Periodicity of Retzius lines in fossil Pongo from South China

Periodicity of Retzius lines is a key factor in dental development. In this study, we examined the periodicity of Retzius lines in fossil Pongo from South China using polarized light microscope observation of dental ground sections. The periodicities all of the 15 teeth were 9 d. Comparisons of periodicity were made with extant primates, fossil apes and hominins. Periodicity of fossil Pongo from South China was relatively long but fell within the variation of extant Pongo, Gorilla and modern human, and longer than periodicity of Pan and other extant primates. Fossil Pongo from South China was similar to Lufengpithecus and Sivapithecus, shorter than Gigantopithecus and longer than European and African fossil apes and most early hominins in periodicity. Generally, the periodicities of Asian large-body fossil apes were longer than the periodicities of European and African large-body fossil apes in Miocene. Difference among species and trend of evolution in periodicity were analyzed and discussed. We found that periodicity might gradually increase from Proconsul in early Miocene to several fossil apes in Miocene and then Gigantopithecus in Pleistocene. In addition, this study made correlate analysis between periodicity and body mass respectively in males and females of six extant apes and five fossil apes, and found that periodicity positively correlated with body mass.

Incremental dental features are temporal records of development preserved in interior and on the surface of teeth and reflect the process of dental development. Studies of incremental dental development provide valued information for the reconstruction of life history and phylogeny of fossil species. Traditionally, enamel incremental features ( Figure  1) include: (1) cross-striations and (2) Retzius lines and their surface manifestation, known as perikymata. Crossstriations are short-period features, of which periodicity is 24 h. Retzius lines are referred to as long-period features because their periodicity is longer than 24 h. Retzius lines were first found and described by Retzius in 1837 [1] and today form an important part in dental growth and development studies [2]. For example, they are a key variable for accurate estimation of the dental formation time in primates.
However, they can also vary between species and have different features. Retzius lines can also be used to determine the age of juveniles and to characterize individual development, providing information on taxonomy [3]. Previously studied material has included extant and fossil primates, early hominins and modern humans. In China, Zhao et al. researched dental growth and development of Lufengpithecus lufengensis and Gigantopithecus blacki [4][5][6]. In all the achievements of dental development, the determination of periodicity of Retzius lines is an important content, and many data have already been accumulated. But it is vacant in the dental development of Pongo from South China.
Pongo is the only great ape in Asia, living in the tropical forest of Kalimantan and Sumatra now. Fossil records [7][8][9] indicate that Pongo had once widely distributed in Southeast Asia and South China in Pleistocene. In most studies, fossil Pongo from South China were classified into a subspecies of Pongo pygmaeus, Pongo pygmaeus weidenreichi, to distinguish them from those of Indonesian Pongo.
In recent years, many fossil materials of Pongo have been found in South China including thousands of Pongo teeth from more than 30 sites dating from early to late Pleistocene. These fossil Pongo from South China in Pleistocene have contributed to our understanding of the evolution and classification of Asian great apes since late Miocene and the origin of early human. They are important materials for studying the origin, evolution and classification of extant Pongo. Literature concerning fossil Pongo, however, is scarce. Gu et al. [9] and Zhou [7] researched some Pongo teeth from a few sites in South China, and discussed the classification and evolution of Chinese Pongo. Wang et al. [8] discussed their classification of Pongo from South China simply based on measuring a large amount of Chinese fossil Pongo teeth and statistical analysis. Zhao et al. [10] described fossil Pongo teeth from Mulanshan cave, Chongzuo Guangxi and thought that fossil Pongo from South China was different from Indonesian Pongo. All the previous studies about fossil Pongo from South China were based on dental morphology.
In this study, we made dental ground sections of fossil Pongo from South China and observed dental incremental features under polarized light microscope to determine periodicity of Retzius lines, which provide some clues for further study of crown formation time in future, individual development and systematic taxonomy. In addition, periodicities were compared between fossil Pongo from South China with other fossil apes, early hominins, extant primate and modern human. We investigated the differences and correlations among different species of primates. Additionally, we analyzed the correlation of periodicity and body mass, which might provide some information to recover the characters of individual development of fossil hominoids and hominins.

Materials
Fossil Pongo teeth examined in this study were obtained from Guangxi by a Guangxi field investigation team from the Institute of Vertebrate Paleontology and Paleoanthropology during the 1950s-1960s. From those samples, we selected fifteen teeth with complete crowns and with little or slight worn edges and crests. Two of those samples were from the Daxin Gigantopithecus cave, and the geological age was early Middle Pleistocene [11]. In addition, five teeth were bought from a drug store in Liuzhou. Those teeth were mainly from Laibin, Liujiang, Liuzhou, Yishan and Liucheng. Three other teeth were bought from a drug store in Nanning (Guangxi, mainly from Fusei and Chongzuo). Another five teeth were bought from drug stores in Guangxi, but the sites where they originated could not be determined. Considering that there was no Holocene material of fossil Pongo from in South China, and that the geological time of the fossil Pongo were all Pleistocene (because of associated fauns), we estimate that the date of five fossil Pongo teeth with no definite site were also of Pleistocene age.

Production of dental ground sections
All the teeth were cut across the mesial cusps in a buccallingual plane using an EXAKT wire saw. Procedures of making these sections are: The teeth were ultrasonically cleaned in water and etched in 5% phosphoric acid for 20 s. Granular DPX mounting media was paved onto the bottom of an embedded box. The teeth were put onto the DPX mounting media, and liquid DPX mounting media was then poured into the box until the teeth were submerged. The box was transferred to a light curing instrument (EXAKT 530) and exposed in blue followed by yellow light each for 6 hours. Longitudinal cuts were made using a thick wire saw (EXAKT E300CP) after which approximately 120-150 m thick thin sections were cut using a thinner wire saw. The samples were then ground and polished to an 80-100 m thickness using an EXAKT 400CS grinder-polisher. The sections were ultrasonicated, cleaned with ethanol, and mounted onto cover slips using DPX mounting media.

Determination of periodicity
Photomontages were generated with 5× and 20× objectives lenses, using a Leica polarizing light microscope and digital images were captured with a Leica DMRX camera. Retzius lines were observed firstly at 50× magnification. An area with clear cross-striations was then selected to count the number of cross-striations between successive Retzius lines, which was the periodicity of Retzius lines.

Results and discussions
Retzius lines were seen clearly in every section examined under polarized light (Figure 2). In some areas clear prism and cross-striations were observed (Figure 3). The periodicity  obtained from all 15 fossil Pongo teeth in this study was 9 d (Table 1).

Comparison with periodicity of extant primate, modern human, fossil apes and early hominins
The periodicity of the fossil Pongo from South China is 9 d, falling within the variations of extant Pongo and Gorilla [12], and longer than the periodicity of extant Pan, Hylobates, Symphalangus, Semnopithecus, Papio, Macaca and Malagasy. The periodicity of Pongo from South China falls within the variation of modern human and is comparable to the mean of modern human [13].
The results show that the Pongo from South China is similar to Lufengpithecus and Sivapithecus, shorter than Gigantopithecus and longer than Afropithecus turkanensis, Ouranopithecus macedoniensis and Dryopithecus laietanus in periodicity. Periodicities of early hominins such as Australopithecus afarensis, Australopithecus africanus, Paranthropus robustus and Paranthropus robustus are mostly 6-8 d, which is shorter than Pongo from South China. Generally, we find that the periodicities of fossil great apes in Asia were longer than those in Africa and Europe. This may be associated with radiation, spread and area isolation of great apes since the Miocene. The South China Pongo may be more closely related to Lufengpithecus, Sivapithecus, and Gigantopithecus.
In Table 2, Malagasy lemur which belongs to Prosimii (lower primates) has the shortest periodicity (only 3 d). This is shorter than great apes, Hylobates, Symphalangus, Semnopithecus, Papio and Macaca which belong to Anthropoidea (higher primates). The periodicities of great apes (Pongo, Pan and Gorilla) are longer than those of small apes (Hylobate and Symphalangus). The periodicities of Macaca and Papio (Cercopithecinae) are longer than those of Semnopithecus (Colobinae). Additionally, the periodicities of fossil apes with different geological ages were compared. We found that periodicity might gradually increase from Proconsul in early Miocene to several fossil apes in Miocene and then Gigantopithecus in Pleistocene. The periodicity of modern human has a large range of variation. Its maximum (12 d) is much longer than the periodicity of Austalopithecus (6-8 d). Paranthropus robustus [27] 1 9 Paranthropus boisei [28] 1 7 Homo neanderthalensis [29] 1 7

Correlation analysis of periodicity and body mass in living primates and early hominins
Life history traits are a combination of interrelated events, including the timing of key maturational events such as weaning, menarche, age at first reproduction, adult brain size, body mass and longevity. Dental development closely correlates with an individual's life history, and periodicity is an important variable in dental development. Reid et al. [30] reported a positive correlation between periodicity and body mass in hominoids, but did not discriminate between genders. In this study, we analyzed the correlation between periodicity and body mass in six extant apes (Pan troglogytes, Pan paniscus, Pongo pygmaeus, Gorilla gorilla, Hylobates lar and Symphalangus syndactylus) and five fossil apes (Australopithecus afarensis, Australopithecus africanus, Paranthropus robustus, Paranthropus boisei and Homo neanderthalensis). Considering gender difference of body mass, we analyzed males and females separately. Body mass data was taken from the Fleagle [31]. Where a range of periodicity was given, we used the median value.
Our results show that periodicity positively correlates with adult body mass ( Figure 4). The correlation coefficient for females is 0.615 (P < 0.05) and for males is 0.748 (P < 0.01) ( Table 3). It is inferred that body mass and life history of fossil Pongo from South China might be in the variation of extant Pongo.

Conclusions
To summarize, the periodicity of Retzius lines of fossil Pongo from South China is relatively long, when compared to extant primates, fossil apes and early hominins. Periodicity is significantly positively correlated with body mass. It seems that Pongo, Lufengpithecus and Gigantopithecus hominoids from Asia have longer periodicity compared with Miocene fossil apes in Europe and Africa, which have some referenced values to analyze classification, evolution and adaptation of hominoids in different times and areas.