig. 10 (a-b). Coronal sections through the pineal organ of Hypsignathus monstrosus. (a) Note the deep location of the Type-A pineal (P), with respect to the cerebral hemispheres (CH). (b) The proximity of the pineal organ and the subcommissural organ (SCO) is clearly indicated. c. x 16, x 45 respectively. Cresyl violet, 20 ,um. Megachiropteran pineal organ 155

Vt~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... j S J~~~~1Pse PC:

~~~~~~~~~ ~ ~ ~ ~ ~ - 9a -'

~~~~~~~~~~~~~~~~s Jli- __ __

6-2 156 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN

..: 1; "..m.,J9.,z. I I I

t.. Megachiropteran pineal organ 157

+ 1-

a)-

a) E O Z 0. - 0 - ._ _0 -1 -

+

2 3 log body weight

Fig. 14. Double logarithmic plot of pineal volume (mm3) versus body weight (g) in 36 species of megachiropteran bats. The heavy reference line drawn through the points for average body weight and pineal volume is the same as used in Figure 1 in Bhatnagar et al. 1986, and has the formula: log y = -2-094 +0 735 x log body weight. Parallel lines either above or below the reference line indicate the degrees of deviation from this 'average vespertilionid' by a factor of 2. For further explanation see Materials and Methods. Symbols and abbreviations: o, revised data on 19 species from Bhatnagar et al. 1986 (see Table 2); +, additional data on 17 species from this study (Table 1). the three species of Notopterini did not show great variation in pineal size and their indices remained on a low level. Macroglossini and Epomophorini also shared low positions on the scale of pineal indices despite somewhat greater interspecific variation, whereas Cynopterini and Pteropodini had, on the average, clearly larger pineals (Fig. 16). Finally, when the 14 species of the tribe Pteropini were subdivided into three subtribes, members of Rousettina and some Pteropus species were observed in rather low positions with respect to Dobsonia praedatrix. Two species of the Dobsoniina, Dobsonia inermis and Dobsonia moluccensis, had pineal indices which overlapped with Pteropus species (Fig. 17) and two others exceeded the range covered by Pteropus

Fig. 11. Coronal section through the anterior pineal region of Myonycteris torquata. The pineal organ (P) is protruding into the third ventricle (III). The habenular nucleus (HN), and the habenular commissure (HC) are prominent. c. x 57. Cresyl violet, 20 ,um. Fig. 12. Numerous cavities are seen in the pineal organ (P) of Megaloglossus woermanni. BV, blood vessels; C, cavities; CH, cerebral hemispheres; SCO, subcommissural organ c. x 75. C. violet, 20 ,um. Fig. 13. The ring-like base formed by the pineal organ of Syconycteris crassa. Note the close proximity of the subcommissural organ (SCO) with the pineal (P). c. x 70. Cresyl violet, 20 ucm. 158 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN

.( < 0 ~~~~~~~~ CC °-08

I .I U

C)

Ln L CC CV)~ ~ ~ ~ ~ ~ 2 M-0a _ t< C >o 0o o CC c C In 0 La> In .C)N 0=5Z O

$) CC3 C1 ea<

0~Co 0C L1C E v >Q Cd 4)- _o LO >Ci

a. C.) Z U5 z CC 0

C #C-'.0@ eB C 0 C Uf n~ ~ _- C0& - U )In~~~~~~~~~I 0 O d 4X.,) _ *

cis C

N C.

0ci C)C

C4 Cd .c

o 0o 0D 0 0 o 0 0 0 0 0 0 0 0 0 In ) A l (OOLx) ewnloA Ieeu!d senipul Megachiropteran pineal organ 159 species. No matter how the comparisons were made, an extremely large pineal index in Dobsonia praedatrix was the result. The range of pineal size indices in the 36 megachiropteran species was from 33 to 4393 (Tables 1, 2). The lowest index belonged to a small bodied bat, Macroglossus minimus (body weight 14-5 g; SI, 33; volume 0 0190 mm3), but the highest indices were reached in Dobsonia praedatrix (SI, 4393; volume 16 3447 mm3), Dobsonia species (SI, 1123), Pteropus poliocephalus (SI, 789) and Pteropus lylei (SI, 520). Since Dobsonia praedatrix is so exceptional in its pineal size, relevant data on all individuals of this species have been provided in Table 3.

DISCUSSION Morphological characteristics of the megachiropteran pineal organ Structure Classification of the megachiropteran pineal organ As characterised by their shape, size and topography with respect to cerebral hemispheres (Vollrath, 1979), megachiropteran pineal organs can be readily divided into three classes: all small fruit bats exhibit Type A, conical or oval-shaped pineals that are deeply recessed under the cerebral hemispheres (Figs. 10-13). Of the 36 species examined thus far (Bhatnagar et al. 1986; Chang, Bhatnagar, Tseng & Karim, 1987; and this study), the pineals in 25 species belong to this category. Large bats, such as Pteropus tonganus and P. poliocephalus, are endowed with type AB pineals which are longer than their greatest width but still remain recessed under the hemispheres (Figs. 9a, b; Legait, Bauchot & Contet-Audonneau, 1976a). Dobsonia species, which form a medium-sized group among megachiropterans (Table 1), exhibit type a,#C pineals with a wide base straddling the third ventricle, extending dorsally as a solid column becoming superficial to the hemispheres and spreading over the cerebellum in a mushroom-like manner (Figs. 2-4). Thus far, an ,b/C type pineal has been described only in the family Nycteridae (Bhatnagar et al. 1986). The generally deep location of the pineal in the megachiropteran fruit bats appears to be reflected in their roosting behaviour. Most fruit bats roost in trees, during the daylight; even cave dwelling fruit bats inhabit the lighted areas within their caves. Species that remain secluded in the darkest nooks and corners during daylight hours, e.g. hipposiderids, rhinolophids and nycterids, exhibit pineal organs that reach the dorsal surface of the brain. Dobsonia species are similar to this group, since they are cave dwellers (Hamilton-Smith, 1966; Dwyer, 1975). It is interesting to note that the Dobsonia species in this study were collected from deep dark caves of New Guinea in light of the fact that Smith & Hood (1981) did not find any Dobsonia praedatrix roosting in caves. The extremely large pineal in Dobsonia species also is in contrast with the situation in birds where the species with the largest pineal are said to be more diurnal (See Vollrath, 1981, p. 437). However, nothing more can be said about the enormous size and topography of this pineal and its significance until the ecology and biogeography ofDobsonia species are well established. There is hardly any information on these highly intriguing bats from Papua New Guinea. Pineal-third ventricle interrelationships and exposure of the pinealocytes to cerebrospinalfluid (CSF) Developmentally, the mammalian pineal organ is derived from a diencephalic ependymal evagination in the intercommissural zone between the habenular and posterior commissures (Bargmann, 1943). It is no surprise, therefore to find that the 160 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN Table 4. Data on the large pineal organs in some animals Pineal Body Brain weight weight Weight Length Width Volume Size Species (kg) (g) (g) (mm) (mm) (mm3) + CV index*

Bata Dobsonia praedatrix 0-184 303 5 33 4-51 16 345 19 6 4393 Emuc Dromaeus 31 25-10 0 10 1000 700 9653 599 novaehollandiae 34 27-70 - - Walrus Odobenus rosmarus Adult 1260' 30OOJ 18-O0 667d 1126d Neonatal 45-68' 3.44k Seals Common seal Phoca vitulina 90 00' 0-7879k 16-00J 8-00 71'60 52 2156 Elephant seal Mirounga leonina Neonatal 35.00b 1-55b 14'96-10 Adult 35000b 0-70b 25-32' 5-9' 1350-00b 1*90b 183398 711 Weddell seal Leptonychotes weddelli, Neonatal 34900' 3-60b 29 00' 22-00' Adult x 395-00' l0e 20-30e 1-000e 9165-25 - 923 0-50b Tarsier Tarsius syrichta 00868g 4169 0-9969 465 Humanm 60-00g 13709 0-1386 12-00' 8-00' 46-12 176

* The size indices were calculated using the vespertilionid regression line (see Figure 14). a This study; b Bryden et al. (1986); Cobb & Edinger (1962); d Crile & Quiring (1940); e Cuello & 1 Tramezzani (1969); Kopsch (1953); g Legait et al. (1976b); h Legait & Legait (1977); ' Tedman (1977); J Turner (1888); k Vollrath (1981); ' Walker (1983); m Data on human pineal, which is not large compared to other pineals, is included for comparison.

pineal organ in bats is intimately related to the third ventricle and consequently to the CSF. However, this relationship is particularly intimate in megachiropterans, where the pineal parenchyma is exposed directly to the CSF due to a deficient ependymal lining. As a result, pinealocyte clusters protrude into the third ventricle. This situation was observed not only in the four Dobsonia species, but also in other megachiropterans, e.g. Epomops, Scotonycteris, Casinycteris and Pteropus. In the fruit bat (Eidolon helvum), close contact between the pineal tissue and the CSF has been observed (Pevet, 1983). CSF-contacting pinealocytes have been described also in other mammals (Hewing, 1982, 1984; Welsh, 1983; also see Vollrath, 1981). It remains to- be determined whether similar relationships exist in the Microchiroptera. Even though the significance of CSF-contacting pinealocytes is poorly understood, it is necessary to point out here that this aspect of pineal morphology need not and should not be overemphasised. Open communication of pinealocytes with the CSF in a few species does not mean that discharge of pineal secretions into the third ventricle is an important aspect of pineal function, even in these species. Since pineal secretory products are considered to be localised predominantly at the ends of the perivascular extensions of the pinealocytes, the systemic circulation is presumably the principal, Megachiropteran pineal organ 161 and perhaps the only, means by which pineal influence is conveyed to the brain and other target organs. Pineal-brain interrelationships and the feasibility ofpinealectomies in bats Pineal-brain interrelationships in mammals are just beginning to receive attention (Mollgaard & M0ller, 1973; M0ller & Korf, 1987; Romijn, 1975; Stehle, Reuss & Vollrath, 1987). Most data on the pineal are from rodents and other laboratory animals and relate primarily to structure. In this context only incidental remarks refer to other closely related brain regions such as the third ventricle with its recesses and choroid plexus, the habenular and posterior commissures, the surrounding vascula- ture, including sinuses, and the meningeal coverings. -Bats appear to be an ideal group of animals in which to study pineal-brain interrelationships. Not only has every type of pineal organ from Type A, AB, ABC, a,xC to aC now been reported in bats, but the highly variable nature of the pineal-brain relationship has also been postulated (Bhatnagar et al. 1986). Such relationships range from species in which the pineal organ remains minimally connected to the brain, as in the vampire bat (Bhatnagar, 1988), to those such as megachiropteran fruit bats in which interconnections on a much broader level are apparent. Even within the four Dobsonia species such variation has been observed. Only the most posterior fibres of the habenular commissure pass through the pineal base in D. praedatrix and D. inermis, whereas in D. moluccensis and Dobsonia species the habenular commissure maintains a course anterior to the pineal isolating the two structures. Such variation is also observed in other fruit bats, e.g. Pteropus tonganus, Scotonycteris zenkeri and Megaloglossus woermanni, and in several other micro- chiroptera (see Table 2 in Bhatnagar et al. 1986). A close examination of the pineal-brain interrelation is justified since it is highly relevant to pinealectomy, which is a commonly performed procedure. Regardless of the extent of pineal-commissural interrelation it is necessary to emphasise that the pineal maintains a close connection with the diencephalic roof through the choroid plexus of the third ventricle and through the suprapineal recess. Additionally, the habenular commissure, which also courses through the pineal, frequently is joined in this by the posterior commissure. In yet other species, both commissures remain anatomically separate from the pineal as in the vampire bat (Bhatnagar, 1988). The vasculature and meningeal relations of the pineal add to the importance and intricacy of the pineal-brain connection. Surgical management of pinealectomies ought to include consideration of the intricate topography exhibited by the pineal in relation to other brain structures. Postpinealectomy assessment using histotechniques is imperative to ensure that all pineal tissue has been removed and that no other brain areas have been damaged. A quick scan of the recent literature reveals the irony of the so called 'successfully executed' pinealectomies. Considering pineal-brain topography and especially the generally deep location of the megachiropteran pineal relative to the cerebral hemispheres, the feasibility of pinealectomy appears to be extremely limited and difficult, if not entirely impossible. Intrapineal neurons The history of intrapineal neurons has been chequered and interesting. Statements on the rare occurrence of nerve cell bodies in mammalian pineal organs (Quay, 1965, 1970), and the report of a single neuron in the pineal of Pteropuspoliocephalus (Kenny, 1965) have created the impression that this is an unusual occurrence. Lack of 162 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN comparative data at the time of these investigations and superficial study of the tissue may have led to such conclusions. Recent data on 105 species of bats (Bhatnagar et al. 1986; Table 1) have reported intrapineal neurons in several species of Mega- and Microchiroptera. Among the former, large numbers of neurons were observed in Dobsonia praedatrix. Ganglionic collections of neurons in association with blood vessels have also been noted in Pteropus poliocephalus pineal (unpublished observations). Regardless of their location, intrapineal neurons have always been observed close to blood vessels. Present investigations on chiropteran pineals strongly support the following general conclusions which may apply equally to the intrapineal neurons in other species: (i) presence of intrapineal neurons is to be expected and is not a rare phenomenon; (ii) such neurons are found in the connective tissue closely associated with blood vessels both peripherally and intrinsically; (iii) these nerve cell bodies are ganglionic and appear multipolar; (iv) they resemble neurons in other accessory ganglia such as those reported in the vomeronasal organ of Artibeus (Bhatnagar & Kallen, 1974). Little can be said about the significance of their proximity to vessels at this time. It would be inappropriate to assign to them a vasomotor role based simply on their location. A thorough investigation of their projections will be necessary before any conclusions can be drawn. Whatever their significance may be, the fact that intrapineal neurons are not found in every species, and even when they are, their scarcity speaks against attaching much significance to them. However, intrapineal neurons have been described also in other mammals including monkeys (Ueck, 1979; Vollrath, 1981 for reviews). Recently Ichimura, Arikuni & Hashimoto (1986) reported "about 70 nerve-cell bodies" deep in the central part of one half of a pineal organ of the monkey, Macaca fuscata. No comment on the vascular relations of this 'ganglion-like' structure is made in this report, but it is interesting to note the close association with a blood vessel of the three nerve cells shown in their Figure 1. An important contribution made by Ichimura et al. is their observation of various types of synaptic formations on pinealocytes and pineal neurons. Their conclusion that the monkey pineal neurons are derived from pineal ganglion cells and not from postganglionic parasympathetic neurons is worthy of further inquiry. Seen in this light Dobsonia praedatrix, among bats, is an ideal species for investigating intrapineal neurons. Vascularity of the pineal organ Megachiropteran pineal organs are highly vascularised. In the absence of any detailed study it is safe to assume that the main arterial supply to the megachiropteran pineal is provided by branches of the posterior choroidal arteries with venous return into the great cerebral vein as described for other mammals. Even a quick look at a frontal section of the pineal region of Pteropus tonganus (Fig. 9a) is enough to convince one of the enormous arterial supply of the region. Surgical management of such a 'vascular cuff' would be challenging during pinealectomy. In Dobsonia praedatrix unlike most other chiropteran species, major arterial branches enter the pineal, give off arterioles that follow the connective tissue trabeculae and finally break into capillaries. These connective tissue trabeculae appear to define pineal lobules, especially in the superficial parts of the organ. A detailed investigation of the pineal vascular system in Chiroptera is long overdue. Are there distinct cortical and medullary regions in the megachiropteran pineal? Distinct lobes and cell clusters or cords have been observed frequently in bat pineals Megachiropteran pineal organ 163 (Quay, 1965). In several species of both Mega- and Microchiroptera, morphologically distinct dorsal and ventral subdivisions of the pineal have been observed (Bhatnagar et al. 1986). A wide, clearly demarcated band of cortical tissue was observed in the pineal of Dobsonia praedatrix stained with Masson's or Gomori trichrome procedures. The difference at the gross morphological level was in the packing density of pinealocytes and the amount of connective tissue in the two zones (see Results). Detailed histological and ultrastructural investigation will be necessary to determine whether there are structural differences between the parenchymal cells of this and the inner pineal region. Are the species with large pineals more diurnal and vice versa? It has been reported that the pineal is absolutely larger in several diurnal animals, e.g. birds (Starck, 1955; Quay, 1965, 1972) and pinnipeds, such as seals and walruses (Turner, 1888). Bats, being strictly nocturnal, do not conform to this principle. Pteropus species that roost in tree canopies in bright daylight are not endowed with an absolutely and relatively large pineal organ. On the contrary, the size of bat pineals appears to be governed by the principle that darkness enhances the pineal activity and therefore increases the size of the pineal. The largest bat pineals are observed in Dobsoniapraedatrix (SI, 4393), and the rhinolophids, Rhinolophus trifoliatus (SI, 1966), and R. luctus (SI, 1437) (Bhatnagar et al. 1986), species that roost in dark cave interiors. Volume When discussing differences in pineal volume between species, it should be kept in mind that intraspecific variability of pineal size can be rather large. The extreme variability in human pineal weight (from 28 to 834 mg, n = 500) apparently associated with factors such as stress, disease processes and involution has been pointed out by Legait, Bauchot, Stephan & Contet-Audonneau (1976b; also see Vollrath 1981, p. 38). Moreover, functional activity of the pineal gland has an important effect on pineal volume. In the dormouse (Glis glis), pineal volume is reduced in July and enlarged from October to January, when the animals hibernate (Legait et al. 1975). In our sample such differences were not observed; megachiropterans are tropical or subtropical mammals, where seasonal influences are not pronounced. Furthermore, brains of any one species were often taken within a few days of each other (e.g. Dobsonia praedatrix brains, between 29 December 1981 and 6 January 1982, see Table 4). The coefficients of variability of pineal size in megachiropteran species vary between 3-1 and 50 8 %. Species with small pineals (small SIs) tend toward greater variation in pineal size differences then do species with large pineals. However, the differences in pineal size between species are so large that intraspecific variability does not conceal them. Indices of pineal size in the 36 species of Megachiroptera range from 33 to 4393 relative to the base for vespertilionids, with their average SI of 100; that means there are megachiropteran species that have a relative pineal size only one third that of a typical vespertilionid (Macroglossus minimus, SI, 33), and others with nearly 44 times as much (Dobsonia praedatrix, SI, 4393). The range of vespertilionid pineal SIs was found to be from 18 to 290 (Bhatnagar et al. 1986); eight of the 36 megachiropterans exceed the upper limit reached by vespertilionids. The mean SI of megachiropteran pineals is 323 (n = 36), indicating a threefold enlargement in comparison to vespertilionids. Other microchiropteran families with high pineal SIs were the Rhinolophidae (n = 4; SI,951), Megadermatidae (n = 5; SI, 335) and Emballonuridae (n = 11; SI, 249). Rhinolophids owed their high average SI to two species with 164 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN relatively large pineals: Rhinolophus luctus and R. trifoliatus, but none reaches the extreme of the Dobsonia praedatrix pineal, either absolutely or relatively. Pineal size indices have been reported for a large number of insectivores, prosimians and simians, including man, by Legait et al. (1976b). These indices, however, cannot be used for direct comparison because they are based on a different reference group and are calculated from another slope of the regression line, fitting insectivores and primates. Recalculation of these SIs on the vespertilionid base shows that they range between 0.1 and 108 in Insectivora, between 20 and 465 in prosimians and between 8 and 239 in simians. Man, with a size index of 176 does not exceed the range of non- human primates. The maximum value among the non-human primates is that for Tarsius syrichta, a nocturnal South East Asian prosimian (SI, 465). Reports on exceptionally large pineals in certain vertebrate species (Starck, 1955; Cobb & Edinger, 1962; Turner, 1888; Cuello & Tramezzani, 1969; Tedman, 1977; Bryden et al. 1986), however, have been deficient in providing data on body weight, brain weight and pineal weight or volume, parameters that are necessary for the evaluation of relative pineal size. So the question arises, how do these values in other vertebrates compare with the pineal size in Dobsonia praedatrix? Of course, there are absolutely larger pineals than the one found in Dobsonia (see Table 4). The 1-9 g pineal in an adult elephant seal reported by Bryden et al. (1986) points to a pineal volume of 1834 mm3 (if it is assumed that the specific gravity of pineal tissue is similar to that of the brain tissue, which is 1 036). This would mean that the pineal of an elephant seal is, in absolute size, more than 100 times larger than that of Dobsonia. But since body size also influences the pineal size, one has to take into account the difference between the 1350 kg of body weight in Mirounga leonina and the 184 g of body weight in Dobsonia praedatrix. The slopea (= 0 735) in a pineal volume/body weight plot indicates that a 10-fold increase in body weight is accompanied by a 7 35-fold enlargement of pineal volume, at least in bats. If a similar relation is also valid in Pinnipedia, the pineal SI of an adult elephant seal (1834 mm3 pineal volume, 1350 kg body weight) would be 711, i.e. lower than the relative pineal size in Dobsonia. The highest pineal SI among the species listed in Table 4 is, beside Dobsonia, that of Phoca vitulina. It should be emphasised that intraspecific development of pineal size from the neonatal to the adult may follow different allometric rules. The reference line used in our studies is only valid for a comparison between adult animals. As to the possible functions of such a large pineal organ in an adult bat, it would be unwise to offer even speculative comments before enough data on the neurobiology of Dobsonia praedatrix become available. In general, the size of an organ is directly proportional to its overall activity level. Based on this principle, the absolutely and relatively largest pineals known in any species ought to be able to provide answers to the intriguing question - what does the pineal do? Moreover, the comparative size of the pineal in the four species of Dobsonia investigated in this report offers adequate material for insights into such questions as which selective evolutionary pressures could create a species as distinct as D. praedatrix with such an enormous pineal organ compared to the other three Dobsonia? And finally, why is the pineal organ so large in Dobsonia praedatrix? Only future investigations can resolve the many questions presented by the intriguing pineal morphology of Dobsonia.

SUMMARY This investigation is based upon the pineal organs of 92 specimens of 36 species of the family Pteropodidae (Mammalia, Chiroptera). The size of the megachiropteran pineal correlates well with body size (r = 0 864), confirming the former conclusions Megachiropteran pineal organ 165 that generally larger bodied bats have larger pineals. The range of the pineal size index in 36 megachiropteran species is from 33 to 4393. In most species the pineal organs are small, deeply recessed under the cerebral hemispheres and of Type A (except in Dobsonia and Pteropus, where they are of Type a,xC and AB, respectively). Morphological and volumetric data gathered from serially sectioned brains include body and brain weights, pineal type, dimensions, volume and size index for each species. There are distinct dorsal and ventral subdivisions of the pineal in some species and a clear separation of pineal parenchyma into cortical and medullary regions in others. In several species where overlying ependyma is lacking pinealocyte clusters communicate freely with the CSF. Groups of intrapineal neurons are noted in the connective tissue beside blood vessels. The habenular commissure shows much interspecific variation in its course through the pineal. Detailed examination of pineal-brain relationships clearly suggests that, due to the generally deep location of the pineal in relation to cerebral hemispheres, pinealectomies in the species studied may be extremely difficult, if not entirely impossible. The absolutely and relatively largest pineal organ among bats, and relatively perhaps among all vertebrates, has been discovered in the New Guinean naked-backed bat, Dobsonia praedatrix, with pineal size index of 4393, and a volume of 16-3447 mm3, which is 0-56 % of the brain. This a,cC-type, mushroom-shaped, solid and compact pineal organ measures 5-33 x 4-51 mm. The cortical and medullary parenchyma are divided into lobes by large calibre blood vessels along which numerous intrapineal neurons are observed. A smaller but similarly shaped pineal is noted in the other three Dobsonia. Data on the largest known pineals in ratitae birds, seals and walruses have been compared with that of D. praedatrix and the human pineal. This study supports the hypothesis that pineal development may reflect dependence on habitat and possibly other related factors. In grateful acknowledgement of the excellent help rendered during this investigation we wish to recognise Dr John Nelson, Victoria, Australia (specimen collection), Clare Roberg, Monika Martin, Sigrid Wadle, Barbara Schmittenbecher, and Glynton Hammond (histology), Helga Grobecker and Indu Bhatnagar (photography), Michael Stephan (computer programming), Helma Lehmann (secretarial work) and Susan Hodge (typing). Professors James B. Longley, Fred J. Roisen and Frederick K. Hilton critically read the manuscript and offered valuable suggestions. Figures 3 and 4 were completed under the guidance of Professor Hilton. The senior author was supported by a sabbatical leave from the University of Louisville and by Max-Planck- Institut fur Hirnforschung, Frankfurt a.M. during the course of this project.

REFERENCES BARGMANN, W. (1943). Die Epiphysis cerebri. In Handbuch der mikroskopischen Anatomie des Menschen (ed. W. v. Mollendorfl), vol. VI, part 4, pp. 309-502. Berlin: Springer. BHATNAGAR, K. P. (1988). Ultrastructure of the pineal body of the common vampire bat, Desmodus rotundus. American Journal of Anatomy 181, 163-178. BHATNAGAR, K. P., FRAHM, H. D. & STEPHAN, H. (1986). The pineal organ of bats: a comparative morphological and volumetric investigation. Journal ofAnatomy 147, 143-161. BHATNAGAR, K. P. & KALLEN, F. C. (1974). Morphology of the nasal cavities and associated structures in Artibeus jamaicensis and Myotis lucifugus. American Journal ofAnatomy 139, 167-190. BRYDEN, M. M., GRIFFITHS, D. J., KENNAWAY, D. J. & LEDINGHAM, J. (1986). The pineal gland is very large and active in newborn antarctic seals. Experientia 42, 564-566. CHANG, N., BHATNAGAR, K. P., TSENG, M. T. & KARIM, K. B. (1987). Ultrastructure of the pineal gland of the tropical bat Rousettus leschenaulti. Acta anatomica 128, 194-203. COBB, S. & EDINGER, T. (1962). The brain of the emu (Dromaeus novaehollandiae, Lath). I Gross anatomy of the brain and pineal body. Breviora (Cambridge, Mass.) 170, 1-18. 166 K. P. BHATNAGAR, H. D. FRAHM AND H. STEPHAN CRILE, G. & QUIRING, D. P. (1940). A record of the body weight and certain organ and gland weights of 3690 animals. The Ohio Journal of Science 40, 219-259. CUELLO, A. C. & TRAMEZZANI, J. H. (1969). The epiphysis cerebri of the Weddell seal: its remarkable size and glandular pattern. General and Comparative Endocrinology 12, 153-164. DWYER, P. D. (1975). Notes on Dobsonia moluccensis (Chiroptera) in the New Guinea Highlands. Mammalia 39, 113-118. HAMILTON-SMITH, E. (1966). The geographical distribution of Australian cave-dwelling Chiroptera. International Journal of Speleology II, 91-104, pls. 20-24. HEWING, M. (1982). Pinealocytes contacting the cerebrospinal fluid of the suprapineal recess in the Mongolian gerbil (Meriones unguiculatus). Cell and Tissue Research 222, 177-185. HEWING, M. (1984). Seasonal variations in the cerebrospinal fluid-contacting area of the pineal gland in the golden hamster (Mesocricetus auratus). Anatomy and Embryology 169, 91-96. ICHIMURA, T., ARIKUNI, T. & HASHIMOTO, P. H. (1986). Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations. Cell and Tissue Research 244, 569-576. KENNY, G. C. T. (1965). The innervation of the mammalian pineal body (a comparative study). Proceedings of the Australian Association of Neurology 3, 133-140. KoPSCH, F. (1953). Lehrbuch und Atlas der Anatomie des Menschen, Bd. III. Nervensystem - Sinnesorgane. 18. Aufl. Leipzig: Georg Thieme. LEGAIT, H., Roux, M., DUSSART, G., RICHOUX, J. P. & CONTET-AUDONNEAU, J.-L. (1975). Donnees morphometriques sur la glande pineale du Loir (Glis glis) et du Lrot (Eliomys quercinus) au cours du cycle annuel. Comptes rendus des seances de la Sociite de biologie 169, 132-136. LEGAIT, H., BAUCHOT, R. & CONTET-AUDONNEAU, J.-L. (1976a). Etude des correlations liant les volumes des lobes hypophysaires et de l'epiphyse au poids somatique et au poids de l'encephale chez les chiropteres. Bulletin de l'Association des anatomistes (Nancy) 60, 175-187. LEGAIT, H., BAUCHOT, R., STEPHAN, H. & CONTET-AUDONNEAU, J.-L. (1976b). Etude des correlations liant le volume del'epiphyse aux poids somatique et encephalique chez les rongeurs, les insectivores, les chiropteres, les prosimiens et les simiens. Mammalia 40, 327-337. LEGAIT, H. & LEGAIT, E. (1977). Contribution a l'etude de la glande pin6ale humaine. Etude faite a l'aide de 747 glandes. Bulletin de l'Association des anatomistes (Nancy) 61, 107-121. M0LLER, M. & KORF, H.-W. (1987). Neural connections between the brain and the pineal gland of the golden hamster (Mesocricetus auratus). Tracer studies by use of horseradish peroxidase in vivo and in vitro. Cell and Tissue Research 247, 145-153. MOLLGAARD, K. & M0LLER, M. (1973). On the innervation of the human fetal pineal gland. Brain Research 52, 428-432. QUAY, W. B. (1965). Histological structure and cytology of the pineal organ in birds and mammals. In Structure and Function of Epiphysis cerebri (ed. J. A. Kappers & J. P. Schade), Progress in Brain Research 10, 49-86. New York: Elsevier. QUAY, W. B. (1970). Pineal organ. In Biology ofBats (ed. W. A. Wimsatt), pp. 311-318. New York: Academic Press. QUAY, W. B. (1972). Infrequency of pineal atrophy among birds and its relation to nocturnality. Condor 74, 33-45. PEVET, P. (1983). Anatomy of the pineal gland of mammals. In The Pineal Gland (ed. R. Relkin), pp. 1-75. Amsterdam: Elsevier. ROMIJN, H. J. (1975). Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.). III. An electron microscopic investigation of the innervation. Cell and Tissue Research 157, 25-51. SMITH, J. D. & HOOD, C.S. (1981). Preliminary notes on bats from the Bismarck Archipelago (Mammalia: Chiroptera). Science in New Guinea 8, 81-121. STARCK, D. (1955). Die endokraniale Morphologie der Ratiten, besonders der Apterygidae und Dinornithidae. Morphologisches Jahrbuch 96, 14-72. STEHLE, J., REuss, S. & VOLLRATH, L. (1987). Electrophysiological characterization of the pineal gland of golden hamsters. Experimental Brain Research 67, 27-32. STEPHAN, H., FRAHM, H. D. & BARON, G. (1981). New and revised data on volumes of brain structures in insectivores and.primates. Folia primatologica 35, 1-29. TEDMAN, R. A. (1977). The pineal gland of the Weddell seal and the southern elephant seal. Journal of Anatomy 124, 524. TURNER, W. (1888). The pineal body (Epiphysis cerebri) in the brains of the walrus and seals. Journal of Anatomy and Physiology 22, 300-303. UECK, M. (1979). Innervation of the vertebrate pineal. In The Pineal Gland of Vertebrates Including Man (ed. J. A. Kappers & P.Pevet). Progress in Brain Research 52, pp. 45-88. New York: Elsevier. VOLLRATH, L. (1979). Comparative morphology of the vertebrate pineal complex. In The Pineal Gland of Vertebrates Including Man (ed. J. A. Kappers & P. Nvet). Progress in Brain Research 52, pp. 25-38. New York: Elsevier. VOLLRATH, L. (1981). The Pineal Organ. In Handbuch der mikroskopischen Anatomie des Menschen (ed. A. Oksche & L. Vollrath), vol.VI, part 7. Berlin: Springer. WALKER,E. P. (1983). Mammals of the World, 4th ed., vols. 1-2. Baltimore: Johns Hopkins. WELSH, M. G. (1983). CSF-contacting pinealocytes in the pineal recess of the Mongolian gerbil: A correlative scanning and transmission electron microscopy study. American Journal of Anatomy 166, 483-493.