Evolution of vertebrate postcranial complexity: axial ...

[Palaeontology, 2018, pp. 1–13]

EVOLUTION OF VERTEBRATE POSTCRANIAL COMPLEXITY: AXIAL SKELETON REGIONALIZATION AND PAIRED APPENDAGES IN A DEVONIAN JAWLESS FISH by MARION CHEVRINAIS 1 , 2 , ZERINA JOHANSON 3 , KATE TRINAJSTIC 4 JOHN LONG 5 , CATHERINE MOREL 1 , CLAUDE B. RENAUD 6 and RICHARD CLOUTIER 1

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1 Laboratoire de Paleontologie et Biologie Evolutive, Universite du Quebec a Rimouski, Rimouski, QC G5L 3A1, Canada; [email protected], [email protected] 2 Laboratoire de Planetologie et Geodynamique, UMR CNRS 6112, Universite de Nantes, Nantes, 44000, France; [email protected] 3 Department of Earth Sciences, Natural History Museum, Cromwell Road, London, SW75BD, UK; [email protected] 4 Department of Environment and Agriculture, Curtin University, Perth, WA 6102, Australia; [email protected] 5 Faculty of Science and Engineering, Flinders University, 2100, Adelaide, SA 5001, Australia; [email protected] 6 Research & Collections, Canadian Museum of Nature, Ottawa, ON K1P 6P4, Canada; [email protected]

Typescript received 19 December 2017; accepted in revised form 22 April 2018

the axial skeleton is also described for the first time in an extant jawless fish, the sea lamprey Petromyzon marinus Linnaeus. Our data indicate that regionalization of the axial skeleton occurred earlier in vertebrate evolutionary history than previously appreciated. This regionalization is coupled with modifications of the appendicular skeleton in Euphanerops. These new observations combined with a new phylogenetic analysis of early vertebrates provide a more precise understanding of how the appendicular and axial skeletons developed and evolved within vertebrate evolutionary history.

Abstract: One of the major events in vertebrate evolution involves the transition from jawless (agnathan) to jawed (gnathostome) vertebrates, including a variety of cranial and postcranial innovations. It has long been assumed that characters such as the pelvic girdles and fins, male intromittent organs independent from the pelvic girdles, as well as a regionalized axial skeleton first appeared in various basal gnathostome groups if not at the origin of gnathostomes. Here we describe the first occurrence of pelvic girdles and intromittent organs in the Late Devonian jawless anaspid-like fish Euphanerops longaevus Woodward (Miguasha Lagerst€atte, eastern Canada), associated with a morphologically differentiated region of the axial skeleton. Morphological differentiation of

Key words: early vertebrate, intromittent organ, axial skeleton regionalization, evolution.

O U R understanding of the evolution of the vertebrate postcranial skeleton has advanced significantly in recent years, including new morphological and developmental data on the vertebral column of hagfish, a basal living craniate (Ota et al. 2011, 2014); although the vertebral column of the lamprey has been described for many years (Marinelli & Strenger 1954), its development is poorly known. Paired pectoral fins with a supporting girdle first evolved in jawless vertebrates (osteostracans; Janvier 1981, 1985), while pelvic girdles and intromittent organs (paired male reproductive structures) have been reported in antiarch placoderms (Long & Young 1988; Long et al. 2009, 2015; Zhu et al. 2012; Trinajstic et al. 2015), the most phylogenetically basal gnathostomes (Qiao et al.

2016; but see King et al. 2016). This confirms these characters as primitive for jawed vertebrates, but with pelvic girdles and intromittent organs apparently absent among jawless vertebrates. However, the postcranial skeleton of most fossil jawless vertebrates is poorly known and open to conflicting interpretations (Wilson et al. 2007; Johanson 2010). The anaspid (or anaspid-like) Euphanerops longaevus, preserves what is perhaps the most complete postcranial skeleton in fossil jawless vertebrates (Janvier & Arsenault 2007). Janvier & Arsenault (2007) made the last exhaustive description of the Euphanerops skeleton, describing several anatomical elements composing the axial skeleton (i.e. two series of arcualia, ‘haemal series’) and paired ventral

© The Palaeontological Association

doi: 10.1111/pala.12379

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fins representing the paired appendages. Later, Sansom et al. (2013) proposed a new interpretation of the anal fin of Euphanerops. Based on the presence of two distinct series of radials in the anal fins separated by 100 lm of sedimentary matrix, Sansom et al. (2013) proposed that a pair of fins was present, the first observation of paired anal fins in vertebrates (but see below). Our re-examination of the Euphanerops postcranial skeleton, and new data on the axial skeleton of the lamprey Petromyzon marinus, provides new insights into the evolution of paired fins and regionalization of the axial skeleton, previously thought to be characteristic of jawed vertebrates, including axial skeleton regionalization in both taxa, and pelvic girdle structures, including intromittent organs, in Euphanerops.

MATERIAL AND METHOD Euphanerops longaevus Woodward, 1900 Specimens of Euphanerops longaevus come from the Upper Devonian fossil-fish Lagerst€atte of Miguasha (Quebec, Canada) (Cloutier et al. 1996) and are housed in the Musee d’Histoire Naturelle de Miguasha, Canada (MHNM) and in the Natural History Museum, UK (NHMUK). Interpretative drawings of specimens (MHNM 01-123, MHNM 01-02A and NHMUK P6813) under water immersion were realized using a camera lucida (Fig. 1). Elemental composition analysis was performed on two immature specimens (Fig. 1B, C) using an INCA X-sight (Oxford Instruments) energy dispersive X-ray spectrometer coupled to a JEOL 6460LV SEM at Universite du Quebec a Rimouski (Quebec, Canada). Samples were observed without conductive coating. Each spectrum was acquired with a 10 lm spot size, for 100 s (process time 5, spectrum range 0–20 keV, 2000 channels) at an accelerating voltage of 20 kV. The detection limits of chemical elements are about 1000 ppm or 0.1 wt%. Quantitative optimization of the system was done using copper as a standard. Elements were automatically identified and quantified in weight by the INCA software and results were normalized to 100%.

Petromyzon marinus Linnaeus, 1758 Specimens of Sea Lamprey Petromyzon marinus used in this study are housed in the Fish Collection of the Canadian Museum of Nature (CMNFI). They were sampled from Lake Huron basin (ON, Canada) and the SainteAnne River (Quebec, Canada). In addition, ammocoetes freshly collected from the Old Woman River, Lake Superior basin (Ontario, Canada) were provided by the

Bayfield Institute (July 2012, Fisheries and Oceans Canada). The Maurice-Lamontagne Institute also provided a fresh adult collected from the Saint Lawrence River (Canada) during the summer of 2010. Fresh specimens were fixed in 4% buffered formalin and transferred to 70% ethanol. Total length (TL), the distance between the tip of the snout and the posteriormost part of the caudal fin (Renaud 2011), was measured with digital calipers. Whole-mount specimens of ammocoetes (four populations, n = 79, TL = 19–129 mm) and young adults (four populations, n = 21, TL = 119–153 mm) were cleared and stained (C&S) following standard protocol (Potthoff 1984). Alcian blue (0.3%) in acid solution was used to colour cartilaginous structures. A structure was considered formed when it took the blue stain. Specimens were examined under a Leica MZ16A binocular microscope equipped with a Qicam digital camera with a CCD sensor (Meyer Instruments, TX, USA). The branchial, predorsal, second dorsal and caudal regions of an ammocoete (Old Woman River, AMPMH01, TL = 132.9 mm) and an adult (Saint Lawrence River, ADPMH-01, TL = 270.5 mm) were selected for histological preparation. The samples were processed in a Shandon Citadel 2000 automated tissue processor. They were dehydrated in graded ethanol solutions (20–100%), transferred to xylene/ethanol and finally pure xylene. Samples were then transferred to a 50/50 xylene/paraffin solution and impregnated with melted paraffin under a vacuum. Samples were embedded in Paraplast Plus and sectioned at 7 lm intervals. Sections were processed through regressive staining using standard haematoxylin and eosin (H&E). Sections were observed with a Leica DMLB microscope and images were taken with an AmScope MU1000 microscope digital camera using ToupView software (v. 3.7; AmScope 2013; http://www.amscope.com/sof tware-download).

Phylogenetic analysis of vertebrates We used 43 taxa of jawless and jawed vertebrates in a revised data matrix (Chevrinais et al. 2018). Our matrix included 308 characters including 298 characters from previous analyses (Chevrinais et al. 2018). The data matrix was analysed with PAUP version 4.0b10 (Swofford & Sullivan 2003) using two outgroups (Cephalochordata and Tunicata). All characters were unordered and unweighted. We used an heuristic search; the branch-andbound search did not yield trees. Maxtrees was set at 100 000. ACCTRAN and DELTRAN options were used. We performed 1000 bootstrap replicates using heuristic searches. We set the maximum of trees saved for each random sequence addition to 100 000.

CHEVRINAIS ET AL.: POSTCRANIAL COMPLEXITY IN A FOSSIL JAWLESS FISH

Euphanerops longaevus. A, MHNM 01-123, nearly complete specimen photograph and drawing; black asterisks show the ventral shift of anatomical elements, probably due to post-mortem release of decompositional gas. B, MHNM 0102A immature specimen, photograph in water immersion, and drawing. C, NHMUK P6813 (holotype) immature specimen photograph and drawing. Black squares indicate positions of close-ups in Figure 4. Anatomical abbreviations: an.fin rad, anal fin radial; an.fin ray, anal fin ray; ann.cart, annular cartilage; a-v fins, paired anteroventral fins; branchial app, branchial apparatus; d.fin, dorsal fin; int. org, intromittent organs; neuro.el, neurocranial elements; pelv.d, pelvic discs; sk, skull. Large arrows indicate anterior. Scale bars represent 10 mm (A); 5 mm (B, C). Colour online.

FIG. 1.

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RESULTS Euphanerops displays three rounded neurocranial elements, a head with calcified cartilage, an annular cartilage, a lamprey-like branchial apparatus, a series of arcualia and other cartilages surrounding the notochord, paired anteroventral, median or paired anal fin(s), a median dorsal fin, and a hypocercal caudal fin (Janvier & Arsenault 2007; Sansom et al. 2013) (Fig. 1).

Fins in Euphanerops longaevus Fin rays are defined as structures internally strengthening and supporting the fins (Francillon-Vieillot et al. 1989; Arratia et al. 2001; Witten & Huysseune 2007). Fin rays can consist of cartilaginous, mineralized and osseous tissues. Four kinds of fin rays have been described. Lepidotrichia, present in actinopterygians and sarcopterygians with the exception of dipnoans, are flexible rays composed of mineralized hemisegments connected by ligaments (Francillon-Vieillot et al. 1989; Witten & Huysseune 2007). With few exceptions in actinopterygians and derived actinistians, actinotrichia are present between the most distal hemisegments of the lepidotrichia and consist of short, tapered rods of a special type of collagen called elastoidin that are generally distally branched (Francillon-Vieillot et al. 1989; Witten & Huysseune 2007). Ceratotrichia are flexible, unsegmented cartilaginous rods present in the fins of chondrichthyans. These fin rays are longer and thicker than actinotrichia and are usually branched distally (Kemp 1977; Geraudie & Meunier 1982; Francillon-Vieillot et al. 1989). Camptotrichia, present only in dipnoans, consist of straight cylindrical rods arranged in two asymmetrical rows (Geraudie & Meunier 1982; Arratia et al. 2001). This kind of fin ray is usually branched and is composed of acellular fibrous tissues and mineralized bones (Arratia et al. 2001). The paired anteroventral fins of Euphanerops originate directly behind the annular cartilage and extend posteriorly just anterior to the anus (Fig. 1A). The fin radials are described as proximal cartilaginous endoskeletal elements (composed of spherulitic chondrocytes (Fig. 2B, D) with thin extracellular matrix comparable to the axial skeletal elements (Fig. 3H) and an alveolar superficial layer (Fig. 2D)), whereas the fin rays (most closely resembling lamprey fin rays) are more distal, and composed of stacked chondrocytes (Fig. 2D–F). Previous interpretations of Euphanerops (Janvier & Arsenault 2007) called

the distal fin elements ‘radials’ because of the purported homology with the ‘Radii pterygiales’ of lampreys (Marinelli & Strenger 1954). But in lamprey, the distal elements of the dorsal fin are branched distally and composed of stacked chondrocytes; thus we identified those elements as fin rays (Fig. 3D, black asterisk; Chevrinais et al. (2018, fig. 1) (Vladykov 1950; Jollie 1962). Despite the term that has been used in previous studies (i.e. fin radial; Goodrich 1958; Janvier & Arsenault 2007; Sansom et al. 2013) we use the term ‘fin ray’ for Euphanerops in the present study, based on the position of these elements and the presence of stacked chondrocytes. The organization of the radials and fin rays suggests that the paired anteroventral fins correspond to a series of repeated anatomical units along the ventral flank (Fig. 2F, unit). MHNM 01-123 preserved the best information relative to these fins, with left and right sides identified based on the tissues composing radials and rays and the superimposition of these elements (right elements are preserved in internal view and left in external view, Fig. 2C). Radials are more or less sickle-shaped with a large base, whereas rays are rod-shaped showing parallel orientation (Fig. 2B, C, F). Furthermore, curved elements showing stacked chondrocytes (Fig. 2C, D) are observed at the base of each paired radial. Based on these observations, we suggest that the fin unit is composed of one radial, one meso- or metapterygium-like structure and several fin rays (more than four, but the number is difficult to determine). Because there is more than one ray per radial, and the units are formed as repetitive anatomical structures, we hypothesize that instead of having one unique extended ventral fin on each side of the body (e.g. Wilson et al. 2007), Euphanerops possesses multiple fins ventrally, that could be arranged along the body in a manner comparable to acanthodian intermediate or pre-pelvic spines (Wilson et al. 2007). Nevertheless, there is no indication of (a) supporting girdle(s). Anal fin(s) are considered either as median (Janvier & Arsenault (2007) or paired (Sansom et al. 2013) (Fig. 1). An elongate median dorsal fin is composed of a series of rays composed of stacked chondrocytes (contra Janvier & Arsenault 2007), some bifurcated distally (Fig. 1A) as also observed in the lamprey Petromyzon (Chevrinais et al. 2018, fig. 1).

Axial skeleton Petromyzon marinus. In the caudal region, chondrocytes form irregular agglomerates of cartilage dorsolaterally to

Paired anteroventral fins of Euphanerops longaevus. A, MHNM 01-123. B–C, close-up of rectangle in A showing fin radial and ray. D, alveolar structure of radial tissue. E, close-up of fin ray showing stacked chondrocytes. F, interpretation of the paired anteroventral fin elements, one body side is represented. Abbreviations: a-v fins, paired anteroventral fins, L, left; R, right. Large arrows indicate anterior. Scale bars represent 3 mm (A–C, F); 1 mm (D); 200 lm (E). Colour online.

FIG. 2.


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basiventrals mediodorsal vertebral elements notochordal cartilages left side arcualia

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Vertebrate axial skeleton. A, Petromyzon marinus body regions and location of arcualia (grey bar). B–C, transverse sections of Petromyzon marinus ammocoete (AMPMH01, 132.9 mm TL) from dorsal region of the caudal fin. C, chondrocytes located between the neural tube and the notochord as indicated by the arrows in B and C. D, posterior region of the caudal fin of a metamorphosing ammocoete (S3-1, 125.6 mm TL); white asterisks, large pentagonal cells present in the median rods of the caudal fin; black asterisk, regularly stacked cells with a rectangular shape forming the fin rays; black arrows, pentagonal chondrocytes lying dorsally to the notochord. E, general morphology of the axial skeleton in Petromyzon marinus. F, Euphanerops longaevus MHNM 01-123, axial skeleton. G, MHNM 01-123, notochord and notochordal cartilages. H, MHNM 01-123, basiventral with shape of element highlighted in black. I, axial skeleton of Tarrasius (Mississippian, Scotland) showing regionalization (five regions) in early actinopterygians (modified from Sallan (2012)). Abbreviations: a, arcualium; c, centrum; dmr, dorsomedian rod; ee, elastica externa; ha, haemal arch; hs, haemal spine; mde, mediodorsal element; n, notochord; na, neural arch; nc, notochordal cartilage; ns, notochordal sheath; nsp, neural spine; nt, neural tube; rb, rib; va, ventral arch; vmr, ventromedian rod. Large horizontal arrows point anteriorly. Scale bars represent 0.1 mm (B); 0.05 mm (C); 0.25 mm (D); 1 mm (E, G, H), 3 mm (F). FIG. 3.

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the notochord (Fig. 3B–D black arrows). These cartilaginous elements are notochordal cartilages; such cartilages have never been described in lampreys before (Marinelli

notochord

& Strenger 1954). Histological sections revealed the presence of two dorsolateral rods positioned dorsally to the notochord and encompassing both sides of the neural


tube (Fig. 3B). The dorsolateral rods are fused to the ventral parts of the dorsomedian rod, thus forming a continuous arch encompassing the neural tube. Notochordal cartilages were also observed in the posteriormost region of ammocoetes (i.e. small amounts of chondrocytes dorsal to the notochord in the posteriormost region of the caudal fin) indicating that chondrogenesis for this cartilage has occurred in earlier stages of development. However, whether these chondrocytes are the results of a notochordal chondrogenesis or the result of the extension of the dorsomedian rod is not clear. Arcualia are defined as the primary cartilaginous elements forming dorsally along the notochord in vertebrates (Parker 1883; Arratia et al. 2001), distinct from the more dorsolateral notochordal cartilages. During embryonic development, the first mesenchymal cells gather around the notochord, forming discrete blocks of cartilage (Arratia et al. 2001). These structures, mainly formed of hyaline cartilage, develop into neural arches in derived groups of fishes (Arratia et al. 2001; Grotmol et al. 2003, 2006). This term is also used to designate the dorsal vertebral elements in the Petromyzontiformes (Damas 1944; Janvier 2003; Richardson et al. 2010; Renaud 2011). In Petromyzon marinus, the development of arcualia was categorized in terms of their position along the body axis (Fig. 3A; Chevrinais et al. 2018, figs 2, 3, table 1). Two centres of development of arcualia have been recorded. The first is located in the branchial region where the arcualia developed anteroposteriorly, decreasing in size posteriorly (Fig. 3E; Chevrinais et al. 2018, table 1). The number of arcualia formed in this region varied from 6 to 12 regardless of the metamorphosing stage. The morphology of the first arcualium is distinctive relative to the others; it is noticeably larger and its distal extremity is always bifid rather than undivided. The second centre of development is located in the second dorsal region where the arcualia developed bidirectionally (Fig. 3E; Chevrinais et al. 2018, fig 2, table 1). In adults, the first 7–8 arcualia located in the branchial region are always the largest and most developed. Besides the size and the typical hook-shaped arcualia, the general morphology of arcualia in the branchial region is variable (Fig. 3E; Chevrinais et al. 2018, figs 2b, 3). The distal extremity is either unifid, bifurcated or trifurcated; it can either point anteriorly or posteriorly. The proximal base of the arcualium is generally well rounded and perforated, as also observed in metamorphosing specimens. Unlike the dorsal region, the caudal region contains arcualia with variable morphologies and irregularly spaced along the notochord (Chevrinais et al. 2018, figs 2c–d, 3d). No arcualium was present in the terminal section of the caudal region. The dorsal arcualia of the lamprey axial skeleton show regional differentiation anteroposteriorly (Fig. 3E; Chevrinais et al. 2018, fig. 2), being larger and

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more complex anteriorly (bifurcated), with a foramen at the base. We defined the mediodorsal vertebral elements as the cartilaginous structures medial to the arcualia and sitting dorsally on the notochord (mde; Fig. 3E; Chevrinais et al. 2018, fig. 2). Their shape is either elongated, short, crumpled, notched or twisted, while their size varies from the same height as the arcualia, to small, stocky and barely visible along the notochord. These elements were present only in the branchial region, developing anteroposteriorly, mostly interspersed between the third to the seventh arcualia. Euphanerops longaevus. The exceptional preservation of slightly mineralized or non-mineralized tissues shows that Euphanerops possesses a notochord, and a series of associated dorsal and ventral cartilages. These include notochordal cartilages, left and right-side arcualia and mediodorsal vertebral elements dorsally, and basiventrals ventrally (Fig. 3F–H). The element previously identified as an elongated ‘white line’ in Euphanerops (Janvier & Arsenault 2007) is reinterpreted as a diagenetic mineralization of the notochord (Fig. 3F, G). The unsegmented and longitudinal fibrous texture of the notochord of Euphanerops recalls the ultrastructural condition of the notochord in the cephalochordate Branchiostoma (Welsch 1968) and conodonts (Aldridge et al. 1993). Dorsolateral to the notochord, paired notochordal cartilages extend posteriorly from behind the head region to the pelvic complex (described below), and appear to have cupshaped surfaces that would have rested against the notochord (Fig. 3F, G). The paired notochordal cartilages have become disrupted anterior to the anal fin(s) (MHNM 01-123), and shifted ventrally, probably due to the post-mortem release of decompositional gas from the gut. The arcualia and mediodorsal elements are similar to those present in the anterior axial skeleton of Petromyzon marinus (Fig. 3E; Chevrinais et al. 2018, figs 2, 3). Dorsal to the notochord and notochordal cartilages in Euphanerops, a row of paired arcualia and mediodorsal cartilages extends along the trunk. The paired arcualia are preserved as distinct rows on MHNM 01-123 (Figs. 1A, 3F), and were originally interpreted as ventral arcualia and intermuscular elements (Janvier & Arsenault 2007). However, we suggest instead that these represent left and right dorsal arcualia that have been shifted slightly from their original positions post mortem (although the right arcualia extend along the body, more anterior left dorsal arcualia are either not preserved or superimposed against the right arcualia). Between these larger arcualia are smaller cartilaginous mediodorsal elements (Fig. 3F). More posteriorly, the arcualia become simplified in shape, and smaller towards the caudal fin. Large, rectangular elements

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interpreted as basiventral/haemal elements are present ventral to the notochord; they begin just dorsal and anterior to the anal fin(s), extending posteriorly (Fig. 3F, purple, H). The presence of these basiventrals in this region suggests that the axial skeleton of Euphanerops shows differentiation into anteroposterior regions, described further below.

Pelvic complex in Euphanerops longaevus In MHNM 01-123, a distinct anatomical gap is present between the posterior end of the paired anteroventral fins (which decrease in size posteriorly toward the gap) and the anterior end of the anal fin(s) (Fig. 1A). Within this gap, a pair of rounded, disc-like elements can be recognized, as well as a more distal pair of rod-like structures (Fig. 4A, B). The discs were first identified as ‘carbonaceous imprints’ (Janvier & Arsenault 2007). These elements are composed of calcified cartilage (Fig. 4C, D) and positioned between the anteroventral fins and anal fin(s), but also near the level where the basiventrals first occur in the axial skeleton (Fig. 3F, I, ha + hs). Because of their composition and their relative position, we suggest these discs represent the pelvic girdles (Fig. 4B–D). Paired, rod-like structures are associated with the distal margin of the pelvic girdles (Fig. 4B, D, int.org). These show large cytoplasmic chondrocyte vacuoles covered by a mineralized alveolar superficial layer (calcium phosphate; Fig. 4D) suggesting they are also endoskeletal. Previously, these elements were described as ‘diffused mineralized matter’ (Janvier & Arsenault 2007). The calcium phosphate signature suggests the presence of mineralization at least on the surface of these endoskeletal elements, such as in perichondral ossification or in calcified cartilage with calcium phosphate crystals deposited in the extracellular matrix (Janvier & Arsenault 2002, 2007). These 8.5-mm long (MHNM 01-123), rod-like structures show a constricted area (neck) at two-thirds of their length, with the distal end slightly curved anteriorly (Fig. 4B). These elements differ from generalized gnathostome pelvic fins (i.e. presence of multiple fin radials, rather than a pair of enlarged elements); as well, their shape is similar to that of the intromittent organ of arthrodiran placoderms (Ahlberg et al. 2009; Trinajstic et al. 2015). Given their shape, composition and position

relative to the pelvic girdles, we suggest these elements represent paired intromittent organs in Euphanerops. Intromittent organs are identified in two individuals and pelvic girdles in three specimens of Euphanerops (Fig. 1). In the smallest Euphanerops (90 mm total length; Figs 1B, 4C), only the pelvic discs are present, suggesting either that the intromittent organs form ontogenetically after the pelvic girdles, or that this specimen is a female. In a slightly larger specimen (96 mm total length, Figs 1C, 4D), a single intromittent organ and one pelvic disc are preserved. The presence of only one intromittent organ, rather than a pair, is probably a preservational artefact. In the largest specimen (distance between the anterior margin of the head to the insertion of anal fin (s) = 170.2 mm; Figs 1A, 4B) paired intromittent organs extend from the body between the paired anteroventral fins and anal fin(s).

Phylogenetic analysis The phylogenetic analysis of 43 early vertebrates resolves Euphanerops at a similar position to that found by a recent analysis (Sansom et al. 2010) (Fig. 5). Euphanerops is resolved as sister-group of Jamoytius, together in a monophyletic group of anaspids (i.e. Rhyncholepis, Pharyngolepis, Birkenia, Lasanius). Anaspids are resolved as the sister group of (lamprey + Ciderius). Cyclostomes are paraphyletic in our phylogenetic analysis which agrees with the results obtained in previous phylogenetic analyses based on anatomical characters (Gess et al. 2006; Sansom et al. 2010; Keating & Donoghue 2016). Osteostracans (i.e. Ateleaspis, Escuminaspis) are monophyletic and resolved as the sister-group of jawed vertebrates.

DISCUSSION Euphanerops possesses the best-preserved postcranial axial skeleton among fossil jawless vertebrates (MHNM 01-123, except for some disruption as noted above). The only other descriptions of fossil jawless axial skeletons include: (1) the presence of fibrous notochord sheath in conodonts (Aldridge et al. 1993); (2) the presence of a notochord in the enigmatic agnathan Gilpichthys greenei (Bardack & Richardson 1977) and in the lamprey Mesomyzon mengae (Chang et al. 2006, 2014); (3)

Euphanerops longaevus (Miguasha Lagerst€atte, Canada) pelvic complex. A, MHNM 01-123, nearly complete specimen. B, close-up of area outlined in A; pelvic discs and intromittent organs. C, MHNM 01-02A, pelvic discs and elemental analysis (representative spectrum). D, NHMUK P6813, pelvic disc and intromittent organs, and elemental analysis (representative spectrum). Chemical elements: Al, aluminum; C, carbon; Ca, calcium; F, fluorine; O, oxygen; P, phosphorus; Si, silicon. Anatomical abbreviations: an.fin ray, anal fin ray; int.org, intromittent organ; pelv.d, pelvic disc. Large arrows indicate anterior. All scale bars represent 3 mm. Colour online.

FIG. 4.


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Phylogenetic relationships among early vertebrates, 50% majority rule consensus tree based on 5 trees at 651 steps (43 taxa including jawed vertebrate species gathered into classes, 308 characters). Numbers on branches show percentage bootstrap support (100 iterations). Schematic representations of taxa are given in ventral view. Abbreviation: int.org is for intromittent organs. List of characters and coding matrix at Chevrinais et al. (2018). Colour online.

FIG. 5.

impressions of the anterior skeleton in heterostracans (Janvier 1993); (4) an indeterminate portion of vertebral skeleton in the osteostracan Ateleaspis (Ritchie 1967) and Escuminaspis laticeps (Belles-Isles (1989) contra Janvier et al. (2004)); and (5) ‘subunits of some form of axial skeleton’ in the anaspid-like Jamoytius (Sansom et al. 2010). In extant jawless vertebrates, the axial skeleton has been known for many years in lampreys (Marinelli & Strenger 1954), but only recently described in hagfish (Ota et al. 2014), comprising a notochord with simple dorsal arcualia in lampreys and ventral post-cloacal cartilaginous nodules along with median cartilaginous caudal bars dorsal and ventral to the notochord in hagfish (Ota et al. 2011). However, for the first time in jawless vertebrates, morphologically distinct regions can be recognized

within the axial skeleton in Euphanerops and Petromyzon. In Euphanerops, differentiation of the axial skeleton is represented by the restriction of the large basiventrals posteriorly (Fig. 3F), indicating an anteroposterior transition similar to that between the lumbar and sacral regions in tetrapods, but also recognized in the placoderm Holonema (Trinajstic 1999) and the fossil ray-finned fish Tarrasius (Sallan 2012) (Fig. 3I). This transition in Euphanerops is associated with the pelvic region including girdles and intromittent organs between the paired anteroventral and anal fin(s). By comparison, Petromyzon shows morphological differentiation anteriorly, involving larger, bifid dorsal arcualia with a foramen through the base and mediodorsal cartilages (Chevrinais et al. 2018, figs 2, 3). These are more complex and morphologically distinct with respect to the much simpler posterior dorsal arcualia, with a distinct point at the junction between the branchial and predorsal regions where these arcualia themselves change (Fig. 3A, E). Anterior regionalization of the vertebral column is well known in jawed vertebrates, including differentiation into a cervical region and fusion and modification of vertebral elements in the synarcual (Johanson et al. 2013) and Weberian apparatus (Bird & Hernandez 2007). The caudal fin of Petromyzon and of the hagfish Eptatretus burgeri also represent a discrete skeletal region, with the development of rods in Petromyzon and cartilaginous plates in Eptatretus dorsal and ventral to the notochord (Fig. 3B, D) (Ota et al. 2011). The presence of axial skeletal regionalization in lampreys and Euphanerops indicates that this differentiation evolved prior to the origin of jawed vertebrates. A series of paired anteroventral fins in Euphanerops, with proximal and distal supporting endoskeletal radials and rays, but lacking supporting girdles, extends anteriorly along the body flank beneath the branchial arches (elongate paired fins are also in this position in certain thelodonts (Wilson et al. 2007)). This series shows that in Euphanerops, developmental mechanisms involved in the formation of paired appendages are expressed along the body, including the anal fins if Sansom et al.’s (2013) interpretation is accepted. More posteriorly, the position of a pelvic region corresponds to the anatomical gap between the paired anteroventral fin series and the anal fin(s) (demarcated by the end of the digestive tract; Sansom et al. 2013), coupled with the differentiation and regionalization of the axial skeleton. The pelvic region is composed of two sets of morphologically distinct paired structures, representing small pelvic girdles and intromittent organs. There are some similarities to the anal fin rays (Fig. 1A), but the most anterior of these rays is clearly associated with the anteriormost anal fin radials, suggesting none had been displaced into the pelvic region. True pelvic fins of jawed vertebrates have more radials, so


alternatively, we interpret these elements as intromittent organs. Intromittent organs facilitate internal fertilization via transfer of sperm from males to females and their presence in vertebrates is currently dated to the Middle Devonian (c. 390 myr) (Long et al. 2009). While intromittent organs are absent in extant jawless fish (hagfishes and lampreys; Marinelli & Strenger 1954; Patzner 1998), they are undocumented (absent or not fossilized) in extinct jawless fishes (heterostracans, galeaspids, thelodonts, osteostracans). In extant jawed vertebrates, intromittent organs show a range of morphologies, including modifications of the pelvic fin metapterygium in chondrichthyans (claspers/mixopterygia) (O’Shaughnessy et al. 2015) and the anal fin lepidotrichia in some teleost fishes (gonopodium) (Turner 1941). Several placoderms, representing the basal phylogenetic nodes of the jawed vertebrate clade, also possess intromittent organs. In contrast to chondrichthyans, these appear to lack any association with the pelvic fins, instead articulating with the posterior trunk shield (Antiarchi) or being clearly separated posteriorly from the pelvic fin/girdle (Ptyctodontida, Arthrodira) (Long et al. 2009, 2015; Trinajstic et al. 2015). In Euphanerops, paired intromittent organs are preserved in close association with the pelvic girdles. It is difficult to determine whether the intromittent organs articulate to the pelvic girdles, but pelvic fins are absent. This suggests that the intromittent organs develop independently from the pelvic fins, as in placoderms, and that this represents the plesiomorphic condition for jawed vertebrates.

CONCLUSION The examined specimens of Euphanerops and Petromyzon show that regionalization is present in the axial skeleton, both anteriorly and posteriorly, with the position of the pelvic girdles and intromittent organs in Euphanerops appearing coincident with the anterior extent of the basiventrals (Fig. 5). The association of paired appendages with certain regions of the axial skeleton has long been appreciated (Burke et al. 1995; Nishimoto & Logan 2016). Although Petromyzon and Euphanerops are resolved to phylogenetically distant regions of the vertebrate cladogram, the presence of this association in Euphanerops, along with differentiation in the lamprey axial skeleton, and the presence of pelvic structures in Euphanerops provide evidence for complexity in the postcranial skeleton prior to the origin of jawed vertebrates. Putative absence of these features in several groups of the jawless vertebrate phylogeny may be due to lack of mineralization and preservation in these fossil groups.

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Authors contributions. The project was designed by MC and RC with material examined and described by CM, CBR, MC, RC and JL. The list of characters was defined by RC, MC, JL, KT, CBR and ZJ, the matrix coding and phylogenetic analysis were performed by MC, JL, KT, CBR and ZJ. Illustrations were made by MC and CM with input from RC, ZJ and KT. All authors contributed to data interpretation, figures and writing of the paper. Acknowledgements. We thank J. Kerr, O. Matton and F. Charest (MHNM) and E. Bernard (NHMUK) for access to collections. We are grateful to C. Belzile (UQAR) for SEM and EDX analyses and V. Roy (UQAR) for help with photographs. We are grateful to D. Potvin-Leduc (UQAR), H.-P. Schultze (U Kansas), P. Janvier (MNHN), M.V.H. Wilson (U Alberta) and O. Larouche (UQAR) for helpful discussions and comments. We are grateful to anonymous reviewers for their comments and suggestions on the first version of the paper.

DATA ARCHIVING STATEMENT Data for this study are available in the Dryad Digital Repository: https://doi.org/10.5061/dryad.1r634nj

Editor. Lionel Cavin

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