Hibernation, Puberty and Chronic Disease in Troglodytes from Spain half a million years ago

Antonis Bartsiokas Corresp., 1 , Juan Luis Arsuaga 2

1 Department of History & Ethnology, Democritus University of Thrace, Komotini, Greece 2 Departmento de Paleontología, Universidad Complutense de Madrid, Madrid, Spain

Corresponding Author: Antonis Bartsiokas Email address: [email protected]

Both animal hibernation (heterothermy) and human are characterized by high levels of serum .To test the hypothesis of hibernation in an extinct human species, we examined the hominin skeletal collection from Sima de los Huesos, Cave Mayor, Atapuerca, Spain, for evidence of .We studied the morphology of the fossilized bonesby using macrophotography, microscopy, histology and CT scanning.We found trabecular tunneling and fibrosa, subperiosteal resorption,‘rotten fence post’ signs,brown tumours, subperiosteal new bone, chondrocalcinosis, rachitic osteoplaques and empty gaps between them, craniotabes, and beading in ribs mostly in the adolescent population of these hominins. Since many of the above lesions are pathognomonic, these extinct hominins suffered annually from renal , secondary hyperparathyroidism, and renal osteodystrophy associated with -Mineral and Bone Disorder(CKD- MBD). We suggest these diseases were caused by non-tolerated hibernation in dark cavernous hibernacula.This is evidenced by the rachitic osteoplaques and the gaps between them mainly in the adolescent individuals along with the evidence of healing mainly in the adults. The sublayers in the rachitic osteoplaquespoint to bouts of arousal from hibernation. The strong projection of the external lip of the femoral trochlea, the rachitic osteoplaques with the empty gaps between them,the “rotten fence post sign”, and the evidence of annual healing caused by non-tolerated hibernation in adolescent individuals, also point to the presence of annuallyintermittent puberty in this population.The hypothesis of hibernation is consistent with the genetic evidence and the fact that the SH hominins lived during a glacial period. The presentwork will provide a new insight into the physiological mechanism of early human metabolism which could help in determining the life histories and physiologies of extinct human species.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 1 Hibernation, Puberty and Chronic Kidney Disease in Troglodytes from Spain half a million 2 years ago 3 4 Antonis Bartsiokas1, Juan-Luis Arsuaga2,3

5 1Laboratory of Physical Anthropology, Department of History and Ethnology, Democritus 6 University of Thrace, 69100 Komotini, Greece

7 2Centro Mixto Universidad Complutense de Madrid-Instituto de Salud Carlos III de Evolución y 8 Comportamiento Humanos, 28029 Madrid, Spain

9 3Departmento de Paleontología, Universidad Complutense de Madrid, Madrid, Spain,

10 11 12 Corresponding Author: 13 Antonis Bartsiokas, 14 P. Tsaldari 1, Komotini, 69100, Greece 15 Email address: [email protected] 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 43 44 45 46 Abstract 47 48 Both animal hibernation (heterothermy) and human renal osteodystrophy are characterized by 49 high levels of serum parathyroid hormone. To test the hypothesis of hibernation in an extinct 50 human species, we examined the hominin skeletal collection from Sima de los Huesos, Cave 51 Mayor, Atapuerca, Spain, for evidence of hyperparathyroidism. We studied the morphology of 52 the fossilized bones by using macrophotography, microscopy, histology and CT scanning. We 53 found trabecular tunneling and osteitis fibrosa, subperiosteal resorption, ‘rotten fence post’ signs, 54 brown tumours, subperiosteal new bone, chondrocalcinosis, rachitic osteoplaques and empty 55 gaps between them, craniotabes, and beading in ribs mostly in the adolescent population of these 56 hominins. Since many of the above lesions are pathognomonic, these extinct hominins suffered 57 annually from renal rickets, secondary hyperparathyroidism, and renal osteodystrophy associated 58 with Chronic Kidney Disease - Mineral and Bone Disorder (CKD-MBD). We suggest these 59 diseases were caused by non-tolerated hibernation in dark cavernous hibernacula. This is 60 evidenced by the rachitic osteoplaques and the gaps between them mainly in the adolescent 61 individuals along with the evidence of healing mainly in the adults. The sublayers in the rachitic 62 osteoplaques point to bouts of arousal from hibernation. The strong projection of the external lip 63 of the femoral trochlea, the rachitic osteoplaques with the empty gaps between them, the “rotten 64 fence post sign”, and the evidence of annual healing caused by non-tolerated hibernation in 65 adolescent individuals, also point to the presence of annually intermittent puberty in this 66 population. The hypothesis of hibernation is consistent with the genetic evidence and the fact 67 that the SH hominins lived during a glacial period. The present work will provide a new insight 68 into the physiological mechanism of early human metabolism which could help in determining 69 the life histories and physiologies of extinct human species. 70 71 Key words: Hibernation; Sima de los Huesos (SH); Atapuerca; Chronic Kidney Disease (CKD); 72 Renal Rickets; Secondary Hyperparathyroidism (2HPT); Renal Osteodystrophy (ROD); Rachitic 73 osteoplaques (ROPs).

74

75

76 Abbreviations used

77 Renal Osteodystrophy ROD; Parathyroid Hormone PTH; Secondary Hyperparathyroidism 78 2HPT; Primary Hyperparathyroidism 1HPT; Osteitis Fibrosa Cystica OF; 79 OM; Sima de los Huesos SH; Rheumatoid arthritis RA; Chronic Kidney Disease - Mineral and 80 Bone Disorder CKD-MBD; Brown Adipose Tissue BAT; Beige Adipose Tissue 81 BeAT; Gastrointestinal GI; Subperiosteal new bone PNB; Rachitic Osteoplaque 82 ROP ; pyrophosphate dihydrate disease CPPD; Subperiosteal resorption SPR; 83 BT; Lamina dura LD. 84 85

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Renal Osteodystrophy ROD Brown Adipose Tissue BAT Parathyroid Hormone PTH Beige Adipose Tissue BeAT Secondary Hyperparathyroidism 2HPT Gastrointestinal GI Primary Hyperparathyroidism 1HPT Subperiosteal new bone PNB Osteitis Fibrosa Cystica OF Rachitic Osteoplaque ROP Osteomalacia OM Calcium pyrophosphate dihydrate Sima de los Huesos SH disease CPPD Rheumatoid arthritis RA Subperiosteal resorption SPR Chronic Kidney Disease - Mineral and Brown Tumor BT Bone Disorder CKD- Lamina dura LD MBD

86

87 1. Introduction

88 Many researchers recently support the view that hibernation (heterothermy) is possible in 89 humans after some molecules - such as 5’-AMP and hydrogen sulfide1 - were identified as 90 inducing a hypometabolic state in non-hibernators analogous to hibernation1,2,3. Their 91 experiments show that under certain circumstances non-hibernators can hibernate. In addition, 92 many adult and non-adult humans have Beige Adipose Tissue (BeAT)4 and/or Brown Adipose 93 Tissue (BAT)5, a tissue used by hibernators during arousals from hibernation bouts6. Both animal 94 hibernation and human renal osteodystrophy (ROD) are characterized by high parathyroid 95 hormone (PTH)7,8. To test whether there is hibernation in extinct humans we searched for 96 evidence of ROD in their skeletal material. To this end, we studied the enormous hominin 97 skeletal collection from Sima de los Huesos (“Pit of bones” known as “SH”), Cave Mayor, 98 Atapuerca. The SH site is a deep shaft in the Cave Mayor of Sierra de Atapuerca (Spain) with 99 thousands hominin bones belonging to at least 28 individuals. They are around 430 thousands 100 years old (430 Ka) and they belong to a stem group for Neanderthals and modern humans9 101 (Homo heidelbergensis?). The site can be regarded as a mass grave in the sense that it is a place 102 of deliberate disposal of multiple dead10,11. The bones are disarticulated and commingled but 103 were deposited during the same sedimentation period12 and are in a good state of preservation to 104 allow the methods we used. 105 We present a histologic, paleopathologic, and ontogenetic analysis of the SH hominins and its 106 effects on the physiology of this species. Results herein emphasize the possibilities for derivation 107 of growth patterns from bone microstructure and pathology of extinct human species, and 108 elucidate important aspects of the life history strategies employed by an extinct human species 109 (see below e.g. 4.1). The present work will provide a new insight into the physiological 110 mechanism of early human metabolism during hibernation which could help not only in 111 determining the life histories and physiologies of extinct human species, but could also have 112 profound implications for understanding the pathophysiological basis of various diseases13. 113 Hibernation is best regarded as a physiologic adaptation to anticipated famine that involves 114 cessation of food intake and a decrease in metabolic rate, body temperature, and heart rate in a 115 niche of constant darkness138-141. In nearly all hibernators, torpor is interrupted by bouts of 116 arousal161,162. Because the decrease in metabolic rate, body temperature, and heart rate was not 117 possible to measure in this population, we tried to find evidence for cessation of food intake and

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 118 bouts of arousal. We found this evidence from its effects, namely in the form of renal rickets 119 ( deficiency), bone growth arrest and sublayers of the rachitic osteoplaques. 120 It should be noted that here we studied an extinct human species and a fossilized population with 121 no soft tissues preserved. So, our diagnoses of the lesions and hibernation relied on bone 122 histological characteristics. The science of paleopathology relies mainly on bone histology and 123 bone external morphology to obtain diagnosis. Thus, it was not possible to measure parameters 124 such as fat content, PTH, and other blood parameters to determine the levels of prehibernating 125 fattening and the stress that followed. 126 We have to emphasize that hibernations are not always healthy. There are cases where all of fat 127 and vitamin D stores are depleted resulting in CKD and its associated pathologies. The adults in 128 the present case of course underwent hibernation but they didn’t show any pathologies - only the 129 healing effects of it - as they didn’t have so great energy demands as the adolescents. Here, are 130 some examples of non-tolerated hibernation in seasonal hibernators: 131 The case of Mixnitz cave where the remains of cave bear (Ursus spelaeus) belong to individuals 132 that died during hibernation as they developed various pathologies including rickets, kyphosis, 133 enamel hypoplasia, and OF because they were unable to build up a sufficient store of fat 73,75. OF 134 is actually not reported by Breuer73 but is shown in a trabecular bone section in his Fig. 2 of Taf. 135 XCII, misidentified as vascular gaps. The juvenile-dominated population structure in the 136 Mokrica cave (Slovenia) is characteristic of hibernation-related mortality in typical cave bear 137 sites. The majority of deaths occurred during hibernation or in short posthibernation period78. 138 Many cases of rickets and ostemalacia have also been reported in fossil bears165. Small 139 hibernating mammals such as bats158,159, hamsters160 and ground squirrels experience PTH- 140 induced OF and osteoporosis7. In the little brown bat, Myotis lucifugus lucifugus, a marked 141 hyperparathyroidism (HPT) existed during hibernation30. Black bears that die during hibernation 142 are uremic74. McGee-Lawrence et al.7 have noticed that the impairment of renal function in 143 hibernating bears is similar to that of humans with renal failure in that the glomerular filtration 144 rate decreases in both species. Indeed, in small hibernators and American black bears there is 145 reduction or cessation of the glomerular filtration rate166-168 experienced during a hibernation 146 bout169. In hibernating woodchucks, the increased secretion of Adrenocorticotropic Hormone 147 (ACTH) may play a role in the genesis of renal glomerular disease which is an important cause 148 of mortality170. Cortisol levels are increased under the stimulation of ACTH during hibernation 149 as well as in 2HPT perhaps in order to aid in fat catabolism. Excessive secretion of cortisol may 150 explain proteinuria in the hibernating dormouse Muscardinus avellanarius166, uremia in black 151 bears74 and death in the hibernating mouse lemur Microcebus murinus from chronic 152 glomerulonephritis88. Therefore, hibernators may suffer from rickets, HPT, and OF if they do not 153 possess sufficient fat reserves. These diseases are all expressions of ROD consistent with CKD. 154 These bear pathologies occur also in ROD in humans and indicate reduced serum 25OHD 155 because cessation of glomerular filtration rate in ROD and vitamin D deficiency in CKD results 156 in plasma reduction of 25OHD76. When 25OHD decreases, PTH increases149.

157 Although hibernators are capable of reaching physiological states that would be lethal to 158 modern humans, various degrees of hypometabolism and hypothermia might have been tolerated 159 by the latter142. The notion that humans can undergo a hypometabolic state analogous to 160 hibernation may sound like science fiction but the fact that hibernation is used by very primitive 161 mammals (placentals, marsupials, monotremes) and primates (bushbabies, lorises, dwarf and 162 mouse lemurs),144 suggests that the genetic basis and physiology for such a hypometabolism

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 163 could be preserved in many mammalian species including humans2,141. Thus, hibernation may 164 have been part of our primate predecessor’s life history143,144. Hence, the prospect of existence of 165 a hypometabolic state akin to hibernation in extinct human species may seem more realistic now 166 than it has been so far13. This is the first report on hibernation of humans obtained naturally, not 167 experimentally. The effect of hibernation in humans was unknown till the present study.

168 2. Materials and Methods 169 We used most of the SH enormous collection of fossilized bones. Sima de los Huesos (SH) has 170 been excavated since 1983 with every year producing more human fossils. So far, more than 171 5,500 human skeletal remains have been found. Most of them have been studied for the purposes 172 of this work but only a minute fraction was possible to present here. 173 We studied the internal and external morphology of the collection of bones by using 174 macrophotography, microscopy, histology and CT scanning. Bones were first examined under 175 normal lighting conditions and a magnifying glass. A Nikon SMZ 800 Stereozoom microscope 176 was used subsequently in several occasions. 177 Bone histology provides valuable information on aspects of a human’s life such as growth, 178 pathology, biomechanics, and lifestyle when histological integrity is generally maintained after 179 fossilization. It is a more sensitive test than radiography for the presence of bone diseases such as 180 renal osteodystrophy (ROD)14. 181 182 3. Results 183 3.1 Osteitis Fibrosa (OF) 184 Initially, we used the ultimate criterion for bone diagnosis, that of histology14. Fig. 1 shows the 185 spongiosa from a natural fracture of scapula ESC X which belonged to an adolescent. It can be 186 seen that some of the trabeculae are hollowed out by minute tunnels. The histological hallmark 187 of secondary hyperparathyroidism (2HPT) identification is osteitis fibrosa (OF)15,16. OF is an 188 intratrabecular osteoclastic tunneling resorption17 called also dissecting osteitis secondary to high 189 levels of PTH. The tunneling in Fig. 1 fits perfectly the above description of OF and is an 190 indisputable pathognomonic evidence of OF in our sample. Scapula AT-1151 also presents OF. 191 As it turns out, OF is widespread in many juvenile specimens in the SH collection that bear the 192 most severe manifestations of ROD (see below e.g., 3.3, 3.5). 193 194 OF also refers to increased resorption on trabecular surfaces called peritrabecular fibrosis18 or 195 ‘cookie-bite defects’19 which is an osteoclastic resorption evidenced by numerous Howship’s 196 (resorption) lacunae covering the trabecular surface. In thin sections it forms an undulating front 197 containing numerous resorption lacunae20. Such minute depressions (i.e., Howship’s lacunae) are 198 shown in scapulae AT-1151 and ESC X too (Fig. 2) in great numbers. They have a brownish 199 coloration due to the clay of the cave deposited in them postmortem and fit perfectly the 200 description of peritrabecular fibrosis. Because these lesions are formed as residua in treated 201 patients with secondary hyperparathyroidism (2HPT)21, we inferred that these lesions in the 202 aforementioned scapulae are similarly residues of 2HPT. 203 Other histological features of OF include trabeculae of varying width, intertrabecular spaces of 204 various sizes20 and short and thin bony spicules transversal to the trabeculae. Some of these 205 spicules can also be seen in Fig. 1. 206 207 3.2 Rachitic

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 208 Cranial bones. In the early adolescent9 Cranium 9 there is excessive deposition of new porous 209 bone on the external table of the supraorbital area with fine porosity (Fig. 3, Fig. 4). It is limited 210 to the outer table whereas the inner table is normal. The diploic trabeculae are thick and coarse 211 with small marrow cavities i.e., there is absence of diploic . A natural cross section in 212 the area above the left orbit shows 4 layers of bone deposition (Fig. 4). These distinct layers are 213 not in firm contact between each other but are sharply divided by parallel (though undulating) 214 empty gaps. The superficial layer is comprised of 4 sublayers (Fig. 4). 215 This porous bone lesion is typical of rachitic hyperostosis22. It has been variously termed as 216 “tabetic patch”, or “pumice stone-like” skull20 or “cardboard-like” bone surface. The inner table 217 is normal as reported by Caffey22 in rickets. The coarseness of the diploic trabeculae is similar to 218 those described in iliac crest biopsies23 in the most severe cases of ROD evidence also of 219 hypertrophic rickets24 (see below 5.2) and absence of anemia as there is no widening of the 220 diploic spaces. Rachitic hyperostosis supraorbitally has been reported in dialysis adolescents 221 with ROD25. 222 223 The bone layers are termed rachitic osteoplaques (ROPs)26. The 4 parallel and regularly spaced 224 ROPs (Fig. 4) signify 4 chronic relapsing episodes which can characterize recidivism of rickets. 225 ROPs are found in adolescent crania with chronic rickets26. However, they are not as distinct and 226 regular as here and they are mostly represented by one ROP. Since, Cranium 9 shows a rachitic 227 hyperostosis in the frontal bone and large browridges when they occur, as here, grow most 228 rapidly towards the end of the adolescent growth spurt157, it follows that the individual too was at 229 the end of his adolescent growth spurt when it died. This is consistent with its dental evidence 230 that shows it is an adolescent. Since depositional regularity signifies annual periodicity in the 231 edible dormouse,156 fossil amphibians and reptiles,136 these 4 layers are annual and correspond to 232 4 years of episodic renal rickets. Then the growth spurt of Cranium 9 may have lasted for 4 233 years. 234 The gaps between the ROPs were apparently filled by osteoid. Osteoid designates that part of 235 newly deposited bone matrix which is not yet mineralized27. The osteoid was preserved in the 236 fossil bones as an empty gap due to its postmortem decomposition. Some hibernators, such as 237 bats28, ground squirrels121 and hamsters160 lose bone during and immediately after hibernation. In 238 hibernating bats with 2HPT30, lines of arrested growth are formed due to osteoid deposition. In 239 chronic, experimental and severe HPT of guinea pigs, osteoid tissue was laid down soon after the 240 last dose of PTH was administered i.e., during the healing phase69-70. These evidences from the 241 literature seem to explain what happened here. 242 The 4 sublayers of the superficial ROP, apparently correspond to 4 interbout arousals from 243 torpor which may correspond to 4 months of hibernation. Since the contrast and the distinctness 244 between depositional zones is most pronounced in hibernators29,77, where there is an almost total 245 cessation of activity for part of the year, it follows that the sharp contrast between the ROPs and 246 the gaps in Cranium 9 are evidence too of hibernation in this individual. After all, interrupted 247 bone deposition is considered as evidence of hibernation72 and the distinctness of lines of 248 arrested growth may be related to whether the animals hibernate or not137. Therefore, the nature 249 of the gaps, the annuality of ROPs and their distinctness, and the sublayers of the last ROP point 250 to hibernation of the SH population in dark cavernous hibernacula. 251 It is interesting that during the recovery phase in the experimental HPT69,70, BT’s, OF and 252 extensive enlargement of the Haversian canals took place. These lesions are also characteristic of 253 the present collection (see above and below).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 254 255 Radius. Layered structures such as those in Cranium 9 are also found in long bones especially 256 those in growth spurt. Since they are often found in many skeletal species here and especially 257 those of juveniles, this also indicates that they are annual136. Fig. 5 shows radius AT-4216 and its 258 natural cross section at the level of radial tuberosity. It is in puberty as the flake epiphysis for 259 tuberosity is discernible subperiosteally. Note the thick layers of bone deposited every year and 260 the fine trabeculae between them. One can see 4 of those layers; this ties with the fact that the 261 flake epiphysis of the radial tuberosity appears and fuses in puberty that lasts 4 years in modern 262 humans. This histology shows that there is puberty in SH hominins that takes more or less the 263 same time as that in modern humans but it is intermittent (because of hibernation). 264 265 3.3 Craniotabes 266 There is also flattening of the posterior of Cranium 9 at the parietals with a slight depression 267 toward their middle (Fig. 6) giving an angular appearance to the skull (Fig. 3). This is known as 268 craniotabes or caput quadratum that is the clinical hallmark of rickets31. It may lead to posterior 269 flattening of the parietal bones22 and the formation of a large, square31,32 or triangular33 shaped 270 head. Craniotabes may develop a parietal eminence in the sides of the parietal bones following 271 the incessant overproduction of osteoid in these bones34 during a time of rapid skull growth33 as 272 during adolescence. This diagnosis is confirmed by the tunneling (OF) of the diploic trabeculae 273 observed for instance in the right craniotabe close to the parietal eminence (Fig. 7). Indeed, 274 tunneling of the diploic trabeculae occurs in HPT18,33 close to the parietal eminence as it is the 275 area mostly affected by rapid growth. 276 277 3.4 “Rotten fence post” sign

278 In the unfused distal femora (such as in the femur AT-2240+AT-2118+AT208), the metaphyseal 279 trabeculae are exposed (Fig. 8). Many of them show tunneling, evidence of OF. This is known as 280 the ‘rotten fence post’ sign. It forms when the resorptive process at the outer metaphysis 281 continues after longitudinal growth has ceased and creates the sign of the ‘rotten fence post’ in 282 radius, ulna and femur underneath the overriding cartilage plate35 due to OF in 2HPT36. The 283 ‘rotten fence post’ sign is diagnostic for renal rickets’37 and has an alternating strut/slit 284 histology38 that resembles the bristles in a brush22. These “woolly” lesions represent metaphyseal 285 subperiosteal resorption (SPR). The ‘rotten fence post’ sign is also shown in Fig. 1 of Mays et 286 al19 in 2HPT. Thus, the lesion with the trabeculae exposed in Fig. 8 is identical to the ‘rotten 287 fence post’ sign evidence of OF, renal rickets and 2HPT in the SH hominins. The distal fibula 288 AT886+AT1252 is from an adolescent as the epiphysis is not fused (Fig. 9). Note the coarsening 289 of metaphyseal trabecular bone evidence of ‘rotten fence post’ and distal fibular subperiosteal 290 resorption (SPR) as described99 in renal rickets, 2HPT and ROD. Also metaphyseal cupping and 291 frayed margins can be seen (Fig. 9).

292 A special case of “rotten fence post” sign appears in the medial margins of the femoral 293 necks. Fig. 10 shows the inferior parts of three femoral necks (F3, AT-2240+AT-2118+AT208, 294 F5) at different stages of growth. The trabecular columns and osteoid voids run parallel to the 295 bone surface in the femoral neck. The development of this sign depends on the intensity of 296 growth. Thus, the biggest femur without epiphyseal fusion has the most marked manifestation of 297 this sign (middle of Fig. 10). The signs of this feature in the other femora are not so developed

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 298 and in the fully adult it has completely disappeared. There is also a flattening of bone beneath the 299 femoral head (Fig. 10). This lesion is a particular type of ‘rotten fence post’ sign and SPR in 300 ROD in concave areas (‘cutback zones’), such as the medial margins of the femoral 301 necks91,100 especially in adolescents99-101. It results in the diminution in the number of trabeculae, 302 and a compensatory thickening of primary stress trabeculae8 as here. The ‘rotten fence-post’ sign 303 along the shaft of the (inferior) femoral neck24,104 is typical and pathognomonic of 304 hyperparathyroidism (HPT) in femora104. Since this sign occurs in adolescents during growth 305 spurt102,103 and because ROD is directly related to the growth rate46,104, it is deduced that the SH 306 hominins too underwent a growth spurt as shown by the intensity of its manifestation in the 307 necks of the SH femora. The existence of puberty is confirmed by ‘the strong projection of the 308 external lip of the femoral trochlea’ in the late adolescent distal femoral epiphyses of SH (not 309 shown) which is diagnostic of adolescence in hominins39. The above finds are the first 310 conclusive evidence of adolescent growth spurt not only in the SH hominins but in human 311 paleontology. 312 313 3.5 Brown Tumour (BT) 314 The CT scan of the ilium of Pelvis 1 displays an aggregation of multiple without internal 315 structure divided by septa and surrounded by a narrow band of marginal sclerosis (Fig. 11). 316 Another particular type of OF is the brown tumour (BT) that is formed at an end stage of 317 hyperparathyroidism (HPT)40 and CKD41. Brown tumours (BTs) are generally multiple cystlike 318 lytic lesions42 that represent reparative granulomas33 that may be present anywhere in the 319 skeleton43,44. An excessive secretion of PTH leads to OF and BT in order to partially restore 320 serum calcium levels to normal45,46. This excessive leads to large well- 321 demarcated spaces filled partially with osteoclasts47. BTs are especially prominent in the most 322 severe manifestations of ROD during vitamin D treatment24. After therapy, thicker sclerotic rims 323 with no internal structure are formed20,47. 324 The morphology of the cysts in the Pelvis 1 fits exactly the above description of BTs and shows 325 that a lytic process after a severe long-standing secretion of PTH has taken place followed by 326 healing as evidenced by the sclerotic rims. This is consistent with the resorption in the symphysis 327 pubis of the Pelvis 1 as such resorptions in the articular cortices of symphysis pubis are 328 especially subject to shearing forces in chronic renal insufficiency (i.e., CKD) and 329 hyperparathyroidism (HPT)18,44,48,49. It is also consistent with the experimental HPT69,70. 330 Therefore, the BTs here are pathognomonic of severe long-standing rickets, 2HPT, and ROD i.e., 331 CKD that have been healed in this specimen. The presence of erosive changes elsewhere in this 332 collection supports the diagnosis of BT too52.

333 3.6 CPPD

334 The adolescent vertebra VC1850,51 (C3, Fig. 12) shows an elliptical cartilage calcification of the 335 annulus fibrosus. This is a rare example of an ossified soft tissue in paleoanthropology that has 336 been preserved and fossilized. Such a complete calcification of the C2/C3 intervertebral disk is 337 presented in a radiograph provided by Freyschmidt et al.53 (their Fig. 5.211). Similarly, the 338 scapula AT-2563+AT-2471 shows a peripheral ridge in the glenoid cavity (not shown) that is 339 apparently the result of mineralization of the fibrocartilagenous glenoid lambrum. Such a 340 peripheral ridge is also identified in La Chapelle-aux-Saints and Shanidar I Neanderthals54. 341 CPPD (Calcium pyrophosphate dihydrate crystal deposition disease/chondrocalcinosis) is most

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 342 common in annulus fibrosus of the intervertebral disc and glenoid labra among many sites55-57. 343 Enthesopathic calcification and may develop in the annulus fibrosus and capsules of 344 apophyseal and appendicular joints58. There is close association of CPPD with HPT or chronic 345 renal failure55,56,59. Therefore, the lesion in Fig. 12 and the aforementioned scapula is consistent 346 with CPPD in HPT associated with the CKD-MBD identification in these SH hominins. 347 Increased breakdown of adenosine triphosphate (ATP), is thought to be one reason as to why 348 crystals in CPPD may develop and mineralize the articular cartilage60. Brown adipose tissue 349 (BAT) is found only in mammals and differs from white adipose tissue (WAT) in that it 350 generates heat at the expense of ATP61. Thus, lipolysis of BAT resulting in increased production 351 of ATP may have been responsible for the excessive deposition of crystals and formation of 352 CPPD that resulted in the mineralization of annulus fibrosus of vertebra VC18, as BAT generates 353 heat during arousals from hibernation. Therefore, in the present case, CPPD is evidence of 354 lipolysis of BAT during interbout arousals. This is confirmed by the 4 sublayers of the interbout 355 arousals of the ROPs in Cranium 9 (Fig. 4).

356 3.7 Subperiosteal new bone (PNB)

357 A striking lesion in much of the SH hominin collection is the development of thin patches of 358 porous material on cortical bone surfaces (Fig. 13) with vermicular or round pores and oblique 359 orientation of them. Similar lesions in cortical bone with inclined porosity have been reported18 360 in human archaeological remains with 2HPT. They are called subperiosteal new bone (PNB) or 361 periosteal neostosis91. The common occurrence of PNB in the SH bone collection implies that we 362 are dealing with only one population of hominins that was affected by the same disease(s) which 363 probably had the same environmental etiology to affect the whole population. This is consistent 364 with the view that the bones belong to the same biological population because of the relative 365 morphological homogeneity of the SH collection12. PNB is an indicator of intense secondary 366 hyperparathyroidism (2HPT), osteitis fibrosa (OF) and advanced hypertrophic rickets or 367 osteomalacia (OM) in severe renal osteodystrophy (ROD) during healing22,19,92-97. PNB may 368 remain evident for years after healing22 by vitamin D therapy92,98. That is why PNB is common 369 in the SH collection.

370 3.8 Beading in ribs and tibial subperiosteal resorption (SPR).

371 Ribs. Beading of the sternal ends is shown in ribs AT3030, AT1241, and AT-2996+AT-3067 372 (Fig. 14). ‘Mottling’ (which corresponds to the “rotten fence post” sign) is more conspicuous in 373 AT-3030 (Fig. 14). In rickets or osteomalacia (OM) and 2HPT, there is beading (i.e., rachitic 374 rosary) at the sternal ends and “mottling”34,39. Therefore, the beading in the ribs of Fig. 14 is 375 evidence of 2HPT and renal rickets.

376 . A scalloping-like porosity can be seen in the upper medial cortex of adolescent Tibia II 377 (epiphyses unfused, Fig. 15). The posterior and medial surfaces are also affected but the lateral 378 one is the least affected. The porosity reaches the height of the nutrient foramen at its lower end. 379 The lesion is superficial. For comparison, the lesion in the adult Tibia III (Fig. 15) has been 380 largely healed. Only sparse porosity remains.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 381 This description fits well that of SPR in 2HPT as such SPR has been found in the tibiae of 382 dialysis patients with HPT106. So, the SPR in the upper medial cortex of the Tibia II is evidence 383 of 2HPT18,107. The lesion is usually scalloping in humans50,99, as it is in experimental rats with 384 ROD109 and always shallow. Many of the lesions in the SH collection such as scalloping SPR 385 and rachitic hyperostosis are those of dialysis patients with ROD too. Therefore, we are dealing 386 here with the acquired type of ROD with evidence of healing.

387 3.9 Lamina Dura (LD).

388 In the socket of the upper right incisor of Cranium 9, the partial loss of LD expands away from 389 the mouth of the tooth socket towards the root region (Fig. 16). In the older or younger 390 individuals the LD is more preserved. Lamina Dura (known also as lamina cribrosa or 391 cribriform plate) is compact bone that lies adjacent to the periodontal ligament, in the tooth 392 socket. It is pierced by many small openings for blood vessels, lymphatics and nerve fibers109,110.

393 Resorption of LD leads to the loss of LD109 which is a sign of OF; this resorption is another 394 instance of SPR,112 since the periodontal membrane is a specialized periosteum112 and alveolar 395 bone is more sensitive and reactive to outside influences than basal bone113. Depending on the 396 severity of the disease, LD may be partially or completely absent107 and the nutrient canals 397 appear wider than normal and are particularly evident in the incisor-canine area112,114 as here. In 398 vitamin D deficiency rickets and HPT, loss of the LD around the teeth is frequently seen115. OF, 399 chronic azotemic renal failure, and chronic renal insufficiency (i.e., CKD) with 2HPT are all 400 associated with disappearance of the LD44,114,116,117. In up to 47% of cases of ROD there is loss of 401 LD119,120. Similarly, there is loss of LD in both osteomalacic and 2HPT primates109,120. Not 402 surprisingly, loss of LD in small hibernators is effected during hibernation31,121.

403 3.10 Phalangeal resorption.

404 Some phalanges, such as AT 2520 and AT 2521 (Fig. 17), present a ragged porotic shaft with 405 ‘bumpy’ irregularities as a result of an extensive subperiosteal “lacelike” resorption (SPR) 406 laterally, medially (cf.44,54,122) and on the palmar surface (cf.122) that has affected extensively the 407 cortical bone. This is called phalangeal resorption and it is pathognomonic of severe chronic 408 2HPT and (CKD)18,98.

409 Phalangeal resorption is the earliest, the most common and the most sensitive sign of 410 2HPT15,95,102. It results from after vitamin D deficiency95. In advanced cases of 411 2HPT most of the cortical surface may suffer from phalangeal resorption44 as in the present case. 412 The round holes in the cortical bone (Fig. 17) may be due to a cumulative resorptive effect of 413 PTH over the years as it is the case in dialysis osteoarthropathy in 2HPT123. In AT 2520 and AT 414 2521 there are also periarticular resorptions (Fig. 17). While they are not pathognomonic, occur 415 frequently in 2HPT124 and their pattern is unlike that of other erosive arthropathies124-125. In some 416 other phalanges with diaphyseal resorption [e.g. in the left adult middle phalanx 5 (AT-1304) and 417 in the subadult middle hand phalanx 3 or 4 (AT-3154)], PNB has also been formed on the dorsal 418 surface96,97 (not shown). Indeed, PNB is always accompanied by SPR in finger bones92-96. In AT- 419 98 there is resorption of the terminal tufts, not unexpectedly, since in ROD there is a marked

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 420 acro- of distal phalanges of foot24. OF tunneling is also present in some of the 421 phalangeal trabeculae.

422 3.11 Differential diagnosis.

423 Conditions associated with PNB include scurvy, Caffey’s disease, infections and malignancies126 424 but their detailed histology is different. For instance, in vitamin C deficiency the trabeculae of 425 the pores are perpendicular to the bone surface128 whereas here a large part of the pores’ 426 trabeculae are inclined. In hyperthyroidism (HPT), the PNB is spiculated. In rheumatoid arthritis 427 (RA), PNB is notable in its absence128,129 and the diaphyseal bone is commonly unaffected128 428 whereas here the opposite is the case.

429 Lamina dura (LD) is not an infallible diagnostic sign of HPT: loss of LD may also occur in OM, 430 Paget’s disease, fibrous dysplasia, sprue, , Addison’s and Cushing’s syndrome, 431 myelomatosis, diffuse metastasization and leukaemias. Especially in osteomalacia, loss of LD 130 432 occurs because teeth are considered as Vitamin D3 controlled mineralized tissues . The 433 diagnosis of HPT is accurate only when all areas are considered110 for evidence of SPR and 434 OF112,118 as it is the case here. Therefore, the loss of LD in Cranium 9 is consistent with the 435 hypothesis of ROD (2HPT, OM and OF) associated with CKD and hibernation in the SH 436 hominins.

437 The pattern of the SPR at the margins of the phalangeal joints is unlike that of other erosive 438 arthropathies such as RA131. In RA the diaphyseal bone is commonly free of SPR132. Erosive 439 osteoarthritis, distinguished by associated osteophytes and subchondral sclerosis, predominantly 440 involves first carpometacarpal and first metatarsophalangeal joints, but rarely involves 441 intercarpal or metacarpophalangeal joints. Phalangeal resorption is not found in 442 hyperthyroidism133.

443 4 Discussion

444 The constellation of gross and histological attributes described above is distinct to ROD 445 associated with CKD-MBD which is the oldest in the skeletal record: there is conclusive 446 pathognomonic evidence for OF, 2HPT and hypertrophic renal rickets in ROD evidenced at least 447 by the trabecular tunneling, the ‘rotten fence post’ sign, the rachitic hyperostosis, the craniotabes, 448 the BTs, the phalangeal resorption and the extra-skeletal calcification (CPPD). Mottling, SPR 449 and beading of the sternal ends of ribs are pathognomonic of renal rickets and 2HPT. The 450 presence of PNB and the robusticity of the bones show the presence of advanced hypertrophic 451 rickets and 2HPT in severe ROD during healing. That is why many of the lesions in the SH 452 collection such as scalloping SPR and rachitic hyperostosis resemble those of dialysis patients 453 with a chronic acquired type of ROD. The CKD-MBD is confirmed by the presence of ROD 454 lesions and the extra skeletal calcification (CPPD, see below 5.1). Rickets and ROD has only 455 been reported in troglophile hibernators such as cave bears and bats30,73,74. Therefore, the SH 456 hominins present the same diseases as those of animal hibernators.

457 Another human species from Atapuerca, Homo antecessor (MIS 21)145, from the nearby caves 458 (Sima del Elefante cave site) does not bear any evidence of the pathologies associated with

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 459 hibernation such as those in SH even though adolescent frontal bone(s) of it were found. MIS 21 460 corresponds to a warm and humid climate146. In addition, a juvenile skull of a hibernating fossil 461 bear of Ursus deningeri from SH does show rachitic hyperostosis on its cranium. These pieces of 462 evidence show that the lesions reported here have nothing to do with the latitude of Atapuerca 463 and the annual UV radiation. Since the latitude is not the cause, the climate might be the cause 464 for their hibernation. Indeed, the time the SH hominins lived - ca. 430 Ka (MIS 12) - coincides 465 with the glacial period that was the most severe and long of the Pleistocene. If today’s climate in 466 Atapuerca features chilly winters, which always include snow and temperatures below freezing 467 often as low as −10 °C, one can only imaging what would the cold have been like in the glacial 468 times of the SH hominins. Thus, the hibernation hypothesis is consistent with the climate at the 469 time.

470 CKD-MBD is a common complication of CKD caused by abnormalities in vitamin D 471 metabolism such as increased serum phosphate, PTH, and bone turnover, and abnormalities in 472 mineralization, growth and extra-skeletal calcification171. As CKD progresses, vitamin D 473 deficiency increases and results in hypocalcemia and 2HPT171. Therefore, it is possible that the 474 SH individuals developed vitamin D deficiency due only to hibernation as it happens in animal 475 hibernators. This sequence of events may have resulted in their demise.

476 Because rickets and 2HPT are diseases of growth, then in the SH juveniles, the flake epiphysis 477 for tuberosity in the radius (Fig. 5) and the severe expressions of rickets and 2HPT such as the 478 presence of tunneling trabeculae (OF), the resorption of LD, the ‘rotten fence post’ sign, the SPR 479 and their degree of manifestation in the SH adolescents, ‘the strong projection of the external lip 480 of the femoral trochlea’, the evidences of healing and the presence of regularly spaced ROPs and 481 gaps, all show that the SH individuals had an intermittent (seasonal) pubertal growth spurt. This 482 is because the bone manifestations of rickets are most striking at the time of greatest growth 483 speed at puberty62 as its high growth rates result in high metabolic and nutritional demands63. 484 This is even more so in the case of adolescents with renal rickets, HPT, and ROD64,65 caused by 485 inadequate renal production of vitamin D66 which results in growth failure. Therefore, it may be 486 possible that the SH adolescent children developed a severe vitamin D deficiency which caused 487 marked 2HPT and bone morphology identical to patients with ROD associated with CKD-MBD. 488 The intermitted nature of growth spurt was caused by their inability to build up a sufficient store 489 of fat73 for the winter hibernations. The dental and endocranial volume evidence are consistent 490 with this conclusion12. The hibernation hypothesis for the SH hominins is also consistent with 491 the juvenile-dominated bear and hominin mortality in the Mokrica cave, Slovenia78 and SH79 492 respectively (see also Introduction); 64% of humans in SH are between 11-20 years, an abnormal 493 adolescent mortality that reminds famine80. Hence, the non-tolerated hibernation has caused 494 vitamin D deficiency, renal rickets, and 2HPT in ROD associated with CKD-MBD and has 495 induced growth arrest and formation of ROPs and gaps in the adolescents of SH. The above bone 496 evidence shows that the SH hominins used hibernation as a survival strategy to face winter 497 famine and cold. The ROPs and the gaps and their distinctness are very unusual features in the 498 human skeletal record if not unique. Apparently, the SH adolescents did not have enough stores 499 of fat as shown by the diseases they suffered in contrast to the adult ones.

500 We have shown that the lesions described here in the SH hominins are those suffered by cave 501 hibernators in a state of non-tolerated (pathologic) hibernation. Hibernation is characterized by

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 502 bouts of torpor142 and interbout arousals161,162. We have provided evidence for interbout arousals 503 in the SH hominins in the form of sublayers of ROPs (see 3.2) and CPPD (see 3.6). One month 504 for one hibernation bout, as suggested here for the SH hominins, is not as improbable as it may 505 seem: there are primates, like Microcebus griseorufus, that can hibernate without arousals for 506 months144,172. Then, these hominins may well have stayed for a long period of at least some 4 507 months (corresponding to the 4 sublayers of the ROPs) inside a cave to develop such a severe 508 chronic rickets and 2HPT unique for human bones found in caves. A strategy of hibernation 509 would have been the only solution for them to survive being for months in a cave. Indeed, even 510 in modern humans there is ethnologic and historical evidence about a type of hypometabolism 511 called Lotska in Russian famine areas which is huddling together saving energy and warmth in 512 an ‘almost uninterrupted sleep’ that is analogous to hibernation147. Perhaps it is the same state 513 mentioned since ancient times by the father of history, Herodotus, for people north of Scythia 514 (i.e., today’s Russia) ‘who sleep for half a year’147 which is reminiscent of ursine hibernation, a 515 state similar to prolonged sleep148.

516 These inferences are consistent with the fact that, as regards the mitochondrial DNA, the SH 517 hominins are genetically closely related to Denisovans81 who were adapted to high altitude and 518 cold82,83. Indeed, the SH hominins lived in a glacial period and their cave is at an elevation of 519 about 1082 m, i.e., about 400 m higher than that of the Denisova cave. Furthermore, some genes 520 in the latter have been associated with the activation of the adrenal gland and BAT in humans83. 521 BAT is a tissue used by hibernators during arousals from hibernation bouts6 (see also 522 Introduction). The adrenal gland produces cortisol that increases resorption85 and decreases bone 523 formation84 by inhibiting calcium absorption in the intestine thus leading to 2HPT85. Cortisol 524 increases in hibernating animals, such as bears86 and bats87, a condition that in some males of the 525 hibernating mouse lemur Microcebus murinus leads to hypercortisolism and death from chronic 526 glomerulonephritis88.

527 Because: 1) the lesions in the SH hominins are the same or similar to those of hibernators, 2) the 528 arrested growth evidenced by the ROPs and gaps are formed in intermittent chronic rickets37,67,68 529 due to the calcium mobilizing PTH69-71 (see 5.4) and hibernating animals too72 especially in those 530 with ROD such as bears73,74 and bats30 because of their inability to build up a sufficient store of 531 fat73,75 and the reduction of 25OHD76 that is stored in the fat, 3) the regularity, the extreme 532 contrast and the distinctness of ROPs and gaps in cranial and long bones (Fig. 4, 5), even without 533 gaps, are considered as strong evidences of hibernation,72,77 4) the evidence of healing deduced 534 from BT’s, PNB, and extensive enlargement of the Haversian canals are evidence of a post 535 hibernation period, 5) the SH hominins lived during the most severe glacial maximum in the 536 Pleistocene and the genetic evidence is consistent with this whereas H. antecessor lived in a 537 warm climate and has no evidence of the SH lesions such as rachitic hyperostosis, and 6) the 4 538 sublayers of the superficial ROP of Cranium 9 are consistent with 4 interbout arousals, we 539 deduce that the SH hominins were hibernating. Thus, there are strong evidences of puberty and 540 non-tolerated hibernation in the adolescent SH hominins and non-pathological hibernation in the 541 adults. Since the mature individuals do not show any evidence of these lesions (with the 542 exception of healing evidence such as BTs and PNB and enlargement of the Haversian canals), it 543 follows that they had sufficient fat reserves to withstand the ill effects of hibernation. This is 544 because they did not have to face the extra energy demands posed by adolescence. The healing

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 545 that took place after their adolescence has made these lesions largely to disappear. As a result, 546 the adult individuals were undergoing a more or less healthy and normal hibernation.

547 4.1 The physiological mechanism and the Reconstruction of life History

548 The rachitic metaphysis and the gaps are evidence of hypocalcemia. The resultant increases in 549 PTH may account for the pathologies described in fossil bears, living hibernators, and the SH 550 hominins. Thus, the simplified mechanism in non-tolerated hibernation for cave-dwelling 551 hominins during glacial times could be as follows: frigid conditions anticipated for the coming 552 glacial winter, would have made these hominins to use the Atapuerca caves as hibernacula. 553 Initially, lipolysis would have provided endogenous energy and vitamin D for calcium 554 . At the same time, the use of caves would stop their exposure to UV light. This 555 would decrease serum vitamin D due to constant darkness denning conditions, and the failure of 556 the kidney to adequately excrete phosphorous. Thus, vitamin D will be initially substituted by 557 that released from fat to suppress bone resorption and prevent bone loss. The metabolic 558 homeostasis continues until all fat and its fat-soluble vitamin D are depleted earlier than would 559 have normally been thus, reaching a state of vitamin D insufficiency and deficiency that would 560 have caused hyperphosphatemia. This decreases the concentrations of ionized calcium causing 561 hypocalcemia and gives rise to increased PTH secretion in such levels as to cause 2HPT, 562 increased bone resorption, rickets, OF, CPPD, ROD and arrested growth, all associated with 563 CKD-MBD89,90. This is the state of non-tolerated hibernation in which the body temperature 564 cannot be sustained above a critical point and hibernation terminates or the individual is unable 565 to arouse and dies or dies just after arousal from hibernation as it happens in some hibernators. 566 The raw diets and absence of fire of the SH hominins would have made things worse. Among SH 567 hominins, CKD-MBD was a recurrent disease, apparently on a seasonal basis (winter) because of 568 glacial winters in the high seasonality area of Atapuerca and the high altitude of approximately 569 1082m. In this frigid environment, the cold would have affected their physiology and therefore 570 the ability of this human species to adapt to famine. It is therefore likely that they would have 571 developed strategies, such as hibernation suitable for countering the threat of famine periodically 572 posed to their functional integrity and survival.

573 On arousal, the mechanism is reversed: those hominins that survived, get out of the cavernous 574 hibernacula to replenish their stores of fat, vitamin D, and calcium thus suppressing PTH 575 secretion. At the same time, the CKD pathologies are healing and growth takes place (in the 576 adolescent ones). Fat starts to accumulate for the coming winter. However, should the fat 577 reserves are not enough for a successful hibernation to occur, complete healing may not be 578 achieved leading to a vicious cycle. In this case the individuals would enter hibernation with less 579 fat than the previous year, a situation that might have been lethal for them. This is only the core 580 of this pathophysiological mechanism as many aspects are unknown since more skeletal material 581 and studies are required to understand better the physiopathogenesis of hibernation in this extinct 582 human species.

583 4.2 Theoretical background

584 4.2.1 Renal Osteodystrophy (ROD) and CKD-Mineral and Bone Disorder (CKD-MBD) The term 585 ROD refers collectively to the various osseous abnormalities associated with chronic kidney

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 586 disease (CKD) and defined by bone histomorphometry89,90. CKD-MBD is manifested by 587 abnormalities in Ca, P, PTH, Vitamin D, bone turnover, growth, and soft tissue calcification89,90. 588 The bone lesions in ROD range from a low bone turnover to a high bone turnover (osteitis 589 fibrosa, OF). It may be divided into acquired (glomerular) and congenital (tubular) type8. 590 Acquired type of ROD is due to functional renal impairment8 and in humans can occur in cases 591 of protein malnutrition or prolonged immobilization58. It consists of a mixture of OF (tunneling 592 trabeculae, osteoclastic resorption and PNB), 2HPT, rickets or OM, and growth retardation, with 593 a biochemical picture of hypocalcaemia, hyperphosphatemia, elevated parathyroid hormone 594 (PTH), and vitamin D deficiency58,134,135. OF is by far more common in the acquired type of 595 ROD than the congenital one8.

596 4.2.2 Hyperplastic or Hypertrophic rickets. In subjects with vitamin D deficiency which are 597 otherwise well nourished, a hypertrophic form of rickets develops. In this case, bone cortices are 598 thick because of exuberant deposition of wide concentric layers of osteoid by the periosteum and 599 medullary cavities stenosed34,25. That is not to imply that the robusticity of the SH bones is due 600 solely to such hypertrophy, but it may explain the absence of some signs of rickets in the SH 601 collection.

602 4.2.3 Growth Marks. Cortical bone deposition can be temporarily slowed or halted in response to 603 annual rhythms in living and extinct poikilothermic and homeothermic tetrapods, resulting in 604 compact bone being interrupted by dense growth marks150,151. They are all skeletochronological 605 indicators that may reflect cyclical annual growth, or other endogenous rhythms152 as suggested 606 by their occurrence in extant crocodilians, some rodents and polar bears153. The broad laminae in 607 compact bone such as the ROPs, are formed annually only in vertebrates that undergo periods of 608 hibernation72 or experience marked seasonal shifts in their dietary habits. Rickets is caused 609 primarily by a deficiency of fat as fat is rich in vitamin D154,155.

610 6 Conclusions 611 612 Given the lesions’ morphology, the histology, the pathognomonic features, the differential 613 diagnosis, and the glacial environment they lived, we conclude that the SH fossil hominins, 614 especially those in adolescence, are the earliest known sufferers of a disease indistinguishable 615 from the severe, acquired, and annual CKD-BMD that may have been caused only by non- 616 tolerated hibernation in dark cave hibernacula and recurrent periods of famine. While many 617 questions about their life histories and metabolism are still open (see 4.1), there is no doubt as to 618 the immense consequences that hibernation has for hominin/human physiology and life history. 619 Thus, under certain circumstances, some human species may find themselves in metabolic states 620 (heterothermy) that help them to survive for long periods of time in frigid conditions with limited 621 supplies of food. The CKD-BMD, the gaps between the ROPs and their sublayers, the “rotten 622 fence post” sign, and the evidence of healing are a new tool for determining the presence of a 623 pubertal growth spurt and seasonal hibernation in an extinct human population. 624 625 Acknowledgments. Our thanks are due to the late Don Ortner, who confirmed our diagnosis of 626 rickets in Cranium 9. The Department of History & Ethnology of the Democritus University of 627 Thrace for granting one of us (A.B.) a sabbatical for the spring semester of 2006 in order to 628 undertake this study.

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1022 169. Hittel DS & KB Storey 2002. Differential expression of mitochondria-encoded genes in a 1023 hibernating mammal. J Exp Biol, 205:1625-1631. 1024 1025 170. Christian JJ, JA Lloyd & DE Davies 1965. The role of endocrines in the self-regulation of 1026 mammalian populations. Recent Progress in Hormone Research, 21:501-578. 1027 1028 171. Webster A., E.V. Nagler, R.L. Morton 2017. Chronic kidney disease. Lancet, 389:1238- 1029 1252. 1030

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 1031 172. Kobbe S., J.U. Ganzhorn, K.H. Dausmann 2011. Extreme individual flexibility of 1032 heterothermy in free-ranging Malagasy mouse lemurs (Microcebus griseorufus). J. Comp. 1033 Physiol. B, 181:165-173. 1034 1035 1036 1037 1038 1039 Figures 1040 Figure 1. Histology of scapula ESC X. Natural fracture of scapula ESC X in the area where the 1041 spine meets the body of the scapula. It shows tunneling resorption forming intraosseous cavities 1042 in the trabeculae (arrows) which is conclusive evidence of osteitis fibrosa (OF). Some of the 1043 trabeculae are unfocused because this is a natural section. These tunnels are the residua of active 1044 zones of osteoclastic tunneling resorption due to secondary hyperparathyroidism (2HPT). Note 1045 also numerous brown depressions (peritrabecular fibrosis) on the surface of the trabeculae (see 1046 also Fig. 2) and thin bony spicules transversal to the trabeculae caused by OF too20. 1047 1048 Figure 2. Scapula ESC X showing OF. Natural fracture of scapula ESC X showing OF in the 1049 form of peritrabecular fibrosis in the spongiosa. The trabecular surfaces are covered by numerous 1050 minute depressions of brownish staining due to diagenetic clay deposited in them postmortem. 1051 They are caused by osteoclastic resorption and they are leftovers of active 2HPT in the SH 1052 hominins.

1053 Figure 3. Rachitic hyperostosis. Left: Cranium 11 (a juvenile). Middle: Cranium 9 (early 1054 adolescent). Right: Cranium 4 (adult). Note the fine porosity on the Cranium 9 supraorbital area 1055 caused by excessive deposition of new bone (arrow) which is absent in the other crania. This is 1056 typical rachitic hyperostosis with a typical ‘cardboard-like bone’ surface. In either side of 1057 Cranium 9, are the calvaria of a younger (left) and an older (right) individual from SH to 1058 demonstrate the absence of rachitic hyperostosis in them as the Cranium 9 is apparently in 1059 growth spurt: in older or younger individuals where rickets is less active, the cross section of the 1060 cranial bone has a normal histology. The angular appearance of Cranium 9 is due to the rickety 1061 craniotabes.

1062 Figure 4. Cross section from above the left orbit of Cranium 9. A natural cross section from the 1063 area above the left orbit of Cranium 9 shows 4 rachitic osteoplaques (ROPs) of rachitic 1064 hyperostosis (one ROP is indicated by the star) that are evidence of intermittent (seasonal) 1065 adolescent growth spurt due to 4 annual hibernations. The ROPs are separated by gaps. Note the 1066 superficial ROP is comprised of 4 sublayers which correspond to 4 interbout arousals from 1067 torpor indicating that hibernation may have lasted at least 4 months. Scale bar 5mm.

1068 Figure 5. Radius AT-4216 (a) and its natural cross section (b) at the level of radial tuberosity. 1069 The flake epiphysis for tuberosity, indicated by an arrow, shows that it was in puberty. Note at 1070 least 4 annual layers of bone. In the adult radii of SH, the layers have been remodeled and

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 1071 therefore are not discernible. The number of layers ties with the fact that the flake epiphysis of 1072 the radial tuberosity appears and fuses in puberty that lasts 4 years in modern humans. Scale bar 1073 3mm.

1074 Figure 6. Cranium 9. Note the flattening of the parietals giving a squared appearance to the skull 1075 known as craniotabes (cf. Fig. 3) or caput quadratum (upper left and upper right) that is the 1076 clinical hallmark of rickets. Scale bar 5cm.

1077 Figure 7. Cross section of the right parietal of Cranium 9. Natural cross section of the right parietal 1078 of Cranium 9 close to the parietal eminence. Note the tunneling (OF) of the diploic trabeculae 1079 (arrows) in many areas of the section due to the development of craniotabes in renal rickets. Scale 1080 bar 4mm.

1081 Figure 8. Metaphysis of the distal femur AT-2240+AT-2118+AT208. Note the ‘rotten fence 1082 post’ sign. Some of the trabeculae show tunneling, evidence of OF (yellow arrows).

1083 Figure 9. Distal adolescent fibula AT886+AT1252. Note the coarsening of metaphyseal 1084 trabecular bone evidence of distal fibular SPR and the collapse causing cupping due to resorption 1085 of the medial line evidence of hyperparathyroidism (HPT) and renal rickets. Metaphyseal 1086 cupping and frayed margins (arrows) can be seen (grade 2 score of rickets). Widening at the 1087 growth plate is expected to have existed antemortem. The narrowing seen is an artifact of gluing 1088 the two pieces together.

1089 Figure 10. Three femoral necks. The inferior parts of three femoral necks (F3, AT-2240+AT- 1090 2118+AT208, F5) at different stages of growth demonstrating the “rotten fence-post” sign of the 1091 primary trabeculae which is pathognomonic of hyperparathyroidism (HPT). The most marked 1092 manifestation of this sign is in the middle femur which is at the height of its growth spurt.

1093 Figure 11. Brown tumor in the ilium of Pelvis 1. A multilocular brown tumor (BT) in the ilium 1094 of Pelvis 1. Note the empty spaces of the cysts and the sclerotic rims in the middle of the bone 1095 evidence of healing after chronic 2HPT.

1096 Figure 12. Vertebra VC18 (C3) with chondrocalcinosis in annulus firbrosus evidence of CPPD 1097 in hyperparathyroidism (HPT) associated with CKD-MBD.

1098 Figure 13. PNB Humerus AT-742. Note the subperiosteal new bone (PNB) that is typical for the 1099 bones of the SH collection. It is depositional and has a vermicular or round surface porosity. It is 1100 evidence of secondary hyperparathyroidism (2HPT).

1101 Figure 14. Beading of the ribs. Beading of the sternal ends (arrow) is shown in ribs AT-3030 1102 (top), AT1241 (middle), and more characteristically in AT-2996+AT-3067 (lower) consistent 1103 with secondary hyperparathyroidism (2HPT) and rickets diagnosis. Note “mottling” especially to 1104 the left of AT-3030.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 1105 Figure 15. Adolescent Tibia II (left). Note the porosity (SPR) in the medial cortex evidence of 1106 2HPT and ROD. Adult Tibia III (right). Note that the porosity has largely being healed.

1107 Figure 16. Incisor socket of Cranium 9. Upper right incisor socket of Cranium 9. Note that the 1108 LD has been resorbed and partially lost. It is consistent with the diagnosis of ROD and 1109 hibernation in the SH hominins.

1110 Figure 17. Hand phalanx. Lower bone, AT-2520: palmar view of middle hand phalanx 3 or 4. 1111 Subperiosteal lacelike resorption (SPR) and cortex with many holes are present laterally, 1112 medially, and on the palmar surface as evidence of 2HPT. Note loss of normal cortical bone 1113 along the lateral margins15. Note also the juxta-articular subperiosteal resorption (arrow) at the 1114 margin of the proximal interphalangeal joint accounting for the articular manifestation of HPT 1115 simulating the appearance of rheumatoid arthritis163. Top bone AT-2521: palmar view of left 1116 adult middle hand phalanx 5 with the characteristic subperiosteal lacelike resorption at the sides 1117 of the shaft. Note the subperiosteal resorption (arrow) on the ulnar aspect at the corner of the 1118 distal interphalangeal joint18 simulating the appearance of rheumatoid arthritis. The most 1119 common site of subperiosteal resorption in the joints is the distal interphalangeal joint usually the 1120 fourth or fifth164. There is also tunneling in the trabeculae.

1121

1122

1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 1

Histology

Histology of scapula ESC X. Natural fracture of scapula ESC X in the area where the spine meets the body of the scapula. It shows tunneling resorption forming intraosseous cavities in the trabeculae (arrows) which is conclusive evidence of osteitis fibrosa (OF). Some of the trabeculae are unfocused because this is a natural section. These tunnels are the residua of active zones of osteoclastic tunneling resorption due to secondary hyperparathyroidism (2HPT). Note also numerous brown depressions (peritrabecular fibrosis) on the surface of the trabeculae (see also Fig. 2 ) and thin bony spicules transversal to the trabeculae caused by OF too20.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 2

Scapula ESC X showing OF

Scapula ESC showing OF. Natural fracture of scapula ESC X showing OF in the form of peritrabecular fibrosis in the spongiosa. The trabecular surfaces are covered by numerous minute depressions of brownish staining due to diagenetic clay deposited in them postmortem. They are caused by osteoclastic

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 3

Rachitic hyperostosis

Figure 3 . Rachitic hyperostosis. Left: Cranium 11 (a juvenile). Middle: Cranium 9 (early adolescent). Right: Cranium 4 (adult). Note the fine porosity on the Cranium 9 supraorbital area caused by excessive deposition of new bone (arrow) which is absent in the other crania. This is typical rachitic hyperostosis with a typical ‘cardboard-like bone’ surface. In either side of Cranium 9, are the calvaria of a younger (left) and an older (right) individual from SH to demonstrate the absence of rachitic hyperostosis in them as the Cranium 9 is apparently in growth spurt: in older or younger individuals where rickets is less active, the cross section of the cranial bone has a normal histology. The angular appearance of Cranium 9 is due to the rickety craniotabes.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 4

Cross section

Figure 4 .Cross section from above the left orbit of Cranium 9. A natural cross section from the area above the left orbit of Cranium 9 shows 4 rachitic osteoplaques (ROPs) of rachitic hyperostosis (one ROP is indicated by the star) that are evidence of intermittent (seasonal) adolescent growth spurt due to 4 annual hibernations. The ROPs are separated by gaps. Note the superficial ROP is comprised of 4 sublayers which correspond to 4 interbout arousals from torpor indicating that hibernation may have lasted at least 4 months. Scale bar 5mm.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 5

Radius AT-4216

Figure 5. Radius AT-4216 (a) and its natural cross section (b) at the level of radial tuberosity. The flake epiphysis for tuberosity, indicated by an arrow, shows that it was in puberty. Note at least 4 annual layers of bone. In the adult radii of SH, the layers have been remodeled and therefore are not discernible. The number of layers ties with the fact that the flake epiphysis of the radial tuberosity appears and fuses in puberty that lasts 4 years in modern humans. Scale bar 3mm.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 6

Cranium 9

Figure 6 . Cranium 9. Note the flattening of the parietals giving a squared appearance to the skull known as craniotabes (cf. Fig. 3 ) orcaput quadratum(upper left and upper right) that is the clinical hallmark of rickets. Scale bar 5cm.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 7

Cross section of

Cross section of the right parietal of Cranium 9.Natural cross section of the right parietal of Cranium 9 close to the parietal eminence. Note the tunneling (OF) of the diploic trabeculae (arrows) in many areas of the section due to the development of craniotabes in renal rickets. Scale bar 4mm.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 8

Metaphysis of distal femur

Figure 8 . Metaphysis of the distal femur AT-2240+AT-2118+AT208. Note the ‘rotten fence post’ sign. Some of the trabeculae show tunneling, evidence of OF (yellow arrows).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 9

Distal fibula

Figure 9 . Distal adolescent fibula AT886+AT1252. Note the coarsening of metaphyseal trabecular bone evidence of distal fibular SPR and the collapse causing cupping due to resorption of the medial line evidence of hyperparathyroidism (HPT) and renal rickets. Metaphyseal cupping and frayed margins (arrows) can be seen (grade 2 score of rickets). Widening at the growth plate is expected to have existed antemortem. The narrowing seen is an artifact of gluing the two pieces together.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 10

Three femoral necks

Figure 10 . Three femoral necks. The inferior parts of three femoral necks (F3, AT- 2240+AT-2118+AT208, F5) at different stages of growth demonstrating the “rotten fence- post” sign of the primary trabeculae which is pathognomonic of hyperparathyroidism (HPT). The most marked manifestation of this sign is in the middle femur which is at the height of its growth spurt.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 11

Brown tumor of Pelvis 1

Figure 11 . Brown tumor in the ilium of Pelvis 1.A multilocular brown tumor (BT) in the ilium of Pelvis 1. Note the empty spaces of the cysts and the sclerotic rims in the middle of the bone evidence of healing after chronic 2HPT.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 12

Vertebra C3

Figure 12 . Vertebra VC18 (C3) with chondrocalcinosis in annulus firbrosus evidence of CPPD in hyperparathyroidism (HPT) associated with CKD-MBD.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 13

PNB of Humerus AT-742

Figure 13 . PNB of Humerus AT-742. Note the subperiosteal new bone (PNB) that is typical for the bones of the SH collection. It is depositional and has a vermicular or round surface porosity. It is evidence of secondary hyperparathyroidism (2HPT).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 14

Beading of the ribs

Figure 14 .Beading of the ribs. Beading of the sternal ends (arrow) is shown in ribs AT-3030 (top), AT1241 (middle), and more characteristically in AT-2996+AT-3067 (lower) consistent with secondary hyperparathyroidism (2HPT) and rickets diagnosis. Note “mottling” especially to the left of AT-3030.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 15

Tibiae

Figure 15 . Adolescent Tibia II (left). Note the porosity (SPR) in the medial cortex evidence of 2HPT and ROD. Adult Tibia III (right). Note that the porosity has largely being healed.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 16

Incisor socket of Cranium 9

Figure 16 . Incisor socket of Cranium 9.Upper right incisor socket of Cranium 9. Note that the LD has been resorbed and partially lost. It is consistent with the diagnosis of ROD and hibernation in the SH hominins.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 Figure 17

Hand phalanx

Figure 17 . Hand phalanx. Lower bone, AT-2520: palmar view of middle hand phalanx 3 or 4. Subperiosteal lacelike resorption (SPR) and cortex with many holes are present laterally, medially, and on the palmar surface as evidence of 2HPT. Note loss of normal cortical bone along the lateral margins15. Note also the juxta-articular subperiosteal resorption (arrow) at the margin of the proximal interphalangeal joint accounting for the articular manifestation of HPT simulating the appearance of rheumatoid arthritis163. Top bone AT-2521: palmar view of left adult middle hand phalanx 5 with the characteristic subperiosteal lacelike resorption at the sides of the shaft. Note the subperiosteal resorption (arrow) on the ulnar aspect at the corner of the distal interphalangeal joint18 simulating the appearance of rheumatoid arthritis. The most common site of subperiosteal resorption in the joints is the distal interphalangeal joint usually the fourth or fifth164. There is also tunneling in the trabeculae.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27370v1 | CC BY 4.0 Open Access | rec: 20 Nov 2018, publ: 20 Nov 2018