RADIOLOGICAL MICROSCOPY OF THE HAND PHALANX OF A GIRL FROM DENISOVA CAVE*

In 2010, data on the relict genome of Denisova Cave inhabitants were obtained. The source of DNA was the phalanx of the girl's hand. Modern microtomographic techniques open a new page in understanding the processes of morphogenesis in groups of fossil and modern humans, allowing us to work with the most fragmentary material. In this study of the phalanx of a Denisovskaya girl, a non-destructive radiological microscopy method was used to histologically determine the biological age, as well as to identify microstructural features in comparative illumination. It was found that the diaphyseal and metaphyseal sections of this bone were in the stage of active growth. The histological picture of the formation of the diaphyseal wall of the Denisovskaya girl indicates that her biological age corresponds to about 6-7 years of modern man. A completely different, "adult" histological picture was previously revealed in a juvenile Neanderthal from the Okladnikov cave. The similarity of some features of the growth and development of bones of modern humans and Denisovans suggests that the rate of pre-definitive ontogenesis was formed in the Early Paleolithic. The growth rate of Neanderthal children is peculiar, and this specificity probably appeared after the separation of this line of paleoanthropes from the common trunk.

Keywords: Denisova cave, Pleistocene, girl's hand phalanx, radiological microscopy, histomorphometry, biological age, Denisovans, Neanderthals.

Introduction

In recent years, great progress has been made in the field of decoding the genomes of human fossils. In studies conducted at the Max Planck Institute for Evolutionary Anthropology in Leipzig, the features of mitochondrial and nuclear DNA of representatives of different territorial groups of paleoanthropes were determined. It was possible to identify differences in the genomes of Neanderthal and modern humans, indicating the divergence of these evolutionary features.

* "The study was carried out within the framework of the RFBR project No. 13-06-12024 ofi_m.

page 120
At the same time, the genetic relationships of other members of the genus Homo remained unclear for a long time, and few people expected that it would be possible to isolate and sequence ancient DNA from their remains. Homo habilis (H. habilis) and pithecanthropus (H. erectus) settled mainly in the tropics, and in hot climates, paleoDNA is poorly preserved. For this reason, the taxonomic status of the so - called hobbit, a fossil from Flores Island in Indonesia, has not yet been determined. In addition to climate, another obstacle to paleogenetic studies of the genome of early humans was their great antiquity.

In early 2010, the journal Nature published the results of research conducted by Russian archaeologists and German geneticists. Data on the complete decoding of the mitochondrial genome of a human fossil from Denisova Cave in Altai were obtained. This cave has been studied by the expedition of the Institute of Archeology and Ethnography of the Siberian Branch of the Russian Academy of Sciences for many years. Here we trace the evolutionary changes in the culture of primitive man from the early Middle to late Upper Paleolithic, in the range 280 - 10 KA BP (Derevyanko et al., 2003; Derevyanko, 2009, 2011). In stratigraphic layer 11 of Denisova Cave, a phalanx of the hand of a child who lived ca. 50 thousand hp

The morphological features of this bone were studied by B. Viola [Viola et al., 2010, p. 83]. According to the description made before the genetic study, the Denisova-2008-D-2/91 or Denisova-3 find was a proximal epiphysis of the juvenile phalanx of the hand. The proximal articular surface and adjacent bone were preserved. The growth of the proximal epiphysis of the distal phalanx ends at 13.5 years in girls and at 16 years in boys, so the age of the owner of the phalanx was determined as younger. Judging by the overall size of the bone (maximum radioulnar width of the proximal epiphysis 7.5 mm, maximum dorso-palmar height 5.1 mm), it could have been approx. 6-7 years old. However, it was emphasized that the exact age and belonging to a particular beam were not determined.

A molecular genetic study of a sample obtained from the phalanx revealed that it belongs to a female individual [Krause et al., 2010]. A previously unknown hominin mtDNA type was identified that shared a common ancestor with modern humans and Neanderthals, who lived ca. 1 million hp (1 313 500 - 779 300 mtDNA isolated from a male tooth also found in layer 11 of Denisova Cave is very similar to that of a female tooth (Reich et al., 2010). Deciphering the nuclear DNA of this man allowed us to state that this group of hominins, called Denisovans, had a common ancestor with Neanderthals, diverging about 600 thousand years AGO, after which its history developed independently. The genetic heritage of Denisovans has been identified in modern populations of Southeast Asia, Australia, and Oceania (Reich et al., 2010, 2011). This gives rise to the assumption that they have a very wide range. At the same time, the Denisovans remain a mystery in terms of morphology. The tooth from which the DNA was extracted has some extremely archaic features [Reich et al., 2010]. Consequently, other anatomical features of the Denisovian genotype carriers in Southern Siberia could also be far from modern.

Indirect evidence on the level of morphofunctional divergence between Neanderthals and Denisovans is once again provided by genetic studies [Gongbin et al., 2012]. It was found that the driving selection associated with the rapid evolution of microRNAs affected different organs, tissues, and functional systems in these two lines, such as those associated with cognitive abilities in Denisovans or with tissue regeneration in Neanderthals.

Modern methods of radiological and microscopic analysis open a new page in understanding the processes of morphogenesis in groups of fossil and modern humans, allowing us to work with very fragmentary material. The objectives of the present study of the phalanx of a Denisovskaya girl were: description of the preservation of bone material after taking samples for paleoDNA, creation of three-dimensional and planar images of the fragment with magnification, histological determination of biological age, identification of microstructural features in comparative illumination. In this case, a non-destructive radiological microscopy method was used, which is especially important given the uniqueness of the object under study and the degree of its preservation.

After conducting a paleogenetic analysis, it was proved that the representatives of the group called Homo altaiensis (Derevyanko, 2011) are an ancient derivative that dates back to the Early Paleolithic era, associated primarily with the existence of a large - scale polymorphic species Homo erectus.

Previously, specific features of age-related variability in the growth and development of the Neanderthal skeleton were identified (Dobrovolskaya and Mednikova, 2013). They are manifested, in particular, in the early intensive formation of primary and secondary osteons of the periphery of the cortical layer of tubular bones. Based on the description of the histological features of the tubular bones of juvenile Neanderthals, as well as generalization of literature data, it was suggested that their specific hormonal status is manifested in the activation of the parathyroid and thyroid glands. It became obvious that multilevel studies of macro-and

page 121
microstructures allow us to obtain a new layer of information concerning the nuances of the rate of pre-definitive ontogenesis.

Questions of growth and development of fossil forms are of crucial importance in solving the problems of anthropogenesis. In this regard, the analysis of histological parameters of bone associated with the features of the osteon structure, the preservation of areas of lamellar tissue, is extremely interesting. Revealing the similarity of some features of the growth and development of the studied bone and phalanges of the hand of modern humans will allow us to add facts in favor of its convergence with Pleistocene Denisovans. If signs of a peculiar histological structure are found, it will be possible to speak of a separate ontogenetic status, as was done in the study of the Neanderthal skeleton.

Research methods and results

The study examined anthropological remains from the deposits of lithological horizon 11.2 (square D-2) in the eastern gallery of Denisova Cave, discovered in 2008: Homo sapiens, phalanx tertia (V?). This sample served as the source for the first extraction of Denisovian human DNA (Fig.

The survey was performed in the tomography mode on a VersaXRM-500 X-ray microtomograph manufactured by Xradia Inc., USA.

Sample 1. Full-spectrum X-ray point source (< 2 µm) W, without filters, 40 kV / 75 µA. The distance from the source to the sample is 15 mm, from the sample to the detector is 95 mm, and the optical magnification is x0. 4. The tomogram was reconstructed using 4,321 projections with equal angle increments in the range of 0 - 360°angles. The exposure time is constant, 4 s. The camera is 4MP, binnig 2, the projection image size is 1024x1024. The voxel size is 9.2814 microns.

Sample 2. Full-spectrum X-ray point source (< 2 µm) W, without filters, 40 kV / 75 µA. The distance from the source to the sample is 10 mm, from the sample to the detector is 9 mm, optical magnification x 4. The tomogram was reconstructed in 2,000 projections with equal angle increments in the range of 0 - 360°angles. The exposure time is constant, 0.5 s. The camera is 4MP, binnig 2, the projection image size is 1013x1013. The voxel size is 3.5783 microns.

Three-dimensional models (Fig. 2) and virtual sections in the given planes were constructed on the basis of the X-ray microtomograph software.

Two fragments preserved after taking samples for DNA isolation were examined. The first, larger one, represents the proximal surface of the metaphysis, to which, as noted by the first researchers, the epiphysis has not grown. The highest height of this fragment is 3.4 mm (Figs. 3, 4). Another fragment is a thin section of the diaphysis wall.

When considering the virtual cross-sections of the metaphyseal part of the phalanx, the values of osteons were determined. They vary considerably: they are relatively small, with a diameter of about 210 microns, and large - over 514 microns (Figs. 5, 6). In general, the histological picture of the structure of the cross - section through the metaphysis is characterized by an abundance of large primary osteons (more than 350 microns). These structures have a darker shade, which indicates their lower density. Many of them are not formed osteons, but rather large Haversov channels surrounded by a small zone of concentric lamellae. Such structures are large primary osteons with wide Haversov channels and an abundance of Volkman channels.

Fig. 1. Fragments of the distal phalanx of a child's hand from Denisova cave.

2. Three-dimensional reconstruction based on X-ray microtomography: the inner surface of the metaphysis with traces of mechanical perforation.

page 122

3. Proximal surface of the metaphysis, general dimensions.

4. Lateral projection of the bone fragment.

5. Virtual section in the external (proximal) part of the metaphysis with determination of the size of osteons.

Fig. 6. Virtual section in the distal part of the metaphysis with determination of the size of osteons.

7. Virtual section in the region of the diaphysis with determination of the size of osteons.

channels are typical of immature, childlike compacts (Maat, Aarents, Nagelkerke, 2005). In addition, a significant area of the visual field is occupied by areas of unformed bone tissue.

A different picture is observed when considering the osteon structure of the virtual slice in the region of the diaphysis (Fig. 7). Here, the compact is characterized by a significant proportion of lamellar tissue and rare osteons, the diameter of which does not exceed 210 microns. Note that the sizes of osteons are less variable, ranging from 125 to 202 microns.

Discussion

In order to interpret the data obtained, it is necessary to refer to the data on the sex and age dynamics of growth and development of the distal phalanges of the hand of modern humans. According to the accepted terminology, the distal phalanx of the cis-

page 123
ti refers to secondary bones that have more than one ossification center. Primary (punctum ossificationis primarium) appears in the middle of the bone diaphysis at the early stages of intrauterine development (eighth week of embryogenesis), secondary (punctum ossificationis secundarium) are formed at the age of 2 - 3 years. Early ossification or delayed occurrence of secondary ossification centers in the hand is clearly considered as a developmental pathology. According to the reports of M. A. Zhukovsky and A. I. Bukhman [1995], synostosis in the terminal phalanges of the hand in girls is completed by 13.5 - 14 years. However, more recent data [Antonova, 2006, p.40] indicate that epiphysis growth in these bones is complete by the age of 9 years. Thus, it is obvious that depending on the conditions and quality of life, the processes of synostosis of the distal phalanx can vary widely even in modern humans. The age of formation of ossification centers is determined more strictly.

Based on the above, it can be concluded that two areas of bone, the diaphyseal and metaphyseal, are characterized by different stages of osteogenesis. These differences are expected, since the metaphyseal parts of the bone develop much later than the diaphyseal parts. This discrepancy also confirms the reliability of histological research in the study of ontogenesis. The metaphyseal and epiphyseal parts of the bone are in the stage of active growth. This corresponds to macromorphological data indicating that the epiphyseal width and height of the bone will still be significantly increased. Therefore, within the previously defined age range of 6 - 7 years [Viola et al., 2010], the first half of the interval is more preferable. Significantly less active rearrangements will occur in the thin layer of the diaphysis compacta. Most of them will be associated with the formation of trabeculae.

So, the observed histological picture of the formation of the diaphysis wall of the studied bone allows us to conclude quite confidently that the biological age of the Denisov girl corresponded to the category infantilis 2, i.e. approximately 6-7 years of modern man.

Based on previous studies of the histological structure of the tubular bones of juvenile Neanderthals in Altai, it is possible to compare the studied individual not only with modern humans, but also with the fossil form. An important fact is that Neanderthals and Denisovans lived in the caves of the Northwestern Altai at the same time - 50-40 thousand years ago. This makes it possible not to compare the surrounding natural conditions.

The compactness of the diaphysis of Neanderthal tubular bones was characterized either by the absence of a lamellar layer, or by a small fraction of it (less than 20 %). The size of osteons in the most actively growing part varied within 180-350 microns (Dobrovolskaya and Mednikova, 2013). No larger ones, with a diameter of 400 microns or more, were found. Areas with a predominance of primary osteons in the initial stage of formation were also not noted. The proportion of secondary osteons and bone rearrangements in the cortex of the diaphysis of juvenile Neanderthals in Altai is also high. If the macromorphological estimate of the age of the Neanderthal from Okladnikov Cave corresponded to 8-10 years of the Neanderthal child and 6 years of the modern one, then the indicators of internal massiveness of tubular bones were 16 [Mednikova, 2011; Mednikova, 2011], and the histological picture was 50 years [Dobrovolskaya, Mednikova, 2013] of sapiens.

At the same time, macromorphological criteria show that the Denisov girl and the juvenile Neanderthal from Okladnikov Cave belong to the same age cohort. In addition, they share a common territory (the distance between the caves is about 100 km) and belong to the same stage of the Upper Pleistocene. All the more remarkable is the contrast in the internal structure of the diaphysis of the tubular bones of these children: the similar "juvenile" bone tissue of the Denisov girl and the "maturity"of the Neanderthal.

Conclusion

During the study of fragments of the hand phalanx of the Denisovskaya girl using X-ray microscopy, a histological picture was obtained that is in good agreement with the sequence and chronology of processes described for modern humans. Unlike Neanderthals, the individual belonging to the Denisovans group does not show a striking peculiarity in the microstructural parameters of bone growth and development. The revealed general patterns of ontogenesis of modern humans and Denisovans indicate some closeness between them. This suggests that the most important events of pre-definitive ontogenesis occurred in anthropogenesis as early as the Early Paleolithic. The specific growth and development rates of Neanderthals were formed after the separation of this group of ancient people.

List of literature

O. A. Antonova Age-related anatomy and physiology. Moscow: Vysshee obrazovanie Publ., 2006, 192 p. (in Russian)

Gunbin K. V., Afonnikov D. A., Kolchanov N. A., Derevyano A. N. The Important role of microRNA changes in the Evolution of Homo neanderthalensis and Homo denisova.,

page 124
ethnography and anthropology of Eurasia. - 2012. - N 3. - p. 22-29.

Derevyanko A. P. Transition from the Middle to Upper Paleolithic and the problem of formation of Homo sapiens sapiens in East, Central and Northern Asia. Novosibirsk: IAET SB RAS Publ., 2009, 327 p. (in Russian)

Derevyanko A. P. The Upper Paleolithic in Africa and Eurasia and the formation of a modern anatomical type of man. Novosibirsk, IAET SB RAS Publ., 2011, 560 p. (in Russian)

Derevyanko A. P., Shunkov M. V., Agadzhanyan A. K., Baryshnikov G. F., Malaeva E. M., Ulyanov V. A., Kulik N. A., Postnov A.V., Anoikin A. A. Natural environment and man in the Paleolithic of Gorny Altai. Novosibirsk: IAET SB RAS Publ., 2003, 448 p. (in Russian)

Dobrovolskaya M. V., Mednikova M. B. "Adult" children of Neanderthals: a histological study of juvenile individuals from Okladnikov Cave / / Fundamental problems of Archeology, Anthropology and Ethnography of Eurasia: To the 70th anniversary of Academician A. P. Derevyanko. Novosibirsk: Izd-vo IAET SB RAS, 2013, pp. 523-537.

Zhukovsky M. A., Bukhman A. I. Ossification of the hand skeleton and distal forearm in children and adolescents in Moscow // Children's endocrinology: a guide for doctors, Moscow: Meditsina Publ., 1995, p. 40.

Mednikova M. B. Postcranial morphology and taxonomy of representatives of the genus Homo from Okladnikov Cave in Altai. Novosibirsk, IAET SB RAS Publ., 2011, 128 p. (in Russian)

Krause J., Fu Q., Good J.M, Viola B., Shunkov M.V., Derevianko A.P., Paabo S. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia // Nature. - 2010. - Vol. 464, N 7290. - P. 894 - 897.

Maat G.J.R., Aarents M.J., Nagelkerke N.J.D. Age prediction from bone replacement. Remodeling of circumferential lamellar bone tissue in the anterior cortex of the femoral shaft of present Dutch population. - Leiden: Leiden University Medical Center, 2005. - 67 p. - (Barge's Anthropologica; N 10).

Mednikova M. Morphological patterns and developmental age of Neanderthal juvenile postcranial remains from Altai //Abstracts of Meeting of European Society of the Study of Human Evolution, Leipzig, 23 - 24 September, 2011. - Leipzig, 2011. - P. 69.

Reich D., Green R.E., Kircher M., Krause J., Patterson N., Durand E.Y., Viola B., Briggs A.W., Stenzel U., Johnson P.L.F., Maricic T., Good J.M., Marques-Bonet T., Alkan C., Fu Q., Mallick S., Li H., Meyer M., Eichler E.E., Stoneking M., Richards M., Talamo S., Shunkov M.V., Derevianko A.P., Hublin J.-J., Kelso J., Slatkin M., Paabo S. Genetic history of an archaic hominin group from Denisova Cave in Siberia // Nature. - 2010. - Vol. 468, N 7327. - P. 1053 - 1060.

Reich D., Patterson N., Kircher M., Delfin E, Nandineni M.R., Pugach L., Min-Shan Ko. A., Ying-Chin Ko., Jinam T.A., Phipps M.E., Saitou N., Wollstein A., Kayser M., Paabo S., Stoneking M. Denisova Admixture and the First Modern Human Dispersals into Southeast Asia and Oceania //Am. J. of Human Genetics. - 2011. - Vol. 89. - P. 516 - 528.

Viola B., Richards M., Talamo S., Shunkov M.V., Derevianko A.P., Hublin J.-J. Supplementary information 12. Morphology of the Denisova molar and phalanx. Stratigraphy and dating // Nature. - 2010. - Vol. 468, N 7327. - P. 81 - 86.

The article was submitted to the Editorial Board on 11.02.13, in the final version-on 11.02.13.

page 125

Permanent link to this publication: