A new oospecies of Shixingoolithus(Shixingoolithus qianshanensis oosp. nov.)from the Qianshan Basin, Anhui Province, East China

Qing H , Zhong-Ling Chn , Shu-Kng Zhng ,Z-Wn Gui , Y-Ting Chn

a School of Resources and Environmental Engineering,Anhui University,Hefei 230601,Anhui Province,China

b State Key Laboratory of Paleobiology and Stratigraphy, Nanjing Institute of Geology and Paleontology,Chinese Academy of Sciences, Nanjing 210008, Jiangsu Province, China

c School of Resources and Environment Engineering, Hefei University of Technology, Hefei 230009, Anhui Province, China

d Geological Survey of Anhui Province, Hefei 230001, Anhui Province, China

e Key Laboratory of Vertebrate Evolution and Human Origins of Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China

Abstract Here we describe two newly discovered dinosaur eggs from the Upper Cretaceous Chishan Formation in the Qianshan Basin, Anhui Province, East China. These dinosaur eggs can be assigned to a new oospecies of Stalicoolithidae, Shixingoolithus qianshanensis, based on the following combined features: the larger size of eggs,the uniform eggshell microstructure in the radial section,the smaller height and the larger density of radial microstructures at the inner surface of the eggshell.Radial sections of S.qianshanensis show closely arranged columnar eggshell units forming relatively uniform and dense microstructure;some secondary eggshell units and numerous sub-circular radial microstructures appear separately in the middle and inner parts of the tangential sections, respectively. The discovery of S. qianshanensis provides new fossil types of Stalicoolithidae and represents the first dinosaur relative record in the Qianshan Basin, which offer accurate paleontological evidence of Late Cretaceous-Early Paleocene stratigraphic classification in the Qianshan Basin, Anhui Province.

Keywords Dinosaur eggs, Stalicoolithidae, Shixingoolithus qianshanensis, Upper Cretaceous, Chishan

1. Introduction

Dinosaur eggs in the Upper Cretaceous of China are characterized by prodigious quantities,abundant types and wide distribution. Approximately 16 oofamilies and 35 oogenera have been reported in China(e.g., Zhao et al., 2015; Xie et al., 2016; Huang et al., 2017; He et al., 2019; Zhang et al., 2019)and most fossils come from the Xixia Basin of Henan Province,the Nanxiong Basin of Guangdong Province,the Ganzhou Basin of Jiangxi Province, the Tiantai Basin of Zhejiang Province, and the Jiaolai Basin of Shandong Province(Zhao et al.,2002;Li et al.,2009;Wang et al., 2012; He et al., 2017; Zhang et al.,2017). Although more and more dinosaur eggs and eggshell fragments have been reported from Anhui Province, previous studies mostly focused on the Xiuning Basin(Wang et al.,2013;Huang et al.,2017;He et al.,2019,2022).Dinosaur eggs from the Xiuning Basin of Anhui Province are mainly assigned to the eggs with honeycomb-like eggshells, such as Umbellaoolithus xiuningensis (Huang et al., 2017), and Similifaveoloolithus qiyunshanensis (He et al.,2019). Other oospecies of dinosaur eggs, such as ?

Parafaveoolithus xiuningensis (Wang et al., 2013), ?Ovaloolithus weiqiaoensis and ?Ovaloolithus huangtulingensis (Yu, 1998), remain in doubt due to the absence of microscopic photographs (Zhao et al.,2015).

Qianshan Basin is also a Late Cretaceous-Early Paleocene red bed basin in Anhui Province, which is characterized by Paleocene vertebrates of mammals,reptiles and birds(Huang and Zheng,2002;Ding et al.,2011; Dong et al., 2016; Wang et al., 2016). A large number of Paleocene vertebrates were assigned to 45 genus and 61 species (He and Hu, 2016; Wang et al.,2016) in the Qianshan Basin, such as rats (Huang and Zheng, 2002, 2003), turtles (Tong et al., 2016), lizards (Dong et al., 2016), but Cretaceous reptiles related to dinosaurs were rarely reported in the vicinity of the Mesozoic-Cenozoic boundary. Recently,three single dinosaur eggs in the Cretaceous strata were firstly discovered in the Qianshan Basin from the Tianzhu Mountain World Geopark. In this paper, we provide the systematic paleontological description of these new specimens and furtherly confirm their systematic position. These studies will also provide an important Mesozoic dinosaur-related evidence for regional stratigraphic classification and a comparison of the Mesozoic-Cenozoic red bed basins in Anhui Province and its adjacent region, East China.

2. Geological settings

The dinosaur egg site is situated near the National Highway 105,Baoling Village,Huangpu Town,Qianshan County, Anqing City, Anhui Province (Fig. 1A), in the Cretaceous strata of Qianshan Basin.Qianshan Basin is a typical Late Cretaceous-Early Paleocene continental sedimentary basin with approximately 100 km length in the east-west direction and 25 km width in the north-south direction, which is located in the southeastern margin of Dabie Orogenic Belt belonging to the margin basin of orogenic belt(He and Hu,2016;Wang et al., 2016; Xie et al., 2021). According to the former studies and the regional geological survey(Geological Survey of Anhui Province, 2002; Zhou et al., 2017), sedimentary sequences of Qianshan Basin include a set of reddish clastic rocks and are divided into the Upper Cretaceous Chishan Formation,the Lower Paleocene Wanghudun Formation and Doumu Formation in an ascending order (Fig. 1B),overlain by the Lower Cretaceous Yangwan Formation and underlain by the Upper Paleocene Wuxueling Formation. The Lower Paleocene strata in the Qianshan Basin yielded a lot of vertebrate fossils which mainly occurred in the Wanghudun and Doumu formations(Wang et al.,2016).The Upper Cretaceous Chishan Formation is dominated by purplish red medium-thick-bedded conglomerates, lithic sandy conglomerates, pebbly sandstones interbedded with purplish red fine sandstones and siltstones,with a total thickness of approximately 887 m (Geological Survey of Anhui Province, 2002, Fig. 1B). The Paleocene Wanghudun Formation reaches a maximum thickness of 1800 m and is subdivided to the lower, middle,and upper members.The lower member mainly consists of purplish red fine sandstones intercalated coarse sandstones and conglomerates; the middle member is composed of red muddy sandstones interbedded with fine conglomerates,and the upper member comprises brownish red sandstones and conglomerates. The Paleocene Doumu Formation contains purplish red coarse sandstones and conglomerates intercalated few shales with a thickness of about 600 m (Wang et al.,2016). The lower and upper members of the Wanghudun Formation and the Doumu Formation yielded lots of reptile and mammal fossils, such as Testudines,Crocodilia, Anagalida and Pantodonta (He and Hu,2016). The newly discovered dinosaur eggs are recovered from the Chishan Formation (Fig. 1B), which represents a typical fluvio-lacustrine environment in the Qianshan Basin (He and Hu, 2016).

Fig. 1 Map showing the location of the newly discovered dinosaur eggs in the Qianshan Basin, Anhui Province, East China.

3. Material and methods

Fig.2 Holotype of Shixingoolithus qianshanensis oosp.nov.from the Qianshan Basin,Anhui Province,East China.A)Two single dinosaur eggs(QS-01 and QS-02); B) The inner and outer surface of QS-01.

There are totally three dinosaur eggs discovered in the Qianshan Basin, two of which were preserved in the Geological Museum of Tianzhu Mountain World Geopark and the other one was lost and still in the process of collection.The preserved two dinosaur eggs contain an incomplete one and a complete one,which were named QS-01 and QS-02 based on the sequence of discovery. The incomplete dinosaur egg (QS-01) was filled with clusters of calcite crystals,thus the calcites on the inner surface of eggshell were cut off with a slicer prior to the treatment of eggshell. Six eggshell fragments with less calcite crystals from QS-01 and QS-02 were collected and the eggshell thickness was measured several times with a caliper. The loose sediments on the outer surface of the eggshells were removed with small needles and then cleaned in an ultrasonic bath for 10 min at the Geological Laboratory of Anhui University. Eggshells were embedded in Technovit 7200 one-component resin and cut using an EXAKT 300CP automatic microtome. Three tangential and one radial sections for each eggshell were grinded and polished to approximately 30 μm thick using an EXAKT 400CP variable speed grinder polisher and abrasive paper. All section processes were finished at the Institute of Vertebrate Paleontology and Paleoanthropology(IVPP),Chinese Academy of Sciences(CAS),China,and further observations were viewed under an Olympus BX53 polarized light microscope in the Anhui University.

4. Systematic paleontology

Oofamily. Stalicoolithidae Wang et al., 2012.

Oogenus. Shixingoolithus Zhao, Ye, Li, Zhao et Yan (1991).

Revised diagnosis. Eggs are nearly spheroid with the shape index(width×100/length)of 96.40-97.80.The length is 105 mm-137 mm and the width is 99 mm-134 mm. Eggshell thickness ranges between 1.60 mm and 2.60 mm. The height of the radial microstructures in the inner part of eggshells is approximately 1/7-1/4 of the whole eggshell thickness.Growth lines through the radial microstructures are obvious.

Oospecies. Shixingoolithus qianshanensis oosp.nov. (Figs. 2 and 3).

Etymology. “Qianshan” is the Chinese phonetic alphabet of the dinosaur egg locality.

Holotype.Two single dinosaur eggs(QS-01 and QS-02;Fig.2)were preserved in the Geological Museum of Tianzhu Mountain World Geopark,Anhui Province,and four eggshell thin sections(D-01,D-02,D-03 and D-04)were collected in the Anhui University.

Fig. 3 Eggshell microstructures of Shixingoolithus qianshanensis oosp.nov. from the Qianshan Basin, Anhui Province, East China. A) Radial section under polarized light microscope, showing closely arranged eggshell units and the erect or irregular pore canals between eggshell units.White arrow points to pore canal and red arrows point to radial microstructures at the inner end of the eggshell unit;White horizontal line indicates the approximate height of the radial microstructures;B)Tangential section through the outer part of the eggshell,showing the sub-circular or sub-triangular pores;C)Tangential section through the middle part of the eggshell,showing larger pores.Black arrows point to the secondary eggshell units in a pore; D) Tangential section through the inner part of the eggshell, showing radial microstructures; E)Enlarged radial microstructures in the inner part of the eggshell.

Type locality and horizon. Chishan Formation,Upper Cretaceous; Baoling Village, Huangpu Town,Qianshan County, Anqing City, Anhui Province.

Diagnosis. Eggs are spheroids with lengths of 123 mm-137 mm and widths of 107 mm-134 mm.Eggshell thickness is 1.60 mm-2.60 mm and the outer surface is weathered. Eggshell units are tightly arranged in the radial section forming relatively uniform microstructure. The height of the radial microstructure in the inner part of the eggshell unit is 0.21 mm-0.29 mm thick on average, which is approximately 1/7 of the entire eggshell thickness; gaps between radial microstructure eggshell units are small and the density of the radial microstructure is approximately 23-27 per square millimeter. Some secondary eggshell units could be discerned in the middle part of the eggshell.

Description. Two collected eggs (Fig. 2A) are spherical and QS-02 is more complete than QS-01.QS-02 is 137 mm×134 mm with the shape index of 97.80,while that of QS-01 could not be accurately measured due to its incomplete shape. The length and width of QS-01 are larger than 123 mm and 107 mm, respectively. The eggshell of QS-01 and QS-02 are brown in color with the average thickness of 1.60 mm-2.60 mm. The outer surfaces of them are weathered and have no obvious ornaments. The inner surface of the eggshell fragment of QS-01 is covered by a calcite crystal layer and individual calcite crystals are evident (Fig.2B).

The eggshell microstructure in the radial section is composed of closely arranged eggshell units and irregular pore canals (Fig. 3A). Eggshell units are columnar with small widths forming relatively uniform and dense microstructures;the columnar layer and the cone layer and the boundaries between them are not seen in these specimens. Nearly horizontal accretion lines are visible in eggshell units and show dark colors throughout the eggshell. Some upright and irregular pore canals are developed in the eggshell units,especially between the eggshell units or near the outer surface, ranging between 0.13 mm and 0.34 mm in width. Numerous radial microstructures at the inner end of the eggshell units are developed in the radial section.The height of the radial microstructure is 0.21 mm-0.29 mm thick,which is approximately 1/7 of the entire eggshell thickness.

Tangential sections of eggshell units show irregular eggshell units and nearly round, oval, or wormlike pores.In the tangential section through the outer part of the eggshell,pores are sub-circular or sub-triangular in shape with the average diameter ranging between 0.23 mm and 0.43 mm (Fig. 3B). Pores become larger with the maximum width up to 1.55 mm in the middle part of the eggshell; some secondary eggshell units could be seen in the pores (Fig. 3C). In the tangential section through the inner part of the eggshell,there are numerous sub-circular eggshell units with radial microstructures (Fig. 3D and E). The eggshell units are separated from each other with diameters of 0.15 mm-0.31 mm(average:0.23 mm).The density of eggshell unit is approximately 23-27 per square millimeter.Due to a large number of calcite crystals on the inner surface of the eggshell, abundant secondary calcites appear and show clear parallel cleavages.

Comparison and discussion. The dinosaur eggs discovered in the Qianshan Basin are nearly spheroids in shape with a shape index of 97.80(123 mm-137 mm in length, 107 mm-134 mm in width), which differ from the oofamily Elongatoolithidae (Zhao, 1975),Prismatoolithidae (Zhao and Li, 1993; Wang et al.,2011) and Macroelongatoolithidae (Wang et al.,2010) with elongated shapes, and the oofamily Ovaloolithidae with elliptical shape (Zhang and Wang,2010), as well as the oofamily Megaloolithidae with larger diameter (Zhao, 1979). The eggshell microstructure consists of tightly arranged eggshell units,rather than superimposed eggshell units represented by the oofamily Dendroolithidae, Faveoloolithidae,Dictyoolithidae,and Youngoolithidae showing reticular or honeycomb-like organization (Zhao et al., 2015),and rather than relatively looser eggshell units such as Umbellaoolithidae (Huang et al., 2017) and Similifaveoloolithidae (He et al., 2019). The macromorphologies and microstructures of dinosaur eggs in the Qianshan Basin are similar to the oofamily Spheroolithidae, but apparent gaps between wedges developed in the spheroolithid eggshells do not exist in eggshells in the Qianshan Basin. The thickness of the radial microstructure in eggshells in the Qianshan Basin is approximately 1/7 of the whole eggshell thickness, far less than that of spheroolithid eggs, in which the thickness of the radial microstructure is about 1/3 of the whole eggshell thickness. Moreover,the evident secondary eggshell units in the tangential section of dinosaur eggs in the Qianshan Basin are not apparent in the spheroolithid eggs. Therefore, the dinosaur eggs in the Qianshan Basin should be assigned to the oofamily Stalicoolithidae based on the spheroid shapes, tightly packed eggshell units with relatively thin radial microstructures, and the presence of secondary eggshell units in pores (Wang et al., 2012).

So far,the oofamily Stalicoolithidae consists of three oogenera (Stalicoolithus, Coralloidoolithus and Shixingoolithus)with three typical oospecies(Stalicoolithus shifengensis, Coralloidoolithus shizuiwanensis and Shixingoolithus erbeni)(Zhao et al.,1991,2015;Wang et al., 2012; Zhu et al., 2019; Fang et al., 2022). The inner,the middle and the outer parts of the eggshells of known oogenera in the radial sections are distinct. On the contrary, radial sections of dinosaur eggs in the Qianshan Basin are relatively uniform without apparent changes of eggshell unit color and pore canal diameter,which are consistent with the characteristics reflected by the tangential sections. Due to the absence of the outermost part of dinosaur eggshell in the Qianshan Basin,the morphology of the secondary eggshell units in the outermost part is absent,and a detailed comparison of the inner end of the eggshell units is chosen.

The dinosaur eggs in the Qianshan Basin share several same characteristics with the Stalicoolithus shifengensis, including the density (approximately 24-26 per square millimeter) and height (0.20 mm-0.29 mm) of radial microstructures. However,the eggshell thickness of Qianshan dinosaur eggs(1.60 mm-2.60 mm) is generally much less than that of S.shifengensis (3.90 mm-4.00 mm) (Wang et al., 2012;Zhu et al., 2019). Also, the circular and sub-circular shapes of radial microstructures from Qianshan dinosaur eggs are different from that of S. shifengensis,which is characterized by the irregular shapes (Wang

et al., 2012). Although Coralloidoolithus shizuiwanensis also develops similar eggshell thickness, the inner end of the eggshell units of C. shizuiwanensis(20-24 per square millimeter) shows smaller density than that of Qianshan dinosaur eggs, and, gaps between the radial microstructures of Qianshan dinosaur eggs are smaller than those of C. shizuiwanensis(Wang et al., 2012; Zhao et al., 2015; Fang et al.,2022). Furthermore, the length and width as well as the shape index of Qianshan dinosaur eggs (shape index = 97.80) are much larger than S. shifengensis(shape index = 93.19) and C. shizuiwanensis (shape index = 87.50) (Table 1).

In comparison with the known oogenera and oospecies in Stalicoolithidae (Table 1), the Qianshan dinosaur eggs are more similar to Shixingoolithus erbeni in macroscopic and microscopic characteristics,such as length,width,eggshell thickness,the shape of radial microstructures, and the ratio of the radial microstructure height to the whole eggshell. The accretion lines forming the radial microstructures at the inner part of the eggshell from Qianshan dinosaur eggs and from S.erbeni are both distinct compared with S.shifengensis and C. shizuiwanensis. Also, the ratio of the radial microstructure height to the whole eggshell(1/7)of Qianshan dinosaur eggs is much closer to that ratio(1/4)of S.erbeni,bigger than the ratio(1/10)of C.shizuiwanensis and much bigger than that(1/20)of S. shifengensis (Zhao et al., 2015; Fang et al., 2022).

However, there are differences in the arrangement, height and density of radial microstructures at the inner surface of the eggshell between the Qianshan dinosaur eggs and Shixingoolithus erbeni (Zhao et al., 1991, 2015). The closely arranged eggshell units of the Qianshan dinosaur eggs are different from the loosely arranged eggshell units of S. erbeni. The height of radial microstructures of the Qianshan dinosaur eggs(0.21 mm-0.29 mm)is less than that of S. erbeni (0.30 mm-0.40 mm), while the density of radial microstructures of the Qianshan dinosaur eggs(23-27 per square millimeter) is much larger than that of S.erbeni(10-12 per square millimeter)(Zhao et al., 1991, 2015). Therefore, the dinosaur eggs found in the Qianshan Basin, Anhui Province could be assigned to oogenus Shixingoolithus based on the following combined features: the larger size of eggs(the length and width are 105 mm-137 mm and 99 mm-134 mm respectively), the approximate eggshell thickness, the similar shapes of radial microstructures,and the larger ratio between the radial microstructure height to the whole eggshell. Nevertheless, the Qianshan dinosaur eggs are easily distinguished from the known oospecies S. erbeni with the closely arranged uniform eggshell units in the radial section, the smaller height and the larger density of radial microstructures at the inner surface of the eggshell.In consequence,the Qianshan dinosaur eggs could represent a new oospecies Shixingoolithus qianshanensis on the basis of above characteristics.

At present, the classification at oogenus level of Stalicoolithidae is mainly determined by the morphology of the secondary eggshell units in the outmost part of eggshell (Wang et al., 2012; Zhao et al., 2015). Although the microstructure of Shixingoolithus is similar to that of Stalicoolithus and Coralloidoolithus, the secondary eggshell units in the outmost part of Shixingoolithus eggshell are far less developed compared with the latter two oogenera(Zhao et al., 2015). Due to the effects of weathering,the outmost part of the eggshells and the corresponding secondary eggshell units are not preserved in the newly discovered Qianshan dinosaur eggs. Considering the similar macroscopic characteristics to Shixingoolithus and the microstructural characteristics of inner surface in the Qianshan dinosaur eggshell, the newly discovered Qianshan dinosaur eggs could be assigned to the oogenus Shixingoolithus and a new oospecies Shixingoolithus qianshanensis oosp. nov.Based on the different features of Stalicoolithus shifengensis, Coralloidoolithus shizuiwanensis and Shixingoolithus erbeni (Wang et al., 2012; Zhao et al.,2015), the discrepancies of these three oogenera mainly focus on the egg size, eggshell thickness, andmicrostructural characteristics of outer and inner parts of eggshell. As more and more Stalicoolithidae eggs have been discovered, we suggest that the classification criteria at oogenus level should take account of both macroscopic and microscopic characteristics.The macroscopic characteristics (length, width and eggshell thickness)and the microscopic characteristics of radial and tangential sections(the arrangement and width of eggshell units)could be used for determining an oogenus level. Further and specific microscopic characteristics containing the microstructural characteristics of outer and inner parts of eggshell, such as the morphology and characteristics of secondary eggshell units, the height and density of radial microstructures and the ratio of radial microstructure height to the whole eggshell, are useful for oospecies identification. Except the morphology of the secondary eggshell units in the outmost part of eggshell, microscopic characteristics including the gaps, the height and density of radial microstructures at the inner part of the eggshell should be comprehensively considered.

Table 1 Comparison on the different parameters of Stalicoolithidae in China.

At the moment,just one oospecies,Shixingoolithus erbeni, has been referred to Shixingoolithus, which mainly distributes in the Upper Cretaceous Pingling,Tongguling and Yuanpu Formations, Guangdong Province(Fang et al.,2009a;Zhao et al.,2015),the Upper Cretaceous Sangping Formation,Henan Province(Fang et al., 1998; Zhao et al., 2015), and the Upper Cretaceous Wulunguhe Formation, Xinjiang Uygur Autonomous Region (Fang et al., 2009b; Zhao et al., 2015).The holotype locality of S. erbeni lies on Luyuan Village, Mashi Town, Shixing County, Guangdong Province (Zhao et al., 1991, 2015). Other localities are distributed in the Maoming City and Nanxiong City of Guangdong Province (Fang et al., 2009a; Zhao et al.,2015), Sangequan area in Junggar Basin of Xinjiang Uygur Autonomous Region (Fang et al., 2009b) and Xixia Basin of Henan Province (Fang et al., 1998). The newly discovered dinosaur eggs from the Qianshan Basin represent the first discovery of Shixingoolithus in Anhui Province,East China and enrich the diversity and paleogeographic distribution of Shixingoolithus. Moreover, the first description of Shixingoolithus qianshanensis from the Qianshan Basin provides new dinosaur-related fossils for the stratigraphic division and correlation of the Upper Cretaceous-Lower Paleocene strata in the Qianshan Basin, Anhui Province,East China.

5. Conclusions

The dinosaur eggs from the Upper Cretaceous Chishan Formation in the Qianshan Basin, Anhui Province, East China, could be referred to a new oospecies of Shixingoolithus (Shixingoolithus qianshanensis) based on their larger sizes, tight arrangement of eggshell units in the radial section,relatively smaller height and larger density of radial microstructures at the inner surface of the eggshell.

New oospecies Shixingoolithus qianshanensis represents the first discovery of oogenus Shixingoolithus from the Qianshan Basin and extends the paleogeographic distribution of the Stalicoolithidae. S. qianshanensis also provides new paleontological evidence for the identification, division and correlation of the Upper Cretaceous-Lower Paleocene strata in the Qianshan Basin, Anhui Province, East China.

Funding

This research was supported by the National Natural Science Foundation of China (Grant No.41802006); the University Natural Science Research Project of Anhui Province (Grant No. KJ2021A0086);the Foundation from the State Key Laboratory of Paleobiology and Stratigraphy, Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences (Grant No. 213104); and, the Innovation and Entrepreneurship Training Program for College Students of Anhui University (Grant Nos. S202110357128 and S202110357129).

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author.

Authors'contributions

All the authors have actively participated in the preparation of the manuscript.Qing He takes charge of manuscript writing and data processing; Zhong-Liang Chen conducts fossil collection and geological survey;Shu-Kang Zhang conducts fossil processing and manuscript modification;Ze-Wen Gui is responsible for map drawing; Ya-Ting Chen conducts manuscript examination. All authors read and approved the final proof.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported in this paper.

Acknowledgements

We are grateful to Ya-Ming Li from Hefei Mingshuo Instrument Technology Co., LTD. for sample pretreatment, and anonymous reviewers for their helpful comments on earlier versions of this paper.