保存最古老矿物结合多肽的中新世鸵鸟蛋壳化石首次组织化学研究

doi:10.19615/j.cnki.2096-9899.240329

摘要/Abstract

摘要：

古蛋白质比古DNA具有更高的保存潜力，因此蛋白质组学研究可以帮助阐明一些超出古DNA研究领域的灭绝生物群体的生物学特征。迄今为止最古老的多肽发现于中国西北地区晚中新世临夏盆地的鸵鸟蛋壳化石中，是与蛋壳矿化相关的蛋白质struthiocalcin (SCA-1) 的一部分。前人认为SCA-1在蛋壳中均匀分布，并因其与方解石晶体结合的特性而得以在地质历史中长时间保存。本次对同一鸵鸟蛋壳化石进行了组织学、扫描电子显微镜和拉曼光谱分析，发现蛋壳内侧锥体层的晶核含有部分磷灰石，其他部位则完全由方解石构成；这些晶核部分应当是在成岩作用过程中经历了磷酸盐化。在对该化石蛋壳样品脱钙处理后，其锥体层晶核部分存在残留物，呈现网络状纤维结构，其位置和形态与现生鸵鸟蛋壳中脱钙后残留的有机质相似。结果表明，该化石蛋壳中的古多肽可能集中保存在锥体层晶核处，而非在整个蛋壳中均匀分布。磷酸盐化可能是另一个有利于有机物长期保存的埋藏过程。临夏盆地的古气候和埋藏环境可能为该古蛋白分子的保存提供了有利的条件。建议在未来研究中进行更深入的组织化学和矿物学分析，以进一步了解该盆地有机质和古蛋白的保存机制。

关键词: 化石有机质, 生物矿化蛋白, 磷灰石, 磷酸盐化, 鸵鸟蛋壳, 古蛋白

Abstract:

Because ancient proteins have a higher preservation potential than ancient DNA, proteomic studies can help shed light on the biology of some extinct biological groups that are beyond the reach of the field of ancient DNA. The oldest peptide discovered so far is part of the protein struthiocalcin (SCA-1) involved in eggshell mineralization and found within an ostrich egg from the Late Miocene Linxia Basin of Northwest China. It was originally hypothesized that SCA-1 was evenly distributed within the eggshell and was able to enter the fossil record for so long, because it was bound to calcite crystals. We conducted histological, scanning electron microscopy and Raman spectroscopic analyses on this same fossil egg to test if any protein or organic matter could be observed within specific regions of the eggshell and indeed bound to calcite crystals. Our results show that the eggshell is made entirely of calcite except at the base layer, which is made of mammillary knobs at least partially made of apatite. These knobs were secondarily phosphatized during diagenesis. After decalcification of this material, the fossilized mammillary knobs showed fibrous residues consistent in location and morphology with remnants of original organic material forming a network. This network was similar to the organic matrix observed in an extant ostrich eggshell with this same method. The results here suggest that SCA-1 may have been concentrated at the mammillary knobs, rather than evenly throughout the eggshell. Phosphatization may be another taphonomic process that favors organic preservation in deep-time. The paleoclimate and taphonomic environment of the Linxia Basin may have provided favorable conditions for the molecular preservation of this egg. More in-depth histochemical and mineralogical analyses will certainly increase our understanding of organic and ancient protein preservation in this basin.

Key words: fossil organics, struthiocalcin, apatite, phosphatization, ostrich eggshell, ancient proteins

中图分类号:

Q915.21

吴倩, 泮艳红, 李志恒, 周忠和, 艾莉达. 保存最古老矿物结合多肽的中新世鸵鸟蛋壳化石首次组织化学研究. 古脊椎动物学报, 2024, 62(2): 120-134.

WU Qian, PAN Yan-Hong, LI Zhi-Heng, ZHOU Zhong-He, Alida M. BAILLEUL. First histochemical examination of a Miocene ostrich eggshell with the oldest mineral-bound peptides. Vertebrata Palasiatica, 2024, 62(2): 120-134.

图/表 6

Fig. 1 Histology of the fossil and the extant ostrich eggshells A. image of a ground section of the fossil eggshell IVPP V26107; B, C. close-up within the mammillary layer of V26107 (B) and its corresponding polarized light image (C) showing the optical extinction of the mammillary knobs; D. image of the ground section of an extant common ostrich eggshell; E. illustration of the structure of avian eggshells; F, G. close-up of the mammillary layer in D (F) and its corresponding polarized light image (G) Abbreviations: cl. cuticle layer; em. egg membrane; is. internal surface; mk. mammillary knob;ml. mammillary layer; os. outer surface; pc. pore canal; pl. palisade layer, sl. surface crystal layer

Fig. 2 SEM and EDS of the mammillary knobs of the fossil and extant ostrich eggshell ground-section slices A-E. backscattered electron image (A), close-up of the mammillary knob (B-D), and EDS analysis (E) on B of ground-section slice of IVPP V26107, showing a high concentration of phosphorus within the mammillary knob; F-I. backscattered electron image (F), close-up of the mammillary knob (G-H), and EDS analysis (I) on G of a ground-section slice of the extant ostrich eggshell mk. mammillary knob

Fig. 3 Demineralized paraffin section of the fossil (A, D and E) and extant ostrich eggshells (B and C) Image of a paraffin section of fossil eggshell IVPP V26107 left completely unstained (A) and extant ostrich eggshell (B) stained with alcian blue (C). Close-ups of the fossil residues show a fibrous network most likely representing ancient proteins or at least, some fossilized organic matter (D-E) Abbreviations: ag. agar; em. egg membrane; mk. mammillary knob. B and C share the same scale bar

Fig. 4 SEM and EDS of demineralized paraffin section slice of IVPP V26107 Backscattered electron image of demineralized products on a paraffin section slice of IVPP V26107 (A), its close-up (B) and EDS analysis (C) on the same image, showing the fibrous matter rich in carbon and slightly calcified (indicated by the white arrow), attached to crystal of calcite

Fig. 5 Raman spectra point analysis results of the mammillary knob of fossil and extant ostrich eggshell Showing the concentration of apatite in the mammillary knob of IVPP V26107

Fig. 6 Hypothesized diagenetic pathways of the ostrich eggshell from Linxia Basin

参考文献 38

[1]	Arias J L, Mann K, Nys Y et al., 2007. Eggshell growth and matrix macromolecules. In: Bäuerlein ed. Handbook of Biomineralization. Weinheim: John Wiley & Sons, Ltd. 309-327
[2]	Bailleul A M, Zheng W, Horner J R et al., 2020. Evidence of proteins, chromosomes and chemical markers of DNA in exceptionally preserved dinosaur cartilage. Natl Sci Rev, 7: 815-822 DOI PMID
[3]	Bergmann U, Morton R W, Manning P L et al., 2010. Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging. Proc Natl Acad Sci USA, 107: 9060-9065 DOI PMID
[4]	Briggs D E G, Kear A J, 1993. Fossilization of soft tissue in the laboratory. Science, 259: 1439-1442 PMID
[5]	Briggs D E G, Kear A J, Martill D M et al., 1993. Phosphatization of soft-tissue in experiments and fossils. J Geol Soc, 150: 1035-1038 DOI URL
[6]	Collins M J, Riley M S, 2000. Amino acid racemization in biominerals:the impact of protein degradation and loss. In: GoodfriendG A, CollinsM J, FogelM L et al. eds. Perspectives in Amino Acid and Protein Geochemistry. Oxford: Oxford University Press. 120-142
[7]	Cusack M, Fraser A C, Stachel T, 2003. Magnesium and phosphorus distribution in the avian eggshell. Comp Biochem Physiol B Biochem Mol Biol, 134: 63-69 DOI URL
[8]	Demarchi B, Hall S, Roncal-Herrero T et al., 2016. Protein sequences bound to mineral surfaces persist into deep time. eLife, 5: e17092 DOI URL
[9]	Demarchi B, Mackie M, Li Z H et al., 2022. Survival of mineral-bound peptides into the Miocene. eLife, 11: e82849 DOI URL
[10]	Deng T, 2004a. Evolution of the Late Cenozoic mammalian faunas in the Linxia Basin and its background relevant to the uplift of the Qinghai-Xizang Plateau. Quat Sci, 24: 413-420
[11]	Deng T, 2004b. Cenozoic stratigraphic sequence of the Linxia Basin in Gansu, China and its evidence from mammal fossils. Vert PalAsiat, 42: 45-66
[12]	Deng T, 2009. Late Cenozoic environmental changes in the Linxia Basin (Gansu, China) as indicated by cenograms of fossil mammals. Vert PalAsiat, 47: 282-298
[13]	Gautron J, Nys Y, 2007. Eggshell matrix proteins. In: HuopalahtiR, López-FandiñoR, AntonM et al. Bioactive Egg Compounds. Berlin, Heidelberg: Springer Berlin Heidelberg. 103-108
[14]	Gautron J, Stapane L, Le Roy N et al., 2021. Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit. BMC Mol Cell Biol, 22: 11 DOI PMID
[15]	Hendy J, 2021. Ancient protein analysis in archaeology. Sci Adv, 7: eabb9314 DOI URL
[16]	Hendy J, Welker F, Demarchi B et al., 2018. A guide to ancient protein studies. Nat Ecol Evol, 2: 791-799 DOI PMID
[17]	Hincke M T, Tsang C P W, Courtney M et al., 1995. Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin 17). Calcif Tissue Int, 56: 578-583 DOI URL
[18]	Hincke M T, Nys Y, Gautron J et al., 2012. The eggshell: structure, composition and mineralization. Front Biosci Landmark Ed, 17: 1266-1280 DOI URL
[19]	Hincke M T, Da Silva M, Guyot N et al., 2018. Dynamics of structural barriers and innate immune components during incubation of the avian egg: critical interplay between autonomous embryonic development and maternal anticipation. J Innate Immun, 11: 111-124 DOI URL
[20]	Hong M H, Lee J H, Jung H S et al., 2022. Biomineralization of bone tissue: calcium phosphate-based inorganics in collagen fibrillar organic matrices. Biomater Res, 26: 42 DOI URL
[21]	Iline-Vul T, Nanda R, Mateos B et al., 2020. Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites. Sci Rep, 10: 15722 DOI PMID
[22]	Kendall C, Eriksen A M H, Kontopoulos I et al., 2018. Diagenesis of archaeological bone and tooth. Palaeogeogr Palaeoclimatol Palaeoecol, 491: 21-37 DOI URL
[23]	Legendre L J, Rubilar-Rogers D, Musser G M et al., 2020. A giant soft-shelled egg from the Late Cretaceous of Antarctica. Nature, 583: 411-414 DOI
[24]	Li J, Fang X, 1999. Uplift of the Tibetan Plateau and environmental changes. Chinese Sci Bull, 44: 2117-2124 DOI URL
[25]	Li Y, Zhu X, Wang Q et al., 2022. Apatite in Hamipterus tianshanensis eggshell: advances in understanding the structure of pterosaur eggs by Raman spectroscopy. Herit Sci, 10: 84 DOI
[26]	Li Z H, Bailleul A M, Stidham T A et al., 2021. Exceptional preservation of an extinct ostrich from the Late Miocene Linxia Basin of China. Vert PalAsiat, 59: 229-244
[27]	Liu X D, Dong B W, 2013. Influence of the Tibetan Plateau uplift on the Asian monsoon-arid environment evolution. Chinese Sci Bull, 58: 4277-4291 DOI URL
[28]	McCoy V E, 2014. Concretions as agents of soft-tissue preservation: a review. Paleontol Soc Pap, 20: 147-162 DOI URL
[29]	Nys Y, Gautron J, Garcia-Ruiz J M et al., 2004. Avian eggshell mineralization: biochemical and functional characterization of matrix proteins. C R Palevol, 3: 549-562 DOI URL
[30]	Orr P J, 2014. Late Proterozoic-Early Phanerozoic ‘taphonomic windows’: the environmental and temporal distribution of recurrent modes of exceptional preservation. Paleontol Soc Pap, 20: 289-313 DOI URL
[31]	Schiffbauer J D, Wallace A F, Broce J et al., 2014. Exceptional fossil conservation through phosphatization. Paleontol Soc Pap, 20: 59-82 DOI URL
[32]	Schweitzer M H, Zheng W, Zanno L et al., 2016. Chemistry supports the identification of gender-specific reproductive tissue in Tyrannosaurus rex. Sci Rep, 6: 23099 DOI PMID
[33]	Solomon S E, 2010. The eggshell: strength, structure and function. Br Poult Sci, 51: 52-59
[34]	Stein K, Prondvai E, Huang T et al., 2019. Structure and evolutionary implications of the earliest (Sinemurian, Early Jurassic) dinosaur eggs and eggshells. Sci Rep, 9: 4424 DOI PMID
[35]	Thomas B, Taylor S, 2019. Proteomes of the past: the pursuit of proteins in paleontology. Expert Rev Proteomic, 16: 881-895 DOI PMID
[36]	Warren M, 2019. Move over, DNA: ancient proteins are starting to reveal humanity’s history. Nature, 570: 433-436 DOI
[37]	Yang T R, Chen Y H, Wiemann J et al., 2018. Fossil eggshell cuticle elucidates dinosaur nesting ecology. PeerJ, 6: e5144 DOI URL
[38]	Zhang Y, Liu Y, Ji X et al., 2011. Flower-like agglomerates of hydroxyapatite crystals formed on an egg-shell membrane. Colloid Surface B, 82: 490-496 DOI PMID