Supplementary Information for Devonian arthrodire embryos and the origin of internal fertilization in vertebrates John A. Long 1-3, Kate Trinajstic 4, and Zerina Johanson 5 1 Museum Victoria, PO Box 666, Melbourne 3001, Victoria, Australia; 2, Research School of Earth Sciences, The Australian National University, Canberra, A.C.T., Australia, 2600; 3, School of Geosciences, Monash University, Clayton, Victoria, Australia, 3800; 4, School of Earth and Geographical Sciences, The University of Western Australia, Perth 6009, Western Australia, Australia 3, Natural History Museum, Cromwell Rd, South Kensington, London, UK. 1. Description of placoderm embryos: Plate description of ptyctodonts Three embryos, within a female adult Austroptyctodus (WAM 86.9.662), have been recognised ventral to the vertebrae and posterior to the trunk shield (Figs. S1a-e, 2). The most anterior embryo (embryo 1) is positioned behind and slightly under the anterior lateral plate of the adult, and is anteriorly facing being orientated at 180 degrees relative to the mother (upside down). Embryo 2 is posterior to embryo 1 and is located 2.4cm posterior to the adult trunk shield plates, just ventral to the axial skeleton of the mother. This embryo is also orientated 180 degrees in relation to the mother but is facing posteriorly. Embryo 3 is posterior and ventral to embryo 2, lying just above the dermal pelvic plates of the mother. Several plates of embryo three appear to be outside the margins of the mother s body suggesting displacement through the adult body wall, which ruptured through the build up of gases during decomposition. The anterior median ventral plate is the only plate of the trunk shield not recovered. Its absence in all three embryos suggests this plate was not yet ossified. Head shield plates comprise paired preorbital, marginal and paranuchal plates and a single median nuchal plate. The bones most closely associated with feeding and respiration: the tooth plates; www.nature.com/nature 1
marginal; paranuchal; anterior lateral and dorsolateral plates, appear to ossify in utero whereas the pineal plate, postorbital and central plates do not. However, the joint between the paranuchal plate and anterior dorsolateral plate is not fully formed and the jaw joint is also not ossified. A single embryo, in a posteriorly facing curled orientation is present within Materpiscis (WAM 07.12.1, Fig. S2). Fewer plates are present in this specimen with the head shield represented by paired preorbital plates, a single marginal and paranuchal plate and the trunk shield comprising single anterior dorsolateral, anterior lateral and median dorsal plates. All embryos have upper and lower toothplates preserved. There is little anatomic difference between the embryonic plates of the two taxa so the plate descriptions are presented together with individual differences noted. The embryonic preorbital plates are approximately 1/3 the size of the adult plates and are best preserved in Materpiscis. The right preorbital plate is better preserved than the left and is subrectangular in shape. The anterior area is narrow compared with the adult plate. They are also thin in comparison to the adult plate with an open weave ornament. The supraorbital sensory line canal is a short groove extending from the anterior margin of the plate to the level of the ethmoid ridge where it terminates as a large pore opening, which is not raised as in adults. On the right preorbitals of the embryonic Materpiscis two ridges of bone extend posteriorly either side of the pore opening delineating the path of the sensory line canal that will later be enclosed. The medial margin of the plate is irregular unlike the smooth margin of the adult. It cannot be determined if the medial margins of the plates were in contact. The pineal embayment is proportionally smaller in the embryo compared with the adult. In Austroptyctodus the preorbital plate is poorly preserved but part of the outline can be identified anterior to the nuchal plate in embryo 1. It provides no other proportional or morphological details. The nuchal plates from all three Austroptyctodus embryos are preserved in visceral view on the now resin embedded side of the specimen. The plate is square, whereas in the adults it is rectangular in shape with the longer margin in an anterior to posterior orientation. The distinctive cross, made by the bony tubes enclosing the posterior pit line and supraorbital sensory line makes these plates easily identifiable. All plates have convex anterior and posterior margins. In the adult the anterior margin is straight and the www.nature.com/nature 2
posterior margin is slightly pointed. In embryo 1, the dorsal view shows that the sensory canals are deeply incised into the external surface of the lamellar bone as open grooves (Fig. S2), whereas in adults the sensory canals are a visible as a series of pore openings in dorsal view. The external ornament is an open weave allowing the bony base to be visible. In both genera the embryonic paranuchal plate is flatter than the adult plate and the margins appear irregular. The plate appears better ossified than other plates of the headshield with the bony lamina not visible through the open weave ornament (Fig. S1f, g). In posterior view the external endolymphatic opening for the main lateral line is disproportionally large (Fig. S1e). There is no evidence of bony canals on the visceral surface. Compared with the adult, the articular flange is not developed, showing no elevation of the articular area. In external view the central region of the marginal plate is covered by an open meshwork of bone but this does not extend completely to the plate margins (Fig. SI 1c). In Materpiscis the external ornament is not as developed as in Austroptyctodus. In visceral view a thin ridge of bone, corresponding to the mesial laminae in the adult, is preserved in both genera although it is not as developed as in the adult. The adult of Materpiscis has three raised pore openings on the external face of the marginal plate, no pore openings are noted on the embryonic plate, although one opening is present in the centre of the embryonic plate of Austroptyctodus (Fig. SI 1c), which corresponds to the pore opening at the junction between the main lateral line canal and post marginal sensory canal in the adult. The embryonic sensory system differs on the marginal and paranuchal plates compared to the preorbital and nuchal plates. Here external pores opening to the surface from ascending tubes, connecting to the lamina bone, are observed whereas the sensory lines appear as deeply incised grooves on the dorsal head shield plates, but the visceral tubes are not yet formed on any plates. The median dorsal plate is preserved in visceral view with the median keel clearly visible in embryos 1 and 2 (Fig. SI 1a), and in lateral view in embryo 3. It is flatter than the adult plate and the median dorsal ridge, visible in external view in the adult, is not present. The breadth to length ratio of the embryonic plates is 2.25 whereas in the adult the breadth the length ratio is 2. In Materpiscis the plate is not preserved. www.nature.com/nature 3
The anterior dorsolateral plate is identical in shape to the adult with a defined overlap and the groove for the anterolateral plate (Figs. SI 1a, 2). The anterior foramen for the lateral line canal is distinct and a posterior foramen is observed on the exterior of the plate, whereas in the adult it is seen on the posterior mesial margin. The glenoid condyle of the left anterior dorsolateral is narrow and longitudinally oriented and surrounded by a bony lip, whereas the condyle in the adult is transversely oriented. The thickened base (embryo 2) has a network of bone surrounding it. The articular surface does not appear to be fully formed. The embryonic anterior dorsolateral is 1/4 the size of the adult plate. It is flatter than the adult plate rather than being arched transversely. The anterior lateral plate in Austroptyctodus (Figs. S1e, 2) is crescent-shaped, possessing a branchial lamina that is not as extensive as in the adult, appearing as a narrow band of thin bone along the dorsal and posterior margins and expanding only along the ventral margin of the plate. In Materpiscis the branchial lamina is similarly developed. The branchial lamina lacks the dermal denticles characteristic of adult plates (Fig. S1b). The external dermal ornament of the anterior lateral plate appears to be almost identical the adult, the plate merely being thinner; however, the postbranchial lamina consists of a primary bony lamina, with an undifferentiated globular mass present on the surface of the lamina. This difference in structure between the external plate surface and the internal postbranchial lamina supports the hypothesis that the postbranchial lamina denticles are produced internally, through the inductive influence of endoderm, rather than externally as part of the dermal ornament 28. All tooth plates are present in the embryos of both genera. The upper toothplates lack an anterior dorsal process, a feature present in the adults of both taxa. The interior of the upper toothplates of Materpiscis have been visualised through CT scanning. Thin horizontal bony struts are present between a medial bone and each side of the toothplate. The lower toothplates are identical to the adult lower toothplates. Plate Description of embryos of Incisoscutum ritchiei Plates of small individuals of the arthrodire Incisoscutum ritchiei were recognised within the body cavity of two larger specimens during the original description of the species and identified as stomach contents (P50934, P57640 4 ). These individuals were www.nature.com/nature 4
thought to have been ingested by the adult, rather than representing an embryo, because of their orientation within the adult, the disarticulation of the plates and that the plates appeared to be etched by stomach acids. However, by comparing the small Incisoscutum plates present with those in the embryonic ptyctodonts described above, similarities emerge which suggest that these represent Incisoscutum embryos (based primarily on P50934; P57640 has not been completely prepared in acetic acid and so plates remain unexposed). As well, these plates are unbroken and uncrushed which is very different from placoderm plates ingested and found in the preserved gut contents of the arthrodire Coccosteus cuspidatus 12. These gut contents formed an ovoid mass, and included acanthodian and lungfish scales and bones, as well as detrital grains, which were suggested to act as gizzard stones to break up and digest material. This differs from the Incisoscutum adults where only small, unbroken Incisoscutum plates were found, and detrital grains were not seen. In P50934, 23 small plates were found within the adult. These were also ventral to the vertebral column and located just posterior to the trunkshield. Plate identification suggests that only one embryo was present (Fig. 1a, c, e). Some plates could not be identified, and other identifications are tentative. Nevertheless, it appears that most trunkshield plates were present, having been ossified while in utero. In the headshield by comparison, generally only bones associated with the lateral lines were ossified in the embryo. These include the paranuchals (Fig. 1e, f) and preorbitals (Fig. 1e). The postsuborbital, not associated with sensory canals, is also present (Fig. 1e). These are predominantly plates from the lateral parts of the headshield, while plates from the midline such as the nuchal, centrals, pineal and rostral have not been identified. The paranuchals and anterior dorsolateral plates are particularly well preserved and confidently identified, and are described further below. The paranuchals (Fig. S1e, l.pnu, r.pnu) are small plates, and dominated by the large endolymphatic duct. A relatively large process extends medially to run beneath the nuchal plate and a large overlap surface for this plate is present. The lateral line canal runs along the lateral margin of the plate and meets the curved posterior pitline groove, situated above the opening for the endolymphatic duct. These canals are very wide and www.nature.com/nature 5
open. A very faint occipital cross-commisural sensory canal is also present posteriorly, meeting the lateral line canal. The paranuchal is a squarish bone, which is a very different shape from the adult paranuchal (Fig. 1g). The adult paranuchal is more elongate anteroposteriorly, widening anteriorly. The endolymphatic duct openings are proportionately much smaller and the sensory canals are narrower. Relatively speaking, the endolymphatic duct openings, posterior pitline groove and occipital cross-commisural sensory canal are located in the posterior half of the plate, near the margin, while in the smaller individual, these elements were located in the middle of the bone. Ontogenetically, it seems that most plate expansion in the adult has taken place anteriorly. The anterior dorsolateral in the smaller individual is also dissimilar to the adult anterior dorsolateral (Fig. 1e, r.adl). On the smaller anterior dorsolateral, the lateral line canal runs just below the articular condyle (articulating with the PNu) and across the plate to the posterior margin. The articular condyle is proportionately large, occupying about onequarter the length of the anterior margin of the plate. The articular condyle appears to be largely oriented laterally. Ventrally, the overlap for the anterior lateral plate is widest anteriorly and narrows posteriorly. The overlap comprises approximately one-fifth the height of the plate. An overlap for the median dorsal plate is not visible. In an adult anterior dorsolateral the proportions of the plate have changed markedly. The articular process is now relatively smaller, narrower and oriented anteriorly, while the overlap for the AL plate is markedly larger. The posterior extension of this overlap has been lost and the anterior part of the overlap extends for nearly half the height of the plate. The course of the lateral line canal has changed and the canal itself is much narrower. An elongate overlap for the median dorsal is present. The presence of smaller plates within a larger adult individual does not necessarily indicate the presence of an embryo. Beyond the lack of crushing that would be expected in an ingested specimen, there are similarities to the uncontested ptyctodont embryos, including the relatively larger endolymphatic duct opening on the paranuchals and the larger, more open sensory canals on all plates where they occur. www.nature.com/nature 6
Embryonic dermal bone The embryonic bone is composed of fewer layers than adult dermal bones, which commonly comprise three layers, an outer dentinous layer, a middle spongious layer and an inner layer of lamellar bone. Gross 29 noted that antiarch placoderms have multiple superimposed layers forming the middle spongious layer in the dermal bone and these layers increase as the fish ages. Burrow 30 identified four layers within the antiarch Bothriolepis canadensis. The ptyctodont embryos also have a thin, inner layer of lamellar bone and differing amounts of the middle spongious layers (Fig. 1h). The head shield plates have a greater amount of the middle, spongious layer than the trunk shield plates. Juveniles (13-14mm) of the antiarch Asterolepis ornata have lamellar bone present with an irregular network of bony trabeculae on the external surface of the lamina 16. The second stage of development is the formation of the spongious middle layer 16. Larger individuals (longer than 30mm) have fine meshed reticular ornament, which sometimes bears tubercles. The ptyctodont embryos appear to have reached this developmental stage; although, unlike Asterolepis they remain in utero. Endoskeleton Pectoral fins: The scapulocoracoid is preserved in Materpiscis and shows the monobasal condition 31, with a single fin radial articulating to the scapulocoracoid. In Incisoscutum ritchiei a few fin radials are present below the PVL (Fig. 1c, d) and in Austroptyctodus four dermotrichia are present in embryo 1 (Fig. S1d); radials not being present in ptyctodonts. Phylogenetically the fin endoskeleton appears as an undivided oval blade, identified in the ostracoderm Escuminaspis laticeps 32. A monobasal endoskeleton is accepted as the generalised condition for placoderms with proximal dermotrichia present in the ptyctodonts, whereas simple endoskeletal radials support the fins in Rhenanida and the arthrodires. This phylogenetic series appears similar to the development series for Danio rerio 33, where the precursor for the pectoral fin elements is a chondrogenic cellular plate, which persists through the embryonic and early larval stages, with separation of this plate into individual radials later in ontogeny. Additionally, the lepidotrichia (dematotrichia in placoderms) forms prior to the division of the endoskeletal disc into www.nature.com/nature 7
separate radials. 2. Ossification sequences in placoderms Vertebrates as a whole show significant variation in ossification sequence although ossification usually occurs with the formation of the dermal elements, the neurocranium and splanchnocranium generally ossifying later 34,35. As a general rule, it was proposed that the bones involved in the functions of feeding and respiration ossified first 36. The supragnathals in Materpiscis (WAM 07.12.1) meet medially in a symphysis leading to increased structural support for biting ability 1. In addition, the upper toothplates are strengthened through trabeculation. This entails the formation of mineralised struts within the cavity between the bony layers, which transfer and dissipate forces applied to the jaw. Trabeculation is present all extant durophagous stingrays and ontogenetic series show that the struts develop before birth 37. The presence of trabeculae in embryonic ptyctodonts indicates that they were ready to catch and ingest hard prey immediately after birth. All ptyctodont embryos lack the anterior median ventral plate. In juveniles of Asterolepis ornata the medioventral plate is not ossified in the earliest stages of development 18. Although juvenile bothriolepids are known from Antarctic these are not as small as the Asterolepisis and all body plates are present 17. However, in both antiarch taxa there is considerable difference in the shape and proportions of the juvenile plates when compared to the adult form. It is here considered that one of the advantages viviparity provided ptyctodonts was direct development limiting energy expenditure on remodelling plates through different ontogenetic stages. 3. Conclusion The origin of vertebrate viviparity is now well established in the most basal of gnathostomes, the placoderms, although new phylogenies 3 support that the most basal placoderms, and so the most basal gnathostomes (the Antiarchi) were not viviparous and instead spawned externally 18. Blackburn 7 reports over 135 separate evolutionary origins of viviparity, which makes this one of the most significant examples of convergence www.nature.com/nature 8
amongst vertebrates. Having embryonic and juvenile specimens of extinct placoderms gives new insights into the patterns of development and some of the adaptations seen in phylogenetically basal gnathostomes. References, numbering continued from main paper: 28. Johanson, Z. & Smith, M. M. Placoderm fishes, pharyngeal denticles, and the vertebrate dentition. J. Morph. 257, 289-307 (2003). 29. Gross, W. Histologische Studien am Aussenskelett fossiler Agnathan und Fische. Palaeontographica A 83, 1-60 (1935). 30. Burrow, C. Histological structure of the cancellous bone layer in Bothriolepis canadensis (Antiarchi, Placodermi) Lethaia 38, 205-210 (2005). 31.Goujet, D. G. in Major Events in Early Vertebrate Evolution (ed. P. Ahlberg), 209-222 (Taylor & Francis, 2001). 32. Janvier, P., Arsenault, M. & Desbiens, S. Calcified cartilage in the paired fins of the osteostracan Escuminaspis laticeps (Traquair 1880), from the late Devonian of Miguasha (Québec, Canada), with a consideration of the early evolution of the pectoral fin endoskeleton in vertebrates J. Vert. Paleo. 24, 773-779 (2004). 33. Cubbage, C. & Mabee, P. Development of the cranium and paired fins in the zebrafish Danio rerio (Ostariophysi, Cyprinidae). J. Morph. 229, 121-160 (1996). 34. Arratia, G. & Schultze, H. -P. The urohyal: Development and homology within osteichthyans. J. Morph. 203, 247-282 (1990). 35. Mabee, P. M. & Trendler, T. A. Development of the cranium and paired fins in Betta splendens (Teleostei: Percomorpha): Intraspecific variation and interspecific comparison. J. Morph. 227, 248-287 (1996). www.nature.com/nature 9
36. Vanderwalle, P., Focant, B., Huriaux, F. & Chardon, M. Early development of the cephalic skeleton of Barbus barbus (Teleostei, Cyprinidae). J. Fish Biol. 41, 43-62 (1992). 37. Summers, A. P. Stiffening the stingray skeleton An investigation of durophagy in myliobatid Stingrays (Chondrichthyes, Batoidea, Myliobatidae). J. Morph. 243, 113-126 (2000). www.nature.com/nature 10
Supplementary Figure 1 a-d, Austroptyctodus embryos (WAM 86.9.662); e, CT tomogram of Materpiscis paranuchal plate (WAM 07.12.1). www.nature.com/nature 11
Supplementary Figure 2 Sketch showing main features of Austroptyctodus embryo 1 (WAM 86.9.662) as referred to in the text and shown in Supplementary Figure 1a. Abbreviations: AL (ad), adult anterior lateral; l.adl, r.adl, left and right anterior dorsolaterals; l.al, r.al, left and right anterior laterals; IL, interolateral; MD, median dorsal; Nu, nuchal;?pro, preorbital; rad, radialia; r.ig, right inferognathal; r. MG. right marginal; Sgn, superognathal; um.c, umbilical cord remnants. www.nature.com/nature 12
doi: 10.1038/nature07732 Supplementary Figure 3 Detail of Materpiscis attenboroughi WAM 07.12.1. showing embryonic plates and umbilical cord structure. AL, anterior lateral, Ifg, inferognathal; MG, marginal; PNu, paranuchal; Sgn, superognathal; um, umbilical cord. www.nature.com/nature 13