Three-Dimensional Virtual Histology of Early Vertebrate Scales Revealed by Synchrotron X-Ray Phase-Contrast Microtomography

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1213

Three-dimensional Virtual Histology of Early Vertebrate Scales Revealed by Synchrotron X-ray Phase-contrast Microtomography

QINGMING QU

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9128-4 UPPSALA urn:nbn:se:uu:diva-238056 2015 Dissertation presented at Uppsala University to be publicly examined in Lindahlsalen, Norbyvägen 18A, Uppsala, Monday, 2 February 2015 at 14:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Moya Smith (King’s College London).

Abstract Qu, Q. 2015. Three-dimensional Virtual Histology of Early Vertebrate Scales Revealed by Synchrotron X-ray Phase-contrast Microtomography. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1213. 49 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9128-4.

Vertebrate hard tissues first appeared in the dermal skeletons of early jawless vertebrates (ostracoderms) and were further modified in the earliest jawed vertebrates. Fortunately, histological information is usually preserved in these early vertebrate fossils and has thus been studied for more than a century, done so by examining thin sections, which provide general information about the specific features of vertebrate hard tissues in their earliest forms. Recent progress in synchrotron X-ray microtomography technology has caused a revolution in imaging methods used to study the dermal skeletons of early vertebrates. Virtual thin sections obtained in this manner can be used to reconstruct the internal structures of dermal skeletons in three- dimensions (3D), such as vasculature, buried odontodes (tooth-like unites) and osteocytes. Several body scales of early vertebrates have been examined using this imaging method and in situ 3D models of internal structures are created. Andreolepis (an early osteichthyan) scale shows linear growth pattern of odontodes in early developmental stage, which is not observable in traditional thin sections. The scale of another early osteichthyan Psarolepis was studied in the same way. Comparison between Andreolepis and Psarolepis shows that cosmine, a tissue complex in dermal skeleton of early sarcopterygians, originated by a developmental change of odontode shape. Two scales of osteostracans, a group of extinct jawless vertebrates, were studied in 3D and more details have been revealed in comparison to previous results based solely on 2D thin sections. 3D data enables us to compare the vasculature and canal system in different taxa in great detail, which forms the basis of formulating primary homology hypothesis and phylogenetic characters. The new data resulting from this study suggests that vertebrate fossils have preserved much more histological information than we currently appreciate, and provide a new data source of microanatomical structures inside the fossils that can contribute new characters for phylogenetic analysis of early jawed vertebrates.

Keywords: 3D virtual paleohistology, scales, ontogeny, jawed vertebrates, phylogeny

Qingming Qu, Department of Organismal Biology, Norbyv 18 A, Uppsala University, SE-75236 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-554-9128-4 urn:nbn:se:uu:diva-238056 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-238056)

To my parents and Xin (欣)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Qu QM, Zhu M, Wang W. (2013). Scales and dermal skeletal histology of an early bony fish Psarolepis romeri and their bearing on the evolution of rhombic scales and hard tissues. PLoS One, 8: e61485. II Qu QM, Sanchez S, Blom H, Tafforeau P, Ahlberg PE. (2013) Scales and tooth whorls of ancient fishes challenge distinction between external and oral ‘teeth’. PLoS One, 8: e71890. III Qu QM, Sanchez S, Zhu M, Blom H, Tafforeau P, Ahlberg PE. The origin of novel features by changes in developmental mechanisms: a 3D virtual paleohistology study on polyodontode scales of primitive osteichthyans. Manuscript. IV Qu QM, Märss T, Sanchez S, Blom H, Ahlberg PE. Three- dimensional virtual histology of Silurian osteostracan scales revealed by synchrotron radiation microtomography. Submitted, Journal of Morphology. V Qu QM, Haitina T, Zhu M, Ahlberg, PE. New genomic and fossil data illuminate the origin of enamel. Submitted, Nature.

Additional Publications These papers were published during the doctoral studies but not included in the thesis. VI Zhu M, Yu XB, Choo B, Qu QM, Jia LT, Zhao WJ, Qiao T, Lu J. (2012) Fossil fishes from China provide first evidence of dermal pelvic girdles in osteichthyans. PloS One, 7: e35103. VII Zhu M, Yu XB, Ahlberg PE, Choo B, Lu J, Qiao T, Qu QM., Zhao WJ, Jia LT, Blom H, Zhu YA. (2013) A Silurian placoderm with osteichthyan-like marginal jaw bones. Nature, 502, 188-193.

In Paper I, QMQ made all the thin sections, produced all figures and wrote the manuscript with contribution from the other two authors. In Paper II, III and IV, QMQ segmented the data and produced all figures and wrote the

manuscript with contribution from all other authors. In Paper V, QMQ made and analyzed all the thin sections, and wrote the paper with the other authors.

The project was supported by ERC Advanced Investigator Grant 233111 and a Wallenberg Scholarship (both to Per Ahlberg).

Contents

Introduction ...... 9

Paleohistology of early vertebrates ...... 13 A brief history ...... 13 3D synchrotron virtual paleohistology ...... 14 3D virtual paleohistology and phylogeny ...... 16 Phylogeny of early jawed vertebrates ...... 16 Subdivision of the dermal skeleton of early jawed vertebrates ...... 18 Histology of dermal skeletons of early osteichthyans ...... 19 Histology of dermal skeletons of “placoderms” ...... 21 Histology of dermal skeletons of “acanthodians” ...... 22 Histology of dermal skeletons of early chondrichthyans ...... 26 3D paleohistology characters in phylogeny studies ...... 28 Vasculature and canal system within different dermal elements ...... 30 Odontodes in polyodontode scales and dermal bones ...... 31 Distribution and size variation of osteocytes ...... 31 Dentine types ...... 32 Concluding remarks and future perspectives ...... 33

Swedish summary – Svensk sammanfattning ...... 35

Acknowledgments...... 37

References ...... 40

Introduction

This thesis focuses on the histology of dermal skeletal fossils (scales in particular) of long-extinct vertebrates from the Late Silurian to Early Devonian (around 420 million years BP). Like all other paleobiology studies, a good understanding of extant organisms is the foundation and starting point of the work. Hence a brief survey of hard tissues in extant vertebrates is given first, followed by an account of the importance of fossils to understanding the evolution of these tissues. Vertebrates are distinguished from all other animals by having a complicated skeletal system that is made of calcium phosphate (i.e., hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂). The skeletal system has played a key role for the success of vertebrates (including humans) during evolution, by serving as internal support for the body (for example vertebrae), as a physical apparatus for predation in jawed vertebrates (teeth), as a reservoir of phosphate that is essential for the metabolism, and as haematopoietic stem cell niches (bone marrows). The diversity of skeletons in living vertebrates is enormous at the morphological level, from dogfish teeth to turtle shell, from fugu beak to bat thumb, from panda sesamoid bone to zebrafish scales and so forth. Neverthe- less, such a diversified skeletal system is mostly composed of four major categories of hard tissues at the histological level: (calcified) cartilage, bone, dentine and enamel (Hall, 2005). Intermediate tissues such as tooth cemen- tum and chondroid bone, which show developmental and histological similarities to more than one type of hard tissue, also exist; but such tissues are usually taxa-specific or region-specific (Hall, 2005). As connective tissues, all types of vertebrate hard tissues are formed on extracellular matrices (ECM), which behave as scaffolds, and each is secreted by a specific type of cell (i.e., chondroblasts for cartilage, osteoblasts for bone, odontoblasts for dentine and ameloblasts for enamel). Cartilage is characterized by large chondrocytes surrounded by extracellular matrix, which is mainly composed of type II collagens and proteoglycans, and can be stained with Alcian blue for whole mount samples or thin sections. The matrix is secreted by chondroblasts that later become chondrocytes within cartilage (Hall, 2005). In the adult human body, most cartilages have gone through an ossification process called endochondral ossification and been replaced by endochondral bone, such as in humeri and vertebrae. During endochondral ossification, cartilage first becomes globular calcified cartilage and is later replaced by bone, but in certain taxa globular calcified

9 cartilage can be retained at two ends (epiphyses) of long bones (Francillon- Vieillot et al., 1990). Occasionally, cartilage can remain without going through a mineralization process, such as at joints between bones. Unlike in osteichthyans, the cartilage of extant chondrichthyans (sharks and chimaeras) is usually replaced by endoskeletal tesserae made of prismatic calcified cartilage (surface) and globular calcified cartilage (inside) (Dean and Summers, 2006; Hall, 2005; Moss, 1977). Cartilage has also been found in some invertebrates such as cuttlefish and horseshoe crab (Cole and Hall, 2004), and is thus considered a much older tissue than vertebrates themselves. However, vertebrate cartilage ECM is comprised of mainly type II collagen, while invertebrate cartilage is comprised of type I collagen, more similar to the bone ECM of vertebrates (Cole and Hall, 2004). Type II collagen-based cartilage is present in lancelets and cyclostomes, suggesting its early origin before the rise of vertebrates themselves (Zhang and Cohn, 2006; Zhang et al., 2006). Although in vitro calcification has been detected in cyclostomes (Langille and Hall, 1993), their cartilage does not normally calcify (Langille and Hall, 1993), implying that the origin of calcified cartilage occurred after the divergence of extant jawless vertebrates and jawed vertebrates. In contrast to cartilage, the other types of hard tissues are always calcified and only present in living jawed vertebrates but not cyclostomes. Dentine and enamel are considered as dental tissues as they are the major components of our teeth. Canonically, dentine is characterized by numerous thin tubules formed by the retreat of odontoblasts into pulp cavities during the deposition of dentine ECM, which is mainly based on type I collagens and proteoglycans, just as in bone (Butler et al., 1997; Hall, 2005). Such dentine is present only in the teeth of living tetrapods, but has also been described in the scales of extant sharks (Applegate, 1967) and primitive osteichthyan fishes like Latimeria (Smith et al., 1972) and Polypterus (Kerr, 1951; Meinke, 1982), based on the presence of typical dentine tubules. Some catfish also seem to have tooth-like denticles (with normal dentine) on their scales (Sire and Huysseune, 1996), while several other teleost lineages have evolved tooth-like denticles outside the mouth secondarily (Sire, 2001). Be- sides canonical dentine, other peculiar types of dentine have also been reported in living jawed vertebrates. For example, the teeth of many sharks have a root made of osteodentine, with numerous vascular canals rather than a big pulp (Applegate, 1967). Tooth plates of African and South American lungfishes have a special compact dentine called petrodentine, which is compact and less collagenous when fully calcified (Kemp, 2001). Enamel is the hardest vertebrate tissue and mature enamel is made up of more than 95% inorganic mineral (Nanci, 2012). It is the only hard tissue without collagens in its ECM, which is mainly based on a protein called amelogenin (Nanci, 2012). Enamel shows strong birefringence under polarized light and there are usually incremental lines (or growth lines) within the

10 enamel layer because of rhythmic deposition (Risnes, 1998). It is only found inside the mouth of mammals as the superficial layer covering dentine, but the so-called ganoine on scales of living primitive fishes (gars and bichirs) appears to be a primitive type of enamel (Sire, 1995) (Paper V). Bone, as a word, is used to depict both anatomical structure (a long ‘bone’ like our humerus) and the tissue that constitutes the structure. Bone is characterized by an extracellular matrix dominated by type I collagen fibers, with some minor contribution from other non-collagen proteins (Ravindran and George, 2014). Developmentally, bone can be deposited directly on collagen-based matrix to form dermal bone, such as our mandible (lower jaw); or it can be deposited onto a pre-existing scaffold of cartilage to form endochondral bone, such as our vertebrae (Francillon-Vieillot et al., 1990). At the tissue level, abundant cells called osteocytes are usually embedded in bone and they connect with each other through very thin canals called canaliculi (Hall, 2005). However, many teleosts have a special type of bone without osteocytes, called acellular bone (Hall, 2005; Moss, 1963). Although some data seem to support the existence of bone-like tissues on the surface of cartilage in extant chondrichthyans (Bordat, 1987; Eames et al., 2007; Kemp and Westrin, 1979), definitive data for existence of osteoblasts and perichondral ossification is still lacking. A brief phylogeny-based survey of the distribution of hard tissues in extant vertebrates shows that all calcified hard tissues are confined to jawed vertebrates, and living jawless vertebrates possess only uncalcified cartilage (Figure 1). When and how did the skeletal system first evolve? Did all mineralized tissues appear first in jawed vertebrates as suggested by the phylogeny of extant vertebrates? In what form did the earliest vertebrate skeletal tissues appear? Did they have the same histological features as we see in living vertebrates? How did calcified tissues grow when they first appeared in vertebrates? Because there is a significant gap between living jawless vertebrates and jawed vertebrates regarding hard tissues, skeletal fossils of early vertebrates become the only physical evidence to answer these questions (Donoghue and Sansom, 2002). Importantly, since skeletons are usually the only fossil remains of vertebrates, they also serve as the most important material for paleontologists to study vertebrate evolution in general (Benton, 2009). The main purpose of this PhD thesis is to utilize phase-contrast synchrotron imaging to reveal the 3D histological architecture of several early vertebrate scales, and compare the new data with our previous understanding of relevant fossils based on thin sections. It is expected that 3D histology data from early vertebrate skeletons will generate further useful characters to understand the phylogenetic positions of fossil taxa, in particular the early jawed vertebrates. The data obtained from 3D analysis of these tiny scales provides us with innovative perspectives to answer classical questions regarding early vertebrate histology and evolution: 1. How did cosmine and its

11 accompanying pore-canal system originate in early sarcopterygians (Paper III); 2. How did polyodontode scales of early osteichthyans grown in a three- dimensional context (Paper II and III); 3. How is the pore-canal system in the dermal skeleton of various groups of early vertebrates organized in 3D, and what are its possible functions (Paper IV). Notably, it is shown that the early vertebrate microfossils have preserved significant microanatomical and ontogenetic information, which forms the basis for a critical reevaluation of histological characters in phylogenetic studies of early vertebrates. Besides their importance in biostratigraphic studies (e.g., Blieck and Turner, 2000; Märss, 1986; Märss et al., 1995; Soehn et al., 2000), Paleozoic vertebrate microfossils and dermal skeleton fossils probably contain significant biological information for understanding the origin and early evolution of vertebrate hard tissues in particular, and early gnathostome phylogeny in general.

Figure 1. Phylogeny of living vertebrates based on Heimberg et al. (2010). The question marks following ‘bone’ and ‘enamel’ indicate that studies on living vertebrates alone cannot tell when these tissues originated with confidence. There are still on-going disputes about the presence or absence of true bone in living chondrichthyans (see main text). A special hard tissue, enameloid, on shark teeth and enamel on osteichthyan teeth are homologues according to their topology (both on top of dentine), but their developmental processes differ dramatically (Paper V). Fossils can help clarify the ambiguous scenario based on living vertebrates.

12 Paleohistology of early vertebrates

A brief history In his epoch-making monographs on both paleoichthyology and fish taxonomy, Agassiz (1833-44) already noticed the importance of dermal append- ages (mostly scales) for the classification of fishes. He classified fishes into four majors groups according to their scale type: ganoid fish, placoid fish, cycloid fish and ctenoid fish. Although this classification system does not fit with the modern cladistics-based systematics, the four types of scale have been maintained in most zoology textbooks for descriptive purposes (e.g., Kardong, 2006). Notably, Agassiz (1833-44) also studied sections made from dermal skeletons of both living and extinct fishes, which has been considered as the starting point of vertebrate comparative paleohistology (de Ricqlès, 1993). It was demonstrated that ancient fish fossils as old as the Devonian have preserved details of the histology inside the dermal skeleton and can be compared with extant fish (Agassiz, 1833-44). Since then, the study of skeletal histology has attracted attention from many researchers working on primitive (both extant and extinct) vertebrates, mainly for taxonomic purposes (e.g., Goodrich, 1904, 1907, 1913; Hertwig, 1879; Pander, 1856; Williamson, 1849, 1851). de Ricqlès (1993) gave a brief review of paleohistological research. He noted that the early era of paleohistological research was centered on examination of early fish fossils, while paleohistological studies on tetrapods (so called higher vertebrates) flourished much later (de Ricqlès, 1993; de Ricqlès, 2011). Recent years have witnessed a shift of paleohistological studies to tetrapods, focusing on both the biological attributes (e.g., growth rate, body mass etc.) and the ecological context (e.g., environment, lifestyle, biomechanics etc.) of extinct tetrapods using bone histology data (de Ricqlès, 2011; Francillon-Vieillot et al., 1990; Houssaye, 2014). Early vertebrate fossils (particularly before the Devonian) are relatively scarce and fragmentary, and tend to provide limited morphological information; because of this, histological data have been used for studies of both taxonomy and the early evolution of vertebrate hard tissues (Maisey, 1988; Ørvig, 1951; Ørvig, 1967). A general interest in the histological architecture of early vertebrate dermal skeletons is also reflected in previous mono- graphic works, which usually include both anatomical and histological data (e.g., Aldinger, 1937; Stensiö, 1927, 1932). The histology of early vertebrate

13 microfossils such as isolated scales and teeth were also studied extensively, mostly focusing on the tissue composition and growth in different vertebrate groups (e.g., Brotzen, 1934; Gross, 1935, 1947, 1956, 1961, 1967, 1968a, 1971a; Schultze, 1968), and histology characters that have taxonomic implications (Gross, 1966; Schultze, 1977). The mineralization of hard tissues often proceeds following the structure of the extracellular matrix (though not in the case of globular calcified cartilage); hence the organization of apatite crystals generally reflects the organization of matrix proteins such as collagens, which can also be studied in fossils (Ørvig, 1967). Although the chemical components of apatite may have been changed, for example by fluoridation causing a concentration of fluorine in most fossils compared to extant samples (Toots and Parker, 1979), the physical structures at the histological level have often been preserved and allow comparisons with extant animals (Ørvig, 1967). Technically, optical imaging (including polarized and phase contrast) and TEM of thin sections and SEM imaging of acid-etched surfaces have been the prevailing and the most successful methods for studying the histology of vertebrate hard tissues (e.g., Schmidt and Keil, 1971). After making thin sections, the hard tissues in the samples can be determined by comparison with closely related organisms and extant vertebrates (Paper I). Previous researchers also realized that some early vertebrate fossils have complicated canal systems inside, which have been studied by serial thin sections or submerging the samples in xylol to make them almost transparent (Denison, 1947; Gross, 1956). However, such studies only result in idealized 3D models of the structures, with many details missing (Paper IV). Nevertheless, the century-long study using thin sections and SEM has provided important data regarding the early evolution of vertebrate hard tissues and early vertebrate taxonomy (Donoghue and Sansom, 2002; Donoghue et al., 2006; Ørvig, 1967; Sire et al., 2009).

3D synchrotron virtual paleohistology Sanchez et al. (2012) first proposed a new approach to the paleohistology of early vertebrates using phase-contrast synchrotron imaging techniques, which have made significant progress in recent years (Tafforeau et al., 2006; Tafforeau and Smith, 2008). This imaging method has been successfully applied to various fossils to decipher their internal structures (Cunningham et al., 2014; Mondéjar-Fernández et al., 2014; Murdock et al., 2013; Rücklin et al., 2014; Rücklin et al., 2012; Rücklin et al., 2011; Sanchez et al., 2013; Sanchez et al., 2014; Tafforeau and Smith, 2008). A special form of this imaging method, propagation phase contrast X-ray synchrotron microtomography (PPC-SRµCT), was used to study the early vertebrate scales in this PhD project. The advantages of this imaging method include non- destructiveness, fully three-dimensional visualization at resolutions down to

14 a µm or less, and the ability of phase contrast to detect very subtle structures such as growth arrest surfaces (Sanchez et al., 2012). All the taxa examined in this project have been studied by thin sections in the past, and it is confirmed that virtual thin sections reconstructed from PPC-SRµCT can provide almost all the histological information previously gathered from classical thin sections (compare Paper I and Paper III). To identify different tissues using virtual thin sections follows the same princi- ples as real thin sections. However, for each scale scanned in this study, more than two thousand serial virtual sections have been obtained and studied on a graphic computer station, using software VGStudio MAX (Volume Graphics Inc., Germany). Segmentation of different structures of interest generates 3D models after rendering, and subsequent study of their morphology and relative topology to other structures can be carried out in a 3D context. The process is similar to gross anatomy studies of organisms based on CT scan data, but the studied structures are usually much smaller in size, such as the vascular canals and buried denticles (Figure 2). The obtained 3D data forms the basis of this work, and sheds light on the future potential of 3D synchrotron virtual paleohistology as a tool for studying early vertebrate evolution.

Figure 2. Virtual thin section and internal 3D structures of a scale of Andreolepis, an early osteichthyan from the Late Silurian, Sweden. Arrows point to the various structures reconstructed in 3D.

15 3D virtual paleohistology and phylogeny

“….I would like to consider one aspect which, by virtue of its uniqueness, may have considerable impact on future studies of lower vertebrate phylogeny. This single aspect is the information provided by fossils concerning the probable origins and subsequent elaboration of skeletal calcification in early vertebrates.” John Maisey, 1988.

Phylogeny of early jawed vertebrates As explained explicitly by Patterson (1977; 1981), a good understanding of living organisms is always the first step to study fossils. By putting fossil taxa into the phylogenetic tree of living vertebrates we can understand how extant groups originated and gained their synapomorphies. Hence the study of fossil jawed vertebrates starts from the established phylogeny of living jawed vertebrates, which includes two well-supported groups, osteichthyans and chondrichthyans, based on both morphological and molecular data (Blair and Hedges, 2005; Maisey, 1986) (Figure 3). For paleontologists, the problem is the position of two extinct groups of jawed vertebrates: ‘acanthodians’ and ‘placoderms’, both of which were long assumed to be monophyletic (Janvier, 1996). Rapid recent advances have been made in the understanding of the phylogeny of early jawed vertebrates since Brazeau (2009), who first tested the long-assumed monophyly of “acanthodians” and “placoderms” in an explicit cladistic context. New fossil discoveries from China and restudies of some key taxa corroborate the paraphyly of placoderms and provide new frame- works for discussing character state transformations during the early evolution of jawed vertebrates (Davis et al., 2012; Zhu et al., 2013), but many other problems still remain and need further examination (Brazeau and Friedman, 2014). Brazeau and Friedman (2014) give the most up-to-date review of the assumed synapomorphies of acanthodians and placoderms, and the monophyly of each group seems to have weak support. At present the key problem seems to be the phylogenetic position of acanthodians, which have been resolved either as stem chondrichthyans, or distributed on both the chondrichthyan and osteichthyan stems (Brazeau and Friedman, 2014) (Fig- ure 3). As current datasets cannot solve this issue convincingly, additional data or characters are needed.

Figure 3. Phylogenetic scheme of jawed vertebrates based on recent analyses (Brazeau, 2009; Davis et al., 2012), with indication of stem group and crown group. Total group is the combination of stem group and crown group. The position of acanthodians represents only one hypothesis (see text).

It is well recognized that the endocranium and the associating endocast of vertebrate skulls are phylogenetically informative, and endocranial data has been extremely important for understanding the phylogeny of early jawed vertebrates (Brazeau, 2009; Davis et al., 2012; Dupret et al., 2014; Jarvik, 1977; Miles, 1973b). Anatomically, the endocranium is housing the brain, nerves, blood vessels and some important endocrine glands, and is obviously under great functional constraint during vertebrate evolution. The morphological complexity (providing many characters) and evolutionary conserva- tiveness (providing a primary homology framework for comparison among most taxa) make the endocranium an ideal object for generating characters. Perichondral ossification of the endocranium seems to be primitive for jawed vertebrates (Ørvig, 1951), since the jawless vertebrate osteostracans (considered as the closest relative of jawed vertebrates) have perichondrally ossified endocrania, which can be well preserved and amenable to detailed study (Stensiö, 1927). However, perichondral ossification was secondarily lost in many lineages of early jawed vertebrates, whose endocrania seem to have remained cartilaginous, with little potential for fossil preservation (e.g., Downs and Donoghue, 2009). In addition, endocrania of early jawed vertebrates are not always available largely due to unfavorable preservation conditions. The only ‘acanthodian’ with a well-preserved endocranium is the Permian Acanthodes (Davis et al., 2012; Jarvik, 1977; Miles, 1973b), while

17 most other articulated ‘acanthodians’ only have dermal skeletons preserved (Denison, 1979; Hanke and Wilson, 2004, 2010; Hanke et al., 2001; Miles, 1973a). Brazeau (2009) described the second hitherto known acanthodian endocranium, but the material is only preserved in ventral view. Hence a better understanding of ‘acanthodians’ and ‘placoderms’ seems to also de- pend on further examinations of their dermal skeletons, both morphologi- cally and histologically. I will first give a brief review of our current knowledge of the dermal skeletal histology of the four traditional groups, followed by discussions of possible ways to generate new histology-related characters based on the new findings during this PhD work. It is intended to give an introduction to our current knowledge of each group (especially the Paleozoic taxa) in general, in order to know what kind of data can be expected in the future.

Subdivision of the dermal skeleton of early jawed vertebrates In the following brief review, the dermal skeletons are divided into five parts depending on their anatomical positions: scales, spines, fin rays (if present), dermal bones and teeth. For many early jawed vertebrates we often have histology data from only one part (mostly scales), but it is well justified to treat these different elements in different ways. Such treatment is more rea- sonable and has empirical support from fossil data as well as living organisms. For example, the Triassic actinopterygian Ptycholepis bollensis shows different histology between dermal bones and scales (Ørvig, 1978c). Its scales have multiple generations of ganoine (enamel) on the top and canals of Williamson in the base, but the dermal bones lack both features (Ørvig, 1978c). Such differences are also observed in other early actinopterygians (Ørvig, 1978b). In addition, Paper V shows that teeth and dermal odontodes can have different histology in early osteichthyans, showing teeth without enamel but dermal odontodes with enamel. The living primitive fish Polyp- terus shows a similar situation to Ptycholepis, with superimposed ganoine on scales but not on dermal bones (Ørvig, 1978a). The significance of such a difference is difficult to understand, partly due to lack of data based on both dermal bones and scales. However, it highlights the importance of treating dermal skeletons in a region-specific way when histological characters are considered. As a result, the histology of scales in one taxon should not, in principle, be compared with the histology of dermal bones in another taxon. To assume the entire dermal skeleton of one taxon has identical histology has neither empirical nor theoretical support. Theoretically, scales and dermal bones evolved as independent modules and under different selection pressures.

18 Following the discussion of Friedman and Brazeau (2010) and Brazeau and Friedman (2014), the purpose of this brief review is to identify histological characters that may be used to put a fossil in total group osteichthyans, crown group osteichthyans, total group chondrichthyans, crown group chondrichthyans, total group jawed vertebrates and crown group jawed vertebrates. Most of the histological characters are related to the presence or absence of certain types of hard tissues, which is the only conclusion that can be drawn with confidence from thin sections and SEM studies. Tissue terminology regarding fossils is following Ørvig (1967) and Sire et al. (2009).

Histology of dermal skeletons of early osteichthyans Early osteichthyans are better known than the other three groups regarding dermal skeletal histology, and their histology and scale morphology are read- ily comparable with two primitive living fish groups, bichirs (Polypterus plus Erpetoichthys) and gars (Atractosteus and Lepisosteus) (Sasagawa et al., 2013; Sire et al., 2009). Their dermal skeleton has a basic tripartite construction, with multiple odontodes (enamel + dentine) on the top, a cancellous bone layer in the middle with embedded lamellated tissues (elasmodine), and an internal bony base (lamellar or pseudolamellar) (Kerr, 1951; Meinke, 1982; Sire et al., 2009). Early representatives of osteichthyans do not have the lamellated tissues in the middle layer, but the other tissues are almost identical (Paper I) (Figure 4). This tripartite construction is present in the dermal skeleton of most early jawed vertebrates (Sire et al., 2009) as a sym- plesiomorphy.

Figure 4. Thin sections from a scale (PF 5246) (A) and a dermal skull bone (PF 5251) (B) of Cheirolepis canadensis, an early actinopterygian. Note the tripartite construction. Arrow heads indicate the vascular canals, arrows indicate osteocytes. Specimens are housed in the Field Museum of Natural History, Chicago.

Rhomboid-shaped scales with a peg-and-socket structure are only known in total group osteichthyans (contra Friedman and Brazeau, 2010 where An-

19 dreolepis and Lophosteus were considered as lacking peg-and-socket). Paper V supports that the presence of enamel on scales can place a taxon in total group osteichthyans crownward of Lophosteus, and the presence of enamel on teeth can place a taxon in crown group osteichthyans. Paper V also highlights the separate evolutionary history of different dermal modules. As noticed by Schultze (1977), new dentine in early actinopterygian scales is only added laterally to old odontodes, while early sarcopterygian scales add new dentine both laterally and superpositionally (Paper III). This superpositional growth is even found in the scales of the living sarcopterygian fish Latimeria (Roux, 1942). However, dermal bones and dorsal spines of several early actinopterygians and the extant Polypterus exhibit both superpositional and lateral growth (Meinke, 1982; Ørvig, 1978a, c), supporting the division of scales and dermal bones into separate modules regarding histology and growth. Aldinger (1937) described the histology of a scale of Scanilepis sp. (primitive crown actinopterygian), showing the presence of superpositional growth as well. The scales covering the fins of Plegmolepis (primitive crown actinopterygian) shows only one generation of ganoine/enamel. Different species of Cheirolepis (probably stem actinopterygians) also exhibit different growth patterns in scales (Gross, 1973), as represented by C. gracilis that has both superpositional and lateral growth. All these variations should be considered when histology characters are generated, and comparison of histology between taxa should be more explicit with anatomical positions of the skeletal elements under consideration. Cosmine, a tissue complex with a pore-canal system embedded in a sheet of dental tissues, has long been recognized as a synapomorphy of sarcopterygians (Rosen et al., 1981). Paper III gives a detailed review of this histological complex and proposes a developmental hypothesis to explain its origin based on 3D histological data. It is also proposed that 3D histology data (3D vasculature and growth pattern of odontodes) of scales can be used to generate new characters for phylogenetic analysis, but the characters listed in paper III are mostly useful for total group osteichthyans and usually not ap- plicable to scales of the other three groups. It seems that there is no well-recognized dermal histology character related to scales to assign any placoderm or acanthodian to total group osteichthyans. The only candidate character is the presence of ganoine-like tissue on the scales of some acanthodians, but to assign these acanthodians crownward of Lophosteus means that diamond-shaped scales developed in these acanthodians independently of other acanthodians without ganoine. For other skeletal modules, too little is known regarding their histology. Fin spines are commonly found in acanthodians, and recent fossil findings suggest that they are also present in some primitive chondrichthyans (Miller et al., 2003) and osteichthyans (Zhu et al., 1999; Zhu et al., 2009). But the histology of these chondrichthyan and osteichthyan fin spines is largely unknown, hence cannot be compared with acanthodian fin spines (Denison,

20 1979). Dermal bone histology of osteichthyans is only occasionally examined (e.g., Ørvig, 1978a, b, c). Large dermal bones on the head are found in some acanthodians, such as the cheek plate in Culmacanthus (Long, 1983) and the branchiostegal plates in Homalacanthus (Hanke and Wilson, 2004). Histology of these dermal bones in acanthodians may provide comparative data for primary homology assessment and character generation, and contribute to the understanding of the acanthodian-osteichthyan relationship.

Histology of dermal skeletons of “placoderms” Giles et al. (2013) give the most recent review of the histology of placoderms, but no clear distinction is made between dermal bones and scales. The histology data mostly comes from dermal bones (Giles et al., 2013), since scales are not well developed in most taxa and are often not preserved in those taxa that do have squamation (Burrow and Turner, 1999). For example, among all the antiarchs found from the Lower Devonian in Yunnan, China, only one specimen is so far known with squamation preserved (Zhang et al., 2001; Zhu et al., 2012), while the other specimens are mostly skulls and trunk shields. The histology of the scales from this Chinese antiarch remains undescribed. Most placoderms have a dermal skeleton made of three layers: a superficial layer with tubercles made of a dentine-like tissue, a middle layer of cancellous bone with vascular canals, and a basal layer with compact bone (Figure 5). The superficial dentine-like tissue is lost and replaced by compact bone in several lineages (Giles et al., 2013). These three layers are found in most other jawed vertebrates (Donoghue and Sansom, 2002; Donoghue et al., 2006; Sire et al., 2009) as well as in osteostracans (jawless vertebrates, often used as outgroup in studies of gnathostome interrelationships) (Paper IV).

Figure 5. A virtual thin section of the Early Devonian placoderm Romundina, showing buried odontodes (arrow heads).

Burrow and Turner (1999) summarized the histology of placoderm scales and proposed scale-based characters including four histological characters: lamellar base, dentine, dentine types, superposed odontodes. The lamellar

21 base of scales is also found in osteostracans (Denison, 1947; Sire et al., 2009) (Paper IV), as well as in many acanthodian (Denison, 1979; Gross, 1935, 1956) and osteichthyan scales (Paper I). This suggests that the presence of a lamellar base is symplesiomorphic for total group jawed vertebrates. Pres- ence of dentine and superposed odontodes are also symplesiomorphic because they are found in osteostracans (Paper IV), osteichthyans (Paper II), acanthodians (Gross, 1935, 1956) and early chondrichthyans (Williams, 1998; Woodward and White, 1938). The only informative character seems to be the presence of a special type of dentine called semidentine with unipolar cell lacunae (Figure 6), which is one of two characters supposedly uniting placoderms (Brazeau and Friedman, 2014). However, semidentine has a patchy distribution in placoderms, and Burrow and Turner (1999) defined five types of dentine in the dermal skeletons of placoderms, ranging from orthosemidentine to orthodentine. Dentine of dermal bones and scales also show differences in the same taxon (Figure 6). These dentine types are difficult to appreciate based on thin sections alone, and future examination with high-resolution synchrotron imaging or X-ray phase nanotomography (Langer et al., 2012) can be expected to provide a better understanding of these tissues, which are similar to both bone and dentine. At present all these types of dentine involve subjective definitions, and therefore cannot contribute to phylogenetic analysis at this stage.

Figure 6. Thin sections of a scale (S2235) (A) and a dermal plate (S2368) (B) of Romundina, showing the difference of dentine structure. Arrow heads in (A) indicate buried odontodes. Specimens are housed in the Swedish Museum of Natural History, Stockholm.

Histology of dermal skeletons of “acanthodians” Our knowledge of the histology of “acanthodians” comes mostly from isolated scales and fin spines, which are the most common fossil remains of

22 these fishes (Denison, 1979). Many taxa are based only on microfossils such as scales, teeth and fin spines (Denison, 1979; Gross, 1947, 1971a; Märss, 1986; Valiukevicius, 2004; Wang, 1984), and several cosmopolitan taxa have been used as index fossils in biostratigraphic schemes (Blieck et al., 1988; Märss et al., 1995; Valiukevicius, 1998, 2005). Their histology (especially scales) is usually well preserved and studied using thin sections, and taxonomy has been proposed based on histology characters (Valiukevicius and Burrow, 2005). The scale crown is composed of several generations of odontodes made of dentine-like tissues, and the scale base is made of bone with or without osteocyte lacunae (Figure 7). Concentric onion-like growth of scales is rather unique for some acanthodians, but not present in all acanthodian taxa. Many acanthodians have scales showing lateral or areal growth without any sign of superpositional growth, more comparable to the polyodontode scales of early chondrichthyans (e.g., Burrow and Turner, 2013; Williams, 1998). Such lateral growth of scales may unite some acanthodians with chondrichthyans (Brazeau and Friedman, 2014).

Figure 7. A virtual transverse thin section of an unidentified acanthodian scale from Late Silurian of Estonia. There are no osteocyte lacunae in the bony base. Eight odontodes can be recognized, but not in a strict onion-like form.

Most acanthodian scales are polygon- or diamond-shaped with bulging or flattened bases. In this sense none of the known acanthodians can be as- signed to total group osteichthyans crownward of Lophosteus, which seems to be the most basal osteichthyan with rhombic scales (Botella et al., 2007; Friedman and Brazeau, 2010; Gross, 1969, 1971b; Schultze and Märss, 2004). Scales of the early actinopterygian Cheirolepis look similar to diamond-shaped acanthodian scales, but this is just superficial resemblance. In their basal part, Cheirolepis scales have an elongated keel flanked by two

23 ledges (Plate 36 in Gross, 1973), identical to rhombic scales of other osteichthyans (Paper I). The appearance of dentine-like tissues in the scale crown shows significant variation in thin sections (Valiukevicius and Burrow, 2005), but it is generally called mesodentine (with cell lacunae in some forms) to differenti- ate from ‘true’ dentine with regular dentine tubules and without cell spaces. An interesting histological character of scales is the presence of “stranggewebe”, a tissue described by Gross (1971a) as horizontally-oriented elongate lacunae in the mesodentine of many scale-based taxa (Valiukevicius and Burrow, 2005). The stranggewebe is also described in scales from articulated acanthodian specimens (Brazeau, 2012), but not known in scales of placoderms and osteichthyans. However, the 3D structure of stranggewebe has not been examined so far, and its relationship to mesodentine cannot be evaluated based on thin sections alone. Histology of different scales of the same taxon also shows limited distribution of stranggewebe in some acanthodians (Newman et al., 2014), but 3D data is needed to confirm the distribution of this structure. Future studies using X-ray phase nanotomography will be of great interest to decipher this mysterious tissue and may provide a potential synapomorphy for a subgroup of acanthodians. Although it is claimed that the taphonomy favoring preservation of articulated specimens is usually not good for preservation of histology (Valiukevicius and Burrow, 2005), the histology of many articulated specimens seems to be well preserved with cell lacunae and embedded odontodes (Brazeau, 2012; Burrow et al., 2013; Burrow and Turner, 2010; Newman et al., 2014; Valiukevicius, 2003). Most articulated acanthodians from the Early Devonian of Canada seem to have poorly preserved histology, but the vasculature and boundaries between odontodes are still discernable (Hanke, 2008; Hanke and Davis, 2008; Hanke and Wilson, 2004, 2010). Hence further examination of scales and spines of these articulated taxa using synchrotron imaging will be informative as well. Histology of fin spines has been described in some acanthodians, but has largely been based on disarticulated or microfossil remains (Gross, 1947, 1957, 1971a). Histology of fin spines from articulated specimens is sometimes briefly described with other anatomical features, and the preservation seems not to be a problem (Brazeau, 2012; Burrow et al., 2013; Newman et al., 2014; Valiukevicius, 1992, 2003). Since all acanthodians have fin spines, which are also present in early chondrichthyans and osteichthyans, fin spines provide a potential (histological) data source to help resolve the acanthodian problem. In other words, the question whether acanthodian fin spines are histologically similar to osteichthyans or chondrichthyans should be an- swered. However, fin spine histology in other jawed vertebrates, such as the early osteichthyan Psarolepis (Zhu et al., 1999) and the early chondrichthyans Doliodus (Miller et al., 2003), remains unknown and should be examined for comparative analysis.

24 Tooth-whorls are present in many acanthodians and tooth-whorls from microfossil remains are often attributed to acanthodians (Gross, 1947, 1957, 1971a; Ørvig, 1973). They are also found in many early osteichthyans and chondrichthyans but not in any placoderm, suggesting acanthodians belong within crown group jawed vertebrates (Brazeau and Friedman, 2014). His- tology of tooth-whorls from articulated acanthodian specimens is largely unknown, and our knowledge of tooth-whorl histology is usually based on thin sections of isolated tooth-whorls (Gross, 1957, 1971a; Ørvig, 1973). Some acanthodians have dentition-bearing jaw bones of the mandibular arch (Burrow, 2004). Histology of studied jaw bones shows distinct features that are not observed in osteichthyans. For example, growth of the jaw bones seems to follow a polarized direction by adding new dental tissues and bone tissues at the same time, at the anterior end, and teeth are firmly ankylosed to the basal bony bone (Ørvig, 1973). These features show similarities to the jaw bones of some placoderms (Ørvig, 1973). However, 3D histology data is needed to confirm this growth pattern. Potentially the presence of such jaw bones is a synapomorphy of some acanthodians, but is less informative for the relationship of these acanthodians to other jawed vertebrates. All known acanthodians have a reduced dermal skull roof, which is mostly covered by dermal tesserae (Brazeau, 2009; Denison, 1979; Miles, 1973a). Such tesserae look like assemblages of body scales, often with a larger bony base (Miles, 1973a; Valiukevicius, 2003). Dermal cheek bones and branchiostegal plates are occasionally found (Long, 1983; Miles, 1973a; Watson, 1937), but no histology data is available. A recent discovery of an osteichthyan-like placoderm, Entelognathus from the Late Silurian of China, suggests that gulars are probably primitive for crown group jawed vertebrates. Hence the histology of gulars might provide some clues for the relationship between osteichthyans and acanthodians. So far no acanthodian dermal elements have been examined in 3D, unlike the 3D results reported in Paper II, III and IV. One isolated acanthodian scale has been partially modelled during this PhD project, and the preliminary result shows that the growth of acanthodian scales is more complicated in 3D compared to what can be observed in thin sections. The oldest odontode of the modeled scale has a different surface sculpture compared to the surface of a mature scale, and each generation of odontode shows some distinct features. The preliminary result implies that scales from articulated specimens of acanthodians should all be studied in 3D using synchrotron imaging, which is the only way to generate ‘secure’ characters for phylogenetic analysis, as proposed in Paper III.

25 Histology of dermal skeletons of early chondrichthyans Extant chondrichthyans are characterized by a special type of calcified tissue called prismatic calcified cartilage (Dean and Summers, 2006). A few Paleo- zoic endocranium fossils have been attributed to chondrichthyans based on the presence of a prismatic calcified cartilage cover and their morphological similarities to extant chondrichthyans (e.g. precerebral fontanelle present) (Maisey, 2004, 2005). Articulated specimens that can be confidently attributed to the chondrichthyan total group are not common from the Paleozoic. The earliest articulated chondrichthyan with prismatic calcified cartilage is Doliodus from the Early Devonian of Canada (Maisey et al., 2009; Miller et al., 2003).

Figure 8. Thin section (PF 5076) of a scale of Ctenacanthus, a primitive shark from the Carboniferous. Arrow heads indicate different odontodes. The specimen is housed in the Field Museum of Natural History, Chicago.

Unlike living chondrichthyans with placoid (monoodontode) scales that shed constantly during the fish’s lifetime, many early chondrichthyans (represented by articulated or partially articulated specimens) have polyodontode scales (Burrow and Turner, 2013; Miller et al., 2003; Reif, 1978; Williams, 1998; Young, 1982; Zangerl, 1968) (Figure 8). The polyodontode scales of some taxa have a well-defined crown on a convex base (Burrow and Turner, 2013; Zangerl, 1968), while scales of other taxa have almost flat (or even concave) bases (Reif, 1978; Williams, 1998; Woodward and White, 1938). The histology has been described for some of these polyodontode scales using thin sections. The scale crown has been considered as mesodentine (Burrow and Turner, 2013; Ørvig, 1966) or normal dentine (Zangerl, 1968), and the scale base is made of lamellated tissue with osteocytes (Burrow and Turner, 2013). It is notable that most these polyodontode scales from early

26 chondrichthyans show an areal growth pattern, similar to some acanthodian scales. An areal growth pattern is also found in scales of the early osteichthyan Andreolepis based on 3D histology data, although such scales are rhombic with a peg-and-socket structure (Paper II). In this case two characters may be proposed: the presence of polygonal-shaped scales can exclude osteichthyan affinity, and presence of an areal growth pattern can associate some acanthodians with total group chondrichthyans. However, 3D histology is needed to confirm the growth pattern of these polyodontode scales from articulated specimens. Possessing prismatic calcified cartilage, Doliodus is the only known chondrichthyan that has paired fin spines (Miller et al., 2003), which are primitively present in the other three groups of jawed vertebrates. Other articulated chondrichthyans have median fin spines, whose histology has been studied occasionally (Coates et al., 1998; Young, 1982; Zangerl, 1984). Similarities seem to exist between some acanthodian fin spines and chondrichthyan fin spines, with an internal lamellated layer, a middle cancellous layer (either vascular bone or osteodentine) and a surface ornament layer made of dentine (Newman et al., 2014; Young, 1982). As discussed in the last section, the lack of histology data from placoderms and early osteichthyans makes it impossible at present to evaluate the significance of spine histology data. At the same time, spines from articulated chondrichthyans should be studied as well. Isolated teeth and tooth-whorls from microfossil remains have been fre- quently attributed to chondrichthyans based on their multicuspidate crown and distinctive base (e.g., Botella et al., 2009). Histology of teeth from articulated chondrichthyans has been largely unstudied (e.g., Williams, 1998), or briefly reported (Coates and Sequeira, 2001). It seems that some definite early chondrichthyan teeth have a superficial enameloid layer and an inner osteodentine (with numerous vascular canals) layer, sometimes with additional normal dentine (Botella et al., 2009; Coates and Sequeira, 2001; Gillis and Donoghue, 2007). Most examined acanthodian teeth or tooth-whorls also have inner osteodentine with extensive vasculature (Ørvig, 1973), unlike teeth of early osteichthyans with a single pulp (Cunningham et al., 2012; Gross, 1968b, 1969, 1971b). Placoderms seem to have teeth with sim- ple dentine and large pulps (Rücklin et al., 2012; Smith and Johanson, 2003), suggesting osteodentine may be a synapomorphy of acanthodians and chondrichthyans, although some acanthodians do not have any teeth-bearing structures. Histology data of teeth from articulated acanthodians (e.g., Valiukevicius, 1992) is needed to confirm the distribution of osteodentine. None of the known acanthodian teeth or tooth-whorls shows a distinctive layer (enamel or enameloid) covering dentine, suggesting their more basal position relative to those definitive chondrichthyans with enameloid on teeth. A dermal skull roof with large interlocking bones has not been found in any chondrichthyan. If we accept that acanthodians, or at least some acan-

27 thodians, belong to total group chondrichthyans (Brazeau, 2009; Zhu et al., 2013), the loss of a dermal skull roof was possibly a gradual process in the chondrichthyan lineage.

3D paleohistology characters in phylogeny studies The above brief review emphasizes that dermal skeletons of early jawed vertebrates should be treated as different modules, which is the first step for the comprehensive use of histology data in phylogenetic analysis. In recent phylogenetic analyses of early jawed vertebrate, the number of characters related to dermal skeletal histology is rather small (Brazeau, 2009; Zhu et al., 2013; Davis et al., 2012). The histology characters used so far in phylogeny are mostly related to the presence or absence of different types of tissues: 1. Tessellate prismatic calcified cartilage: 0. absent; 1. present 2. Perichondral bone: 0. present; 1. absent 3. Extensive endochondral ossification: 0. absent; 1. present 4. Dentine: 0. absent; 1. present 5. Dentine kind: 0. mesodentine; 1. semidentine; 2. orthodentine 6. Cosmine: 0. absent; 1. present 7. Body scale growth pattern: 0. monodontode; 1. polyodontode 8. Body scale growth concentric: 0. absent; 1. present

All these histology characters do not take into account the anatomical positions of the dermal skeleton, and this treatment will miss potentially useful data related to the histology variation in different regions of body. For example, Paper V distinguishes scales, dermal bones and teeth of the dermal skeleton to show that enamel is only present on scales of Andreolepis. In this case, three more characters can be formulated to include available data: 9. Enamel: 0. absent; 1. present 10. Enamel on scales: 0. absent; 1. present 11. Enamel on dermal skull bones: 0. absent; 1. present 12. Enamel on teeth: 0. absent; 1. present

Logically, taxa without enamel anywhere should be coded as inapplicable for the other three characters (10-12). Histology of fin spines and teeth of chondrichthyans and acanthodians can be included in a similar way, but the lack of data from articulated specimens is an impediment. Hence further histological studies of known specimens become important, as discussed in the review above. The second step to generate potentially useful characters related to histology requires further studies of dermal bones, scales, spines and teeth from articulated specimens using synchrotron X-ray microtomography. 2D thin

28 sections can provide reliable data about what kind of tissues are present, and in which region of the skeletons under study, as reflected in the histology characters in use. Beyond the tissue level, 2D thin sections and surface morphology can only hint at, for example, how polyodontode scales grow by addition of new odontodes. Papers II and III have shown that accurate growth pattern of these polyodontode scales can only be obtained through 3D studies. In such case, the histology data is considered as microanatomical structures inside skeletons. The morphology and topology of internal structures of different dermal elements can be examined in great detail. Order of Approximate Examples Techniques Structure Scale

First Order teeth, scales, fin spines Stereoscopic micro- > 1mm (Gross Anatomy) and dermal bones scope, X-ray micro-CT

organization of vascular canals, pulp cavities or Thin sectioning, canals, embedded odon- transmission (polar- Second Order 100 µm – 1 todes and their distribu- ized; phase-contrast) (Microanatomy) mm tion, organization of bone light microscope, trabeculae, lines of ar- SEM, PPC-SRµCT rested growth (LAGs) Thin sectioning, osteocytes (size, mor- transmission (natural; phology and distribution), Third Order 1µm – polarized; phase- osteocyte canaliculi, den- (Tissue and cell) 100µm contrast) light micro- tine tubules, organization scope, SEM, PPC- of collagen fibers SRµCT molecules (e.g., proteins) Forth Order Immunohistochemis- of the extracellular (Mineral and try, SEM, TEM, X-ray < 1µm matrix and their folding collagen mole- scattering, X-ray dif- structures; apatite crystals cules) fraction and their organization

Table 1: hierarchical categories of vertebrate skeletal structures (focusing on scales and teeth) and corresponding imaging techniques to study different levels of structures. PPC-SRµCT: Propagation phase contrast X-ray Synchrotron Microtomogra- phy. Modified from Francillon-Vieillot et al. (1990).

Obviously the application of PPC-SRµCT to more taxa will become obliga- tory for comparative purposes, but what kind of histology characters can we expect from future 3D analysis? Francillon-Vieillot et al. (1990), following Peterson (1930), proposed a four-level system of vertebrate skeletal tissues (although focusing on bone in particular) based on the size of structures of

29 interest. Such a hierarchical system ranging from molecular to gross anatomy is very useful as guidance of systematic analysis; thus I have adopted the hierarchical system for the early vertebrate skeletal system (Table 1). The second order and third order of structures are of particular interest and form the current focus of 3D synchrotron virtual paleohistology (Cunningham et al., 2014; Sanchez et al., 2012; Sanchez et al., 2013). In principle, dermal skeletal fossils are studied in the same way that endocranium fossils are studied using classical Sollas’ serial grinding method (e.g., Sollas, 1904; Stensiö, 1927) or modern CT scan techniques (e.g., Balanoff et al., 2013; Lu et al., 2012). Based on the 3D data that we already know from recent literature and histology data from traditional 2D thin sections, it can be expected that the following structures are potentially informative for future phylogenetic analysis of early jawed vertebrates.

Vasculature and canal system within different dermal elements The first 3D vasculature pattern of early vertebrate fossils based on synchrotron data was reported by Dupret et al. (2010). In that paper, the entire vasculature inside the premedian plate and the overlaying perichondral bone of the placoderm Romundina stellina was modelled. The 3D data can help explain the ossification pattern of the dermal bone of Romundina. Sanchez et al. (2012) described the 3D vasculatures in the dermal bone of a placoderm (Compagopiscis) and in the cortical bone of the humerus of a sarcopterygian fish (Eusthenopteron). However, the lack of 3D data from other taxa does not allow comparative analysis at present; hence the evolutionary (or phylogenetic) significance is yet to be discussed. Paper III describes the vasculature and canal system of two early osteichthyan scales, and it is shown that the 3D pattern of vasculature itself and its topology within hard tissues can be compared in scales of all early osteichthyans. Such comparative analysis in 3D can generate new observations and potentially useful characters, as discussed in detail in Paper III. Paper IV describes the pore-canal system and vasculature of two osteostracan (Trema- taspis and Oeselaspis) scales, and a notable distribution pattern of different pore canals is observed in the Tremataspis scale for the first time. Such a pattern is impossible to observe by traditional methods and was simply missed in previous examinations. Based on thin sections, it is already known that most acanthodian teeth have a complex of pulp canals (Ørvig, 1973), unlike the single large pulp in early osteichthyans and placoderms (Rücklin et al., 2012; Smith and Johanson, 2003). Such complex pulp canals have also been observed in chondrichthyan teeth (e.g., Botella et al., 2009), but 3D data is still not available. The vasculature of fin spines is even less understood in 3D, and there has been no published study yet. However, the complex vascular canals seen in

30 thin sections of fin spines confirm that they are rich data source for future examination.

Odontodes in polyodontode scales and dermal bones As four groups of jawed vertebrates primitively have polyodontode scales, these scales can be considered as primary homologues and further compared among most taxa of early jawed vertebrates. The morphologies and distribu- tions of buried odontodes described in two early osteichthyan scales (An- dreolepis and Psarolepis) show that the growth pattern of such scales can only be understood in 3D (Papers II and III). The very first, or primordial, odontode looks almost identical in the two scales, but the subsequent odontodes are rather different. Presumably primordial odontodes also exist in scales of other three groups (Reif, 1978, 1982), hence it is possible to compare the morphology and topological position of primordial odontodes in different taxa. The elongated primordial odontode of both Andreolepis and Psarolepis scales may well be another synapomorphy of osteichthyans (Pa- per III), but data from other taxa is needed to confirm this. Some acanthodian and chondrichthyan scales show an areal growth pattern without superpositional addition of odontodes. The presence of this character has been suggested to be a potential synapomorpy for total group chondrichthyans (Brazeau and Friedman, 2014). However, the growth pattern is mostly inferred from the superficial structure of polyodontode scales of early chondrichthyans (e.g., Dick, 1981; Williams, 1998). As shown in Paper II, the first stage of growth of the Andreolepis scale is also a type of areal growth without complete embedment of older odontodes. It is necessary to reexamine the polyodontode scales of acanthodians and chondrichthyans to have a 3D picture of their growth patterns; otherwise it is too early to use the character in phylogenetic analysis. So far too little is known for dermal skeletons other than scales regarding the odontodes, although thin sections show that dermal bones also have buried odontodes in some placoderms (Gross, 1935) and most early osteichthyans (e.g., Ørvig, 1978a, b, c) (Paper I). Dermal bones and fin spines are much larger than scales and teeth, and are more difficult to image at high resolution. However, a multi-scale approach can partially solve this problem and provide sufficient data for pattern generation (Sanchez et al., 2012; Tafforeau and Smith, 2008). Theoretically these larger samples can be examined in the same way as scales, and the distribution of odontodes and growth patterns can be obtained.

Distribution and size variation of osteocytes Sanchez et al. (2012), in one of the first studies with such data, modelled osteocytes in 3D in both fossil and extant samples. Subsequently, statistical

31 analysis was performed to understand how the size of osteocytes changes in different regions of bone, with focus on the functional significance of such distribution (Sanchez et al., 2013; Sanchez et al., 2014). Segmentation and modelling of osteocytes using virtual thin sections of early vertebrate fossils is not as easy as in extant vertebrates, but such data is necessary to understand how bony tissue (scales, dermal bones and fin spines) grows in different taxa. It is claimed that some acanthodian scales do not have osteocytes in their bony base, which should be confirmed with 3D data.

Dentine types This character has been used in recent phylogenetic analyses (Brazeau, 2009; Davis et al., 2012; Zhu et al., 2013), but the different types of dentine are based on rather vague definitions. For example, Ørvig (1975) described the dentine tissue in the dermal skeleton of Romudina as mesodentine approach- ing semidentine, leading to problematic coding of the character. The only way to solve this problem is to examine the 3D pattern of cell lacunae and connecting canaliculi of all the subtypes of dentine in different skeletal elements, and 3D definitions can be proposed subsequently.

The above listed microanatomical structures are an indication of what can be examined using PPC-SRµCT on early vertebrate fossils. None of the structures are new, and researchers using thin sections have routinely described all of them. However, few conclusions can be reached without a 3D context and knowledge of the topological relationship of these internal structures. The ultimate goal of 3D virtual histology studies on these early vertebrate fossils is to build a comparative microanatomical database, similar to what we already have for gross morphology (i.e., endocranium, dermal bone pattern, sensory line pattern etc.). The new dataset, combined with the old one, along with new discoveries of transitional fossil forms (Zhu et al., 2013; Zhu et al., 2009), will give us a better resolved picture of the phylogeny of early jawed vertebrates in the future.

32 Concluding remarks and future perspectives

‘……the results of these century-long investigations are, to say the least, quite chaotic, as far as the dermal skeleton is concerned.’ Philippe Janvier (1996)

Although the fossil record of early vertebrates is mostly based on their dermal skeletons, these dermal skeleton fossils seem to tell of an ‘unstable’ evolutionary history of the organisms. Phylogenetic analysis based on endocranium characters often results in better resolved trees, compared to analysis based on both endocranium and dermal skeleton characters (e.g., Brazeau, 2009). However, the 3D histology data obtained in this PhD project has shown that we are far from understanding the dermal skeletal histology of early vertebrates, or vertebrates in general. Firstly, it has been shown that accurate growth patterns of early vertebrate scales can only be understood using 3D imaging methods such as PPC- SRµCT (Papers II and III). Traditional terminology describing growth patterns based on 2D thin sections can be redefined in s 3D context (Paper II), and new characters can be proposed accordingly. Secondly, 3D organizations of internal structures such as vasculature, pore canal system, bone trabeculae and osteocyte network probably contain significant phylogenetic information that is not available based on studies of 2D thin sections (Papers III and IV). Previous researchers have noticed the importance of studying these structures in a 3D context (e.g., Aldinger, 1937; Borgen, 1992; Denison, 1947, 1951; Goodrich, 1907; Gross, 1956; Ørvig, 1973), but the resulting models are usually too idealized to be used for rec- ognition of patterns and characters, as discussed in Papers III and IV. Thirdly, 2D thin sections often result in controversial opinions of how to define tissues. For example, many subtypes of dentine have been encoun- tered in the dermal skeleton of early jawed vertebrates (Burrow and Turner, 1999; Valiukevicius and Burrow, 2005), ranging from orthodentine as seen in our teeth to meso-semidentine that cannot be found in living vertebrates. The presence or absence of cell lacunae and the pattern of canaliculi are the major criteria to distinguish these subtypes, but 2D thin sections can never show the fine details of the canaliculi web. Future examination of relevant fossils using X-Ray Phase Nanotomography (Langer et al., 2012) will be the only way to redefine all the subtypes of dentine in 3D and provide convinc- ing characters for phylogenetic analysis.

33 Fourthly, 3D microanatomical data is necessary to appreciate individual and intraspecific variation, which is a prerequisite for generating meaningful characters. The current study, based on only four body scales of early vertebrates, cannot give any definitive answer about how to resolve the ongoing debate on early jawed vertebrate phylogeny from a paleohistological perspective. The small number of histology characters in recent phylogenetic analyses of early jawed vertebrates indicates a lack of histology data, especially the lack of microanatomical data that is necessary to recognize patterns and intraspe- cies variations. It can be expected that PPC-SRµCT will become a routine method for further studies on scales, dermal bones, fin spines, and teeth from articulated fossils to generate characters. Whether the current chaotic results regarding dermal skeletons are in fact ‘chaotic’ or not can only be clarified when more 3D data becomes available.

34 Swedish summary – Svensk sammanfattning

Människor och nästan alla andra ryggradsdjur (vertebrater) är utrustade med ett välorganiserat skelettsystem, som hos en vuxen individ inkluderar 32 tänder och 206 ben. Tänder är uppbyggda av två huvudsakliga vävnader: dentin (även kallat tandben, som formar tandens kärna) samt emalj (som bygger upp tandens ytskikt). Övriga ben i skelettet kan delas in i två typer: ett inre sk. endokondralt ben som mineraliserats inuti brosk, samt ett gene- rellt sett yttre sk. dermalt ben som mineraliserats inom en struktur av kolla- genfibrer. Om man undersöker skelettet hos en vanlig benfisk (exempelvis lax) finner man ett liknande skelettsystem, med brosk, ben, dentin och emalj- liknande vävnad. Detta indikerar att nu levande käkförsedda vertebrater (gnathostomer) har ärvt dessa hårdvävnader från sin sista gemensamma för- fader, som levde någon gång efter det att de nu levande käklösa vertebrater- na (pirålar och nejonögon) grenade av från vertebraternas stamträd. Eftersom mineraliserade skelettsystem varit så viktiga för vertebraternas framgång är det tydligt att dessa systems uppkomst och evolution är av största intresse att studera. Skelettdelar har stor potential att bevaras som fossil, och sådana fossil kan hittas i avlagringar som är upp till 400 miljoner år gamla. Dessa fossil ger ledtrådar till hur de skelett vi kan se hos nulevande vertebrater ursprungligen formades och utvecklades. Ett sätt att studera ben är genom att undersöka den inre detaljstrukturen (histologi), vilket ger information om hur skelettets olika delar bildats. Den histologiska arkitekturen är ofta bevarad i fossiliserade skelett och kan därför studeras och direkt jämföras med moderna bens histologi. Detta faktum bil- dar grunden för denna avhandling. Traditionellt har studier av fossila vertebraters skeletthistologi varit base- rade på tunnslip, det vill säga att man skär tvärsnitt genom ett ben och sedan studerar snittytorna. Detta kan ge information om vilka vävnader som finns närvarande i benet, och man kan analysera hur dessa vävnader växt under djurets liv. Eftersom man tittar på en snittyta är man begränsad till att studera strukturen och tillväxten ur ett tvådimensionellt perspektiv. Om man vill studera histologi i ett tredimensionellt perspektiv måste man använda sig av någon slags skiktröntgen (tomografisk undersökning). För den här avhand- lingen har en tomografisk teknik som kallas synkrotronscanning med faskon- trast använts. Den grundläggande principen är lik en vanlig tomografisk undersökning som utförs på ett sjukhus, men den höga energin som används vid synkrotronscanning ger ett mycket högupplöst resultat, vilket kan avslöja

35 extremt små detaljer i fossilen. Samtidigt ger faskontrasten möjligheten att särskilja fossiliserade vävnader med sinsemellan olika densitet och histologi. Fossil av fjäll från flera olika fiskgrupper valdes ut för att scannas med den här tomografiska metoden. Fjäll är små, och kan lätt jämföras mellan olika fiskarter, vilket gör dessa idealiska för att studera med synkrotronscanning. Fjällen scannades vid European Synchrotron Radiation Facility (ESRF) i Grenoble, Frankrike. Data från de högkvalitativa scanserierna analyserades sedan för att förstå fjällens histologi och tillväxthistoria i ett tredimensionellt sammanhang. Ett av de studerade fjällen, som tillhörde en av de tidigaste kända benfis- karna, kommer från cirka 420 miljoner år gamla avlagringar på Gotland, från den senare delen av en period som vi kallar silur. Detta fjäll visade sig växa genom addition av nya tandliknande strukturer i ett linjärt mönster under den tidiga tillväxten, för att sedan övergå till att fylla i mellanrummen som upp- står mellan dessa linjära strukturer (Artikel II). Två olikformade tillväxten- heter, ett linjärt och ett mellanrumsfyllande, har alltså bidragit till uppbygg- naden av det färdiga fjället. Ett annat fjäll tillhörande en annan tidig benfisk från den tidiga delen av devonperioden (omkring 400 miljoner år gammalt) visade sig ha haft en jämförbar tandlik mönsterläggning i början av sin tillväxt, vilket sedan byttes mot bredare tillväxtenheter, som var perforerade av porer (Artikel III). Tredimensionella jämförelser mellan olika tillväxtmönster ger oss möjlig- het att förstå hur olika fjälltyper hos tidiga benfiskar formades genom an- vändning av olikformade tillväxtenheter (exempelvis linjära eller mellanrumsfyllande), samt genom addition av tillväxtenheter i varierande ordning, både rymd- och tidsmässigt. Två fjäll från sk. pansarrundmunnar blev också undersökta på samma sätt som bekrivits ovan (Artikel IV). Det visar sig att vår tidigare förståelse av dessa fiskfjälls histologi är mycket bristfällig. Tillgång till högkvalitativ tredimensionell data kommer därför att kunna leda till en omvärdering av hur vi använder histologisk information för att förstå tidiga vertebraters evolution och släktskap. Användningen av synkrotrontomografi är ett stort steg framåt inom vertebratpaleontologin. Denna metod har visat sig vara det hit- tills bästa redskapet för att studera hårdvävnadernas ursprung och evolution. Bättre förståelse för skelettsystemens ursprung kommer att ge nya insikter om de huvudsakliga nulevande vertebratgruppernas (benfiskar och broskfis- kar) utvecklingshistoria. Nya tomografiska metoder har bidragit till en revi- talisering av ett länge studerat ämne, vertebratpaleontologi, och kommer att fortsätta sprida nytt ljus över vertebraternas – inklusive vår egen – livshisto- ria.

36 Acknowledgments

It has been a rewarding experience to do a PhD in this group. During the last four years I’ve received so much help from many people, without whom this thesis would become impossible. A big ‘Thank you’ to my supervisor Per Ahlberg, who has always been supportive and instructive to my project! You also care about the students’ future career and merits. I really appreciate the trust and freedom you have given to me regarding research, and your understanding of my family situation when my wife was in China. Your insightful comments and suggestions on my work (and papers) are always helpful and your knowledge (on almost everything) is just so impressive. I thank my co-supervisor Henning Blom, who has helped me and provided useful information in various stages of my research. The journey to Estonia with you was memorable. This project fully depends on synchrotron imaging at ESRF (European Synchrotron Radiation Facility), which would be impossible without generous help from Sophie Sanchez, Paul Tafforeau and other local contacts. Even when my requirements for the scan setup were ‘ridiculous’, you guys had the patience to explain every detail to me. Additional gratitude should go to Sophie for your help and suggestions about VG Studio and 3D modeling, for many good discussions on vertebrate hard tissues and imaging methods, for being encouraging all the time, and for being a great co-author and friend. Thank you Donglei, Daniel and Jordi for accompanying me during the trips to ESRF and hard work overnight. In this group I have had my first collaboration with a molecular biologist, Tatjana Haitina. It has been a great experience for me and I have learnt a lot from you, although I think I can never become a molecular biologist myself. Another dimension of knowledge has made this project much more interesting, and challenging. I would like to thank William Simpson, Zerina Johanson, Thomas Mörs, Tiiu Märss, Daniel Goujet, Gäel Clement, Kate Trinajstic and Min Zhu for providing access to fossil material and a lot of interesting discussions during my stay in the museums. Thank you, Rose-Marie and Helena, for helping me with all those admin- istrative and financial things, and taking care of PhD students. The trip to Japan was so pleasant and will be always memorable because of these nice people: Masataka Okabe, Yasuyo Shigetani, Tohru Yano, Nori-

37 fumi Tatsumi, and many other good people from the group. Thanks for supporting this trip, Per! People in IVPP have been always supportive to my work and are great hosts whenever I go back to Beijing: Min Zhu, Meemann Chang, Wenjin Zhao, Zhikun Gai, Jing Lu, Tuo Qiao, You’an Zhu, Zhaohui Pan, Feixiang, Wu, Yemao Hou and Xinxin Hao. In particular, I want to thank Min Zhu for being my master mentor and guiding me into the world of fish fossils, and for allowing me to study the beautiful fossils from Yunnan. I will always appreciate your help in the beginning of my career. 老盖, it was so nice to go to field work with you, and we should definitely continue that in the near future. 飞翔, thanks for designing the signature on the cover page. It is always enjoyable to talk with you, Daniel. Thanks so much for all your help during my stay in Uppsala (or when I was away), for a lot of interesting discussions on science and others, for being patient with me, and for being a good friend. Martin Kundrat, thanks for sharing your research experiences and caring about my research career. You always remind me science is all about the details of data and it has been always nice to talk with you. The thin sections were made at IVPP, with generous help from Wending Zhang and Shukang Zhang. Thank you, Judith, for helping translate some difficult German papers. Jordi, thanks for working on my scale data and you have impressed me by your hard work. I wish you enjoy your PhD in this group. Anna and Donglei, you have ‘saved’ me by providing a roof when I first came to Sweden. Anna, I am so sorry for boiling meat (in the morning, sometimes) at your place! Thanks for sharing cakes and organizing all the great dumpling nights, and for being a good friend. I am not a very social person, but my PhD would have been miserable without social life. The dumpling (or just food) night was always enjoyable, with Anna, Lovisa, Susanne, Oscar, Katarzyna, Sophie, Daniel, Vincent, Aoden and many others. Martin Johansson, thanks for all the interesting discussions and beers at the bar, and for being a great neighbor. En Guo and Qin Peng, thanks for all the dinners shared at your place and for being so nice to me and my wife. Yueling Zhang, thanks for all the discussions about life and research, and the good food you treated me. My further thanks go to all the colleagues for sharing the party time and fikas: Alice, Steve, Grzegorz, Cécile, Johan, Živilė, Helena, Barbro, Arshi, Jordi, Katarina, Shen Lin etc. I would like to thank Per and Alice for helping with the language of the comprehensive summary. Daniel and Henning read the thesis and helped with the Swedish summary. But all the remaining mistakes are my own fault. Lastly, I would like to thank my wife and parents for being supportive whenever I feel lost and unconfident, even though you never know exactly what I am doing. Confucius said, "While his parents are alive, the son may not go abroad to a distance. If he does go abroad, he must have a fixed place

38 to which he goes." I had never thought paleontology is the place to go before entering college, but I will try to share my joy being a paleontologist with you during the rest of my life.我爱你们！

39 References

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