A Paleozoic Stem Hagfish Myxinikela Siroka — Revised Anatomy and Implications for Evolution of the Living Jawless Vertebrate Lineages

Canadian Journal of Zoology

A Paleozoic stem hagfish Myxinikela siroka — Revised anatomy and implications for evolution of the living jawless vertebrate lineages

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2020-0046.R1

Manuscript Type: Article

Date Submitted by the 24-Aug-2020 Author:

Complete List of Authors: Miyashita, Tetsuto; University of Chicago, Organismal Biology and Anatomy Is your manuscript invited for Draft consideration in a Special Zoological Endeavors Inspired by A. Richard Palmer Issue?:

Myxinoidea, cyclostomes, Mazon Creek, Francis Creek Shale, Keyword: Pennsylvanian, Carboniferous, soft tissue preservation

2 A Paleozoic stem hagfish Myxinikela siroka — Revised anatomy and implications

3 for evolution of the living jawless vertebrate lineages1

5 Tetsuto Miyashita*, †

7 *Canadian Museum of Nature, P.O. Box 3443, Station D, Ottawa, Ontario, Canada K1P 6P4

8 †Department of Organismal Biology and Anatomy, the University of Chicago. Chicago, IL 60637 USA

10 Email: [email protected] 11 Draft 12 Running title: Anatomy of a stem hagfish

14 1This article is one of a series of invited papers arising from the symposium “Zoological Endeavours 15 Inspired by A. Richard Palmer” that was co-sponsored by the Canadian Society of Zoologists and the 16 Canadian Journal of Zoology and held during the Annual Meeting of the Canadian Society of 17 Zoologists at the University of Windsor, Windsor, Ontario, 14–16 May 2019. 18

20 Abstract

22 Hagfishes and lampreys comprise cyclostomes, the earliest branching and sole surviving clade of the

23 once diverse assemblage of jawless crown-group vertebrates. Lacking mineralized skeletons, both of

24 the crown cyclostome lineages have notoriously poor fossil record. Particularly in the hagfish total

25 group, Myxinikela siroka Bardack, 1991 from the Late Carboniferous estuarine system of Illinois

26 represents the only definitive stem taxon. Previously known from a single specimen, Myxinikela has

27 been reconstructed as a short-bodied form with pigmented eyes but otherwise difficult to distinguish

28 from the living counterpart. With a new, second specimen of Myxinikela reported here, I re-evaluate the

29 soft tissue anatomy and formulate diagnosis for the taxon. Myxinikela has a number of general features 30 of cyclostomes, including cartilaginous branchialDraft baskets, separation between the esophageal and 31 branchial passages, and a well-differentiated midline finfold. In effect, these features give more

32 lamprey-like appearance to this stem hagfish than previously assumed. Myxinikela still has many traits

33 that set modern hagfishes apart from other vertebrates (e.g., nasohypophyseal aperture, large velar

34 cavity, and cardinal heart) and some intermediate conditions of modern hagfishes (e.g., incipient

35 posterior displacement of branchial region). Thus, Myxinikela provides an important calibration point

36 with which to date origins of these characters.

38 Keywords: Myxinoidea, cyclostomes, Mazon Creek, Francis Creek Shale, Pennsylvanian,

39 Carboniferous, soft tissue preservation, hagfishes, Myxinikela siroka

42 Introduction

44 When Carl Linnaeus described a hagfish as an “intestinal worm” (vermes intestina) (Linnaeus 1758;

45 Fänge 1998), he misclassified the animal but perhaps had a grasp of its strange morphology distinct

46 from any other living fish lineages. Boneless, sightless, and jawless, there is little to the external

47 morphology of hagfish that reveals its vertebrate affinity. The single nasohypophyseal canal has an

48 open aperture at the anterior end of the animal; the excurrent branchial ducts open in the mid-portion of

49 the trunk; and the flaccid skin covers a massive subcutaneous sinus (Marinelli and Strenger 1956;

50 Jørgensen et al. 1998). A hagfish appears even more puzzling internally, having anatomical traits such

51 as: a single semicircular canal; single midline choana; absence of the lateral wall of the braincase; an 52 enormous feeding apparatus occupying theDraft region that would otherwise house the branchial apparatus; 53 and kidneys consisting of segmentally organized glomeruli (Marinelli and Strenger 1956; Jørgensen et

54 al. 1998). The cladistic consensus now has hagfish firmly nested within vertebrates (Mallatt and

55 Sullivan 1998; Kuraku et al. 1999; Near 2009; Heimberg et al. 2010; Miyashita et al. 2019a), and the

56 comparative anatomical, embryological, and physiological research has offered explanations to these

57 peculiar traits (Ota and Kuratani 2006; Oisi et al. 2013b; Miyashita and Coates 2016; Kuratani et al.

58 2016; Higuchi et al. 2019). However, the evolutionary history of the distinct hagfish morphology

59 remains poorly documented or calibrated. This lack of understanding is due to the poor quality of

60 hagfish fossil record.

61 Myxinikela siroka Bardack, 1991 from the Pennsylvanian Francis Creek Shale of Illinois (the

62 Mazon Creek fauna) (Bardack 1991) was the sole fossil hagfish until the discovery of Tethymyxine, a

63 crown-group hagfish from the Cretaceous of Lebanon (Miyashita et al. 2019a). Myxinikela remains the

64 only stem hagfish with cladistic support from multiple datasets (Gabbott et al. 2016; Miyashita et al.

65 2019a). In the original description, the holotype and only specimen of Myxinikela was illustrated with

66 nasohypophsyeal barbels, paired eyes, otic capsules, branchial skeletons, and heart (Bardack 1991,

67 1998). With the exception of the pigmented eyes, the soft tissues identified in Myxinikela have been

68 questioned but conditionally accepted in subsequent analyses (Janvier 1996, 2007, 2008; Sansom et al.

69 2011; Janvier and Sansom 2016; Gabbott et al. 2016; Miyashita et al. 2019a). The apparent rarity of

70 Myxinikela in the Mazon Creek localities presented another challenge to interpreting the anatomy, as

71 no other specimen of the taxon has been found to date. The Mazon Creek fauna has yielded another

72 potential stem hagfish, Gilpichthys greenei Bardack & Richardson, 1977 (Bardack and Richardson

73 1977). The hagfish affinity of Gilpichthys has ambiguous cladistic support. It is typically precluded

74 from cladistic analyses because it can be coded for only a fraction of characters (22.6% in Miyashita et

75 al. 2019a). Gilpichthys was placed on the lamprey stem recently (Miyashita et al. 2019a), but the re- 76 analysis of a modified dataset found it on theDraft hagfish stem, nested immediately outside the clade 77 (Myxinikela + the crown group) (Miyashita et al. in review). Gilpicithys may represent a different

78 preservation mode of the same animal with Myxinikela, which, again, cannot be reliably tested without

79 additional specimens of Myxinikela.

80 In this paper, I report a second specimen of Myxinikela and re-evaluate previous reconstruction

81 of the Carboniferous stem hagfish. The newly identified specimen represents a different mode of

82 preservation in which the overall body proportion appears more slender and the visceral imprints are

83 more prominent than the skeletal remains. The original descriptions of the holotype (Bardack 1991,

84 1998) over-interpreted its hagfish-like morphology, and my revision renders Myxinikela less like a

85 modern hagfish than previously considered. Nevertheless, Myxinikela shows an intermediate stage

86 between the presumed ancestral cyclostome and modern hagfishes and provides calibrations for

87 important characteristics of the crown group Myxinoidea.

89 Institutional abbreviations

90 FMNH, Field Museum of Natural History, Chicago, USA; ROM, Royal Ontario Museum,

91 Toronto, Canada.

93 Systematic Palaeontology

95 Vertebrata Linnaeus, 1758

96 Cyclostomi Duméril, 1806

97 Myxinoidea Müller, 1834

99 Myxinikela siroka Bardack, 1991 100 (Figs. 1–4) Draft 101

102 HOLOTYPE: FMNH PF15373 (Figs. 1, 2). Body length = 73.3 mm.

103 PARATYPE: FMNH PF8472 (Figs. 3, 4). Body length = 62.5 mm.

104 HORIZON: Francis Creek Shale, Moscovian, Middle Pennsylvanian. Both specimens were collected

105 from the site formerly known as Pit 11, Peabody Coal Company, Will-Kankakee Counties,

106 Illinois (Bardack 1997; Hay and Krutty 1997; Ledvina 1997). The abandoned pit is now in the

107 property of Braidwood Generating Station. This locality yields members of the ‘Essex fauna’ —

108 a nearshore estuarine marine habitat in the Pennsylvanian deltaic system of Mazon Creek (Baird

109 et al. 1986; Baird 1997a,b).

110 DIAGNOSIS: The original description (Bardack 1991) did not formulate diagnosis for this taxon. In

111 this paper, Myxinikela siroka is diagnosed as a myxinoid with a unique combination of the

112 following characters: pigmented eyes (symplesiomorphy for myxinoids); eyes close to midline

113 (autapomorphy within myxinoids); eyes and otic capsules close to each other (autapomorphy

114 within myxinoids); branchial region in postotic position (synapomorphy for myxinoids) but not

115 widely separated from the rest of head by lingual apparatus (autapomorphy within myxinoids);

116 branchial region shorter than preoptic length (autapomorphy within myxinoids); branchial

117 basket delineating efferent branchial ducts (symplesiomorphy for myxinoids); extensive midline

118 finfold with a distinct dorsal peak (autapomorphy within myxinoids).

119 In addition to these diagnostic traits, M. siroka differs from all other cyclostomes from

120 Francis Creek Shale (Mazon Creek fauna) in many morphological characters, including: eyes as

121 large as otic capsules and close together on dorsal side (unique among the Mazon Creek

122 cyclostomes); tapering snout supported by nasal skeleton; absence of oral funnel (present in

123 Mayomyzon and Pipiscius); absence of comb-like feeding apparatus (present in Gilpichthys); 124 branchial arches all assuming postoticDraft position (anterior arches assuming infraotic position in 125 Mayomyzon and Pipiscius). As per its monotypic status, diagnosis for the genus follows that of

126 the type and only species, M. siroka.

127

128 Description

129

130 Preservation of soft tissues

131 FMNH PF15373 (Figs. 1, 2) and PF8472 (Figs. 3, 4) are preserved with different sets of structures.

132 Both specimens have the eyes, otic capsules, and visceral structures identified here as the heart,

133 gallbladder, and digestive tract (Table 1). FMNH PF8472 is preserved more three dimensionally than

134 FMNH PF15373. The former required additional preparation in the mid-trunk and tail to expose part of

135 the outline (Fig. 3), whereas the latter lies obliquely compressed at the plane of split. In FMNH

136 PF8472, preserved soft tissues are topographically distinct from the surrounding body enough to cast

137 shadow in low-angle light (Figs. 3, 4). For example, the otic capsules are represented in this specimen

138 as casts or moulds of the cavities lined with an organic-rich dark film (Fig. 4). FMNH PF8472 appears

139 more slender than FMNH PF15373, and shows no evidence for preservation of the midline fin.

140 However, the body outline is not fully exposed in this specimen. The caudal fin, if preserved, would be

141 under the matrix on one part (Fig. 3C). Thus, these differences might turn out to be an artifact of more

142 three-dimensional preservation in FMNH PF8472.

143 The most notable differences in preservation concern cartilages (better preserved in FMNH

144 PF15373) and internal cavities enclosed by a thick, highly vascularized epithelial sheet (more clearly

145 preserved in FMNH PF8472). FMNH PF15373 shows many cartilaginous structures in the head,

146 including the nasal skeleton, parachordal cartilage, orofacial skeleton (potential ‘tooth’ plates), and

147 branchial baskets (Figs. 1, 2). In contrast, no individual cartilages are observed in FMNH PF8472. 148 Instead, the preserved structures clearly delineatedDraft by distinct edges of mineral distributions correspond 149 to large cavities and sinuses that presumably enclosed a significant amount of putrefied matter as the

150 animal decayed: nasohypophyseal, velar, esophageal, and branchial spaces (Figs. 3, 4). The branchial

151 region is preserved as a dark organic stain in FMNH PF 15373, whereas in FMNH PF 8472 it is a patch

152 of infilling within the apparatus. White kaolinite precipitates dominate in the posterior part of the trunk

153 of FMNH PF8472, which are absent in FMNH PF15373. The gallbladder differs in position between

154 the two specimens. It sits in the mid trunk in FMNH PF8472, whereas it is just behind the heart in

155 FMNH PF15373. An amorphous structure anterior to the snout of FMNH PF8472 is likely a decay

156 feature, drainage squeezed out post-burial from the nasohypophyseal canal (Figs. 3, 4). A decay halo is

157 observed for FMNH PF15373 (Figs. 1, 2), but is not apparent in FMNH PF8472. These differences in

158 the set of preserved features between the two specimens suggest different modes of preservation. Thus,

159 the two specimens did not derive from a single taphonomic/decay series.

160

161 Body proportions

162 Overall, FMNH PF15373 and PF8472 are similar to each other in absolute sizes and body proportions.

163 FMNH PF8472 (body length = 62.5 mm) is slightly smaller than FMNH PF15373 (body length = 73.3

164 mm). The former appears substantially slender compared to the latter (maximum body diameter:

165 FMNH PF8472 = 6.2 mm; FMNH PF15373 = 11.0 mm), but this is partly due to preservation and

166 partly due to the fact that the body outline is not fully exposed in the seemingly slender FMNH

167 PF8472. The mid-trunk is the deepest part of the body (Fig. 1), and the body outline is discontinuous

168 precisely in this region for FMNH PF8472 (Fig. 3). Both specimens have a large head relative to body

169 length than modern hagfishes (preoptic head length relative to body length: FMNH PF8472 = 10.8%;

170 FMNH PF15373 = 9.2%) (Fig. 5A). Interestingly, Gilpichthys — another cyclostome from the Mazon

171 Creek fauna (Bardack and Richardson 1977) — has a similarly proportioned preoptic head with its 172 length approximately a tenth of body lengthDraft (Fig. 6A–C). These proportions show in converse that the 173 trunk is relatively short in Myxinikela and Gilpichthys. In modern hagfishes, the preoptic head length

174 typically falls between 2.5 and 4 % the body length, and the body outline of the Cretaceous hagfish

175 Tethymyxine suggests similar proportions in that taxon (Miyashita et al. 2019a).

176 Myxinikela departs markedly from modern hagfishes in having the branchial apparatus in more

177 anterior position. Reflecting this, the prebranchial length in Myxinikela is 12.8% (FMNH PF15373) and

178 16.5% (FMNH PF8472) the body length, respectively. The same metric trait typically falls from 20 to

179 30% the body length in modern hagfishes and is 13% in Tethymyxine, despite having much longer

180 bodies (Miyashita et al. 2019a). Thus, the branchial apparatus sits much closer to the rest of the head in

181 Myxinikela than in the crown-group taxa. Nevertheless, the branchial apparatus of Myxinikela is more

182 posterior in position in comparison with other vertebrates. The branchial apparatus assumes the

183 infraotic or immediately postotic position in other Mazon Creek cyclostomes (Bardack and Zangerl

184 1968; Bardack and Richardson 1977). The length of the branchial apparatus itself is 9.8% (FMNH

185 PF15373) and 10.5% (FMNH PF8472) the body length in Myxinikela, respectively. These branchial

186 proportions compare well with those in the crown group (10–15%) (Miyashita et al. 2019a) and in

187 other Mazon Creek cyclostomes (8–12%) (Bardack and Zangerl 1968; Bardack and Richardson 1977).

188

189 Cranial structures

190 SNOUT. The second specimen of Myxinikela (FMNH PF8472) prompts a close comparison with the

191 holotype (FMNH PF15373), and this helps reinterpreting many cranial structures identified in the

192 original descriptions (Table 1) (Bardack 1991, 1998). Initially, FMNH PF15373 was interpreted to

193 have the mouth and barbels at the anterior terminus of the head (Bardack 1991). In this description,

194 neither structure can be delineated clearly in FMNH PF15373 or FMNH PF8472. Generally,

195 cyclostomes have a subterminal mouth, and hagfishes have a slit-like mouth opening on the ventral side 196 of the snout. This anatomy appears inconsistentDraft with the seemingly terminal position of the mouth 197 reconstructed by Bardack (1991). Neither specimen preserves precise outlines of the terminal snout, so

198 there is no direct evidence for a mouth anywhere in this region. Aside from the mouth, Myxinikela has

199 a large terminal opening for the nasohypophyseal system. A mass of decay matter anterior to the snout

200 in FMNH PF8472 (Fig. 4) suggests a large opening to allow the leakage in the position where, in

201 modern hagfishes, the nasohypophyseal aperture opens with a connection to the pharynx (Strahan

202 1958; Hardisty 1979). The presence of such a large, terminal nasohypophyseal aperture implies a

203 mouth to be positioned subterminally.

204 A large nasohypophyseal aperture also implies the presence of peripheral sensory apparatus.

205 The crown-group hagfishes have four pairs of barbels in this region (two of them peripheral to the

206 aperture; the rest peripheral to the mouth) (Fig. 5A). Indeed, cartilaginous remains extend to terminal

207 positions in FMNH PF15373. At least two extremities are visible, one each for the nasohypophyseal

208 and general perioral position (Fig. 2). However, the outlines are ambiguous. Four pairs of barbels were

209 delineated in the original description of Myxinikela (Bardack 1991), but the proposed outlines cannot

210 be confirmed and likely are an over-interpretation. It is difficult to identify precisely to which barbels

211 of a modern hagfish the two extremities in FMNH PF13573 correspond. The nasohypophyseal barbels

212 are extensions of the tentacular and subnasal cartilages, whereas the perioral barbels are supported by

213 the tentacular and labial cartilages (Fig. 5B) (Miyashita 2012). All except the labial cartilage are

214 similarly decay-resistant (Sansom et al. 2011, 2013).

215 The cartilaginous remains in the snout of FMNH PF15373 are associated with either the

216 nasohypophyseal system or the oral cavity. The single midline rod in the anterior snout is

217 topographically consistent with the subnasal cartilage — a massive, decay-resistant element that forms

218 the nasohypophyseal floor in modern hagfishes (Fig. 5B). In modern hagfishes, the nasohypophyseal

219 canal is dorsally and laterally supported by a series of cartilaginous arches, and the canal leads

220 posteriorly to the olfactory chamber formed by a cartilaginous basket (Fig. 5B). FMNH PF15373 221 indicates cartilaginous structures correspondingDraft spatially with both, but individual elements cannot be 222 delineated in either specimen. Contrary to Bardack’s (1991) original reconstruction, it remains unclear

223 whether or not Myxinikela has specific cartilages homologous to those in the same region of a modern

224 hagfish (Table 1).

225 The preserved feature in the nasohypophyseal region of FMNH PF8472 is a narrow patch of

226 dark stain (Fig. 4). This is not equivalent to the nasohypophyseal skeleton of FMNH PF15373. Like the

227 decay matter pushed out from the aperture, it probably represents putrefied remains within the

228 nasohypophyseal canal (Fig. 5C). This interpretation is also consistent with the similar infillings in the

229 velar, branchial, and esophageal spaces described in the following sections.

230

231 SENSORY CAPSULES. Myxinikela has a pair of pigmented eyes close to the dorsal midline

232 (Figs. 1–4) (Gabbott et al. 2016). The eyes are elliptic, differing from the round eyes or eyespots of

233 other Mazon Creek cyclostomes. Relative to overall body size, the eyes of Myxinikela are smaller than

234 those of the Mazon Creek stem lampreys Mayomyzon pieckoensis (Bardack and Zangerl 1968) and

235 Pipiscius zangerli (Bardack and Richardson 1977), but larger than the pigmented eyespots of

236 Gilpichthys greenei (Bardack and Richardson 1977) (Fig. 6). In Myxinikela, the proximity between

237 eyes is consistent in both FMNH PF15373 and PF8472, which precludes this trait from a taphonomic

238 artifact. The paired skeletal rods underlying the eyes are the parachordal cartilages. The close

239 association between the eyes and the parachordal cartilages suggests that, unlike modern hagfishes but

240 similar to lampreys, the eyes and periocular tissues had a skeletal support. Immediately behind the eyes

241 and fused to the parachordal cartilages, the otic capsules are identified as a pair of dark stains in FMNH

242 PF15373 (Figs. 1, 2) and a pair of cavities (depressions or bulges) lined with dark film in FMNH

243 PF8472 (Fig. 4). In modern hagfishes, the eye and the otic capsule are widely separated from each

244 other, and the hyomandibular fenestra occupies a large area of the face below the sensory capsules (Fig.

245 5B) (Miyashita 2012). In contrast, this ocular-otic distance is short in Myxinikela, leaving little space 246 for — if present — the hyomandibular fenestra.Draft 247

248 ORAL AND FACIAL STRUCTURES. In FMNH PF15373, a skeletal mass is preserved

249 ventral to the nasohypophyseal region. As this element sits within or near the oral cavity, candidates

250 found in a modern hagfish chondrocranium include: keratinous tooth plates or ‘dental’ apparatus;

251 cornual process; palatal cartilage; nasopharyngeal plate; tentacular cartilage; and lingual apparatus (Fig.

252 5B). Its massive profile is inconsistent with rod-like elements such as cornual process, palatal cartilage,

253 or tentacular cartilage, and the limited anteroposterior profile precludes a lingual apparatus. It sits

254 below the subnasal cartilage, so it is unlikely to represent a nasopharyngeal plate, which is always more

255 posterior to the palatal commissure in modern hagfishes. The keratinous tooth plates remain as a

256 possibility. Indeed, keratin is often preserved in the Mazon Creek locality. However, no cusps were

257 identified, and the preserved tissue cannot be differentiated from other cartilaginous structures. Given

258 these uncertainties, this skeletal tissue may represent the keratinous tooth plates, or else a complex of

259 distinct chondrocranial elements in the orofacial region (Fig. 5B, C). FMNH PF8472 shows no trace of

260 the keratinous teeth, but has a dark stain closer to the tip of the snout (Figs. 3, 4). Topographically, it

261 appears close to a barbel-supporting skeleton. However, the area it occupies and the surface texture

262 seems more consistent with other decay infillings in the same specimen.

263 The dark patches surrounding the sensory capsules in FMNH PF8472 are clearly delineated but

264 have no morphological traits diagnostic to any specific structure, and probably represents a decay

265 feature. The amorphous patch behind the otic capsules might be interpreted as the notochordal sheath (a

266 transverse bridge between the parachordal cartilage to receive the anterior tip of the notochord).

267 However, the parachordal cartilages appear well separated from one another in FMNH PF15373, and

268 this is unusually posterior for the anterior end of the notochord. Instead, these patches correspond

269 topographically to the proximal part of the velum — an invaginated portion of the hyomandibular

270 pouch supported by cartilages and muscles to act as a pharyngeal pump and valve for ventilation (Fig. 271 5F–H) (Strahan 1958; Miyashita 2012). In Drafta modern hagfish, immediately lateral to the velum sits a 272 cardinal heart — a blood sinus draining into the anterior cardinal vein and the largest among the

273 ‘accessory hearts’ (Fig. 5G) (Cole 1926; Miyashita 2012, 2016). A cardinal heart, if present in

274 Myxinikela, would correspond topographically to the lower portion of the patch below the otic capsules

275 in FMNH PF8472 (Fig. 4). To support these interpretations, this specimen preserves large fluid-filled

276 cavities. This portion would also correspond to the visceral plate in modern hagfishes, but cartilages are

277 poorly preserved in FMNH PF8472. There is no trace of the visceral plate or notochordal sheath in

278 FMNH PF15373 either. Therefore, I tentatively interpret this circum-otic preservation in FMNH

279 PF8472 as decay matter trapped within the velar cavity and the cardinal heart.

280 Many skeletal elements characteristic to modern hagfishes appear not to have been preserved in

281 the specimens of Myxinikela. Hagfishes use the lingual apparatus to anchor the protractors and

282 retractors of the keratinous tooth plates, and the velar skeleton to pump water into the pharynx (Fig.

283 5B–H) (Strahan 1958; Yalden 1985; Miyashita 2012). In FMNH PF8472, a dark stain sits ventral to the

284 putative visceral plate (Fig. 4). This topographically corresponds to parts of the lingual apparatus based

285 on positions, but there is no further morphological information available to identify this element.

286 Modern hagfishes also have lateral and medial cartilaginous loops coming off the visceral plate, which

287 form a basket for the velum and suspend the lingual apparatus (Fig. 5B). Neither specimen is preserved

288 with any trace of the velar basket. These elements consist of soft cartilages that may be more prone to

289 decay than other hard cartilages (Cole 1905, 1909; Wright et al. 1998; Robson et al. 2000; Miyashita

290 2012). Alternatively, a modern hagfish might be a poor model with which to reconstruct Myxinikela. In

291 modern hagfishes, the velum extends posteriorly and the velar basket forms because the branchial

292 pouches migrate posteriorly during development (Neumayr 1938; Holmgren 1946; Oisi et al. 2013a, b,

293 2015). The branchial region is not far behind the rest of the head in Myxinikela. This configuration

294 precludes the crown-group condition of having a posteriorly developed velar region.

295 296 PHARYNX AND BRANCHIAL REGION.Draft Unlike modern hagfishes, Myxinikela has well- 297 developed cartilaginous baskets supporting the branchial apparatus. In FMNH PF15373, the left

298 branchial basket sits on an amorphous stain of the branchial pouches (Fig. 2). The basket has at least

299 seven — and likely more — branchial segments. The cartilage preserved posteriorly to the basket may

300 be part of the branchial apparatus. If this is the case, there is space enough for at least two and as many

301 as three additional branchial segments. The number of branchial pouches varies (4 to 14) among the

302 crown-group hagfishes, although the most have five to seven (Fernholm 1998; Janvier 2004; Martini

303 and Beulig 2013; Miyashita et al. 2019a).

304 In Myxinikela, each of these segments of the basket surrounds a fenestra, which presumably

305 transmitted the efferent ducts. The branchial arches form a complex latticework of cartilaginous bars

306 dorsally and ventrally to the fenestrae, as apparent from the processes and accessory foramina. This

307 overall morphology is reminiscent of the branchial baskets in lampreys and euphaneropid anaspids,

308 although that of Myxinikela is much smaller in both relative size and number of segments than in

309 anaspids (Fig. 5D; Johnels 1948; Marinelli and Strenger 1954; Bardack and Zangerl 1971; Hardisty

310 1981; Janvier et al. 2006; Janvier and Arsenault 2007). Unlike lampreys, however, there is no evidence

311 for fusion between the branchial baskets and the rest of the skull in FMNH PF15373. Neither does

312 FMNH PF15373 show any evidence for the pericardial cartilage that encloses the heart in lampreys. In

313 comparison, modern hagfishes lack branchial baskets and have small extrabranchial cartilages to

314 maintain open efferent ducts (Fig. 7) (Ayers and Jackson 1901; Miyashita 2012). Homology is

315 uncertain for these cartilaginous elements between Myxinikela and modern hagfishes.

316 With the dark, clearly delineated cartilages, the branchial basket appears differentially

317 preserved than the rest of the chondrocranium in FMNH PF15373. In the original descriptions

318 (Bardack 1991, 1998), these elements were considered ‘branchial vessels’ presumably because in

319 modern hagfishes the branchial skeleton is reduced to a small isolated cartilage attached to the wall of

320 an efferent branchial duct (Ayers and Jackson 1901; Miyashita 2012). Even the dorsal and lateral aortae 321 were identified (Bardack 1998). However, Draftthe general morphology and state of preservation for the 322 specimen contradicts Bardack’s (1998) assessment. The elements only occur in the small branchial

323 region. With the exception of the heart, no other vessels are preserved so clearly in FMNH PF15373.

324 Spatial relationships would be incompatible with respect to the esophageal portion of the digestive

325 tract.

326 Individual branchial pouches cannot be delineated in FMNH PF15373 or FMNH PF8472. I was

327 not able to confirm traces of the branchial epithelia illustrated by Bardack (1998), either. In FMNH

328 PF15373, the branchial region is organic-rich dark imprints topographically closely linked to the

329 baskets (Figs. 1, 2). In FMNH PF8472, the branchial baskets are not preserved. Instead, this specimen

330 has a long strip of clearly demarcated patch in similar position with (but morphologically unlike) that

331 of FMNH PF15373 (Fig. 4).

332 In both FMNH PF8472 and PF15373, the branchial passage appears separate from the main

333 digestive tract, as both specimens are preserved with the esophageal canal dorsal to the branchial

334 apparatus (Fig. 7). Unlike Myxinikela, modern hagfishes draw an afferent branchial duct directly from

335 the pharynx for each branchial pouch (Marinelli and Strenger 1956). This branchial-esophageal

336 separation occurs in the adult phase of lampreys (Fig. 5E) (Marinelli and Strenger 1954; Hardisty 1981)

337 and likely in osteostracans (Fig. 7) (Janvier 1981a, b, 1985, 1996).

338

339 Trunk structures

340 VISCERAL STRUCTURES. Both FMNH PF15373 and PF8472 have visceral imprints that represent

341 the heart, gallbladder, and intestine (Figs. 1, 3). The dark stain posterior to the branchial apparatus and

342 faint relative to the gallbladder was originally interpreted as a heart (Bardack 1991, 1998). Given its

343 position, and given the preservation of other prominent epithelial structures such as the branchial

344 apparatus, no other interpretation is more convincing as to the identity of this structure (Fig. 5A, I). The

345 heart is much more clearly delineated in FMNH PF8472 than in FMNH PF15373. The ‘heart’ in 346 FMNH PF8472 might be alternatively interpretedDraft as a part of the liver. As detailed in the following 347 paragraph, however, it is more consistent with the overall patterns of preservation to consider the liver

348 to not have fossilized than to have become partially preserved while the heart decayed completely. In a

349 decay experiment using modern hagfishes, preservation potentials are lower for the vascular and

350 circulatory structures than for other visceral tissues such as the liver, let alone for skeletal structures

351 (Sansom et al. 2011, 2013). Thus, neither specimen of Myxinikela seems to follow predictions of that

352 particular decay series precisely.

353 Unlike the elongate lobes of the liver in modern cyclostomes (Fig. 5A, I) (Marinelli and

354 Strenger 1954, 1956), the structure initially suspected to be a liver in Myxinikela (Bardack 1991, 1998)

355 is a single bulbous structure in both FMNH PF15373 and PF8472. A vertebrate liver is typically an

356 enormous organ with two distinct lobes, so the morphology in Myxinikela would be highly unusual.

357 Instead, the configuration is more reminiscent of a gallbladder. A gallbladder is prominently sized in

358 modern hagfishes (Fig. 5I) (Marinelli and Strenger 1956), although it is absent in lampreys (Marinelli

359 and Strenger 1954). The decay series of modern hagfishes (Sansom et al. 2011, 2013) reports nothing

360 about the gallbladder, but predicts high preservation potentials of the liver relative to other soft visceral

361 organs. If the bulbous structure in Myxinikela represents a gallbladder, it would imply chemical

362 preservation of bile in this otherwise hollow pouch. That would also indicate that the liver — which

363 would have been closely associated with the gallbladder — is not preserved in either specimen of

364 Myxinikela.

365 The intestine is only partly preserved as dark imprints (Figs. 1, 3). In FMNH PF15373, the faint

366 dark band appears to meet the ventral midline anteriorly to the caudal fin (Fig. 1). Bardack (1991,

367 1998) considered this area as representing the cloaca. This interpretation is likely correct, but would

368 also leave a disproportionately long postcloacal tail (27.0% the body length) for Myxinikela in

369 comparison to modern hagfishes (typically less than a tenth of body length).

370 371 MYOMERES. Myomeres are faintlyDraft visible in FMNH PF15373 behind the head (Fig. 2). 372

373 MIDLINE FIN. The midline fin is only visible in FMNH PF15373 (Fig. 1). The continuous

374 finfold develops over the posterior half of the dorsal midline, has a distinct dorsal peak, wraps around

375 the tail without forming a discrete lobe, and terminates along the ventral midline posterior to the

376 presumptive cloacal region. The fin is distinguished from the decay halo, which is marked by the sharp

377 outline of the body and by slight topographical differences (Fig. 2). In contrast, the fin sits at the same

378 topographical level with the body, and has a soft edge along the base where the carbon-rich soft tissue

379 imprints — while largely following the body outline — extend onto the finbase. There is no evidence

380 for preservation of any endoskeletal support. Bardack (1991, 1998) reconstructed the anterior extension

381 of the midline fin along the ventral outline as a ‘precloacal fin.’ In this description, the feature is

382 interpreted as a decay halo. The finfold likely did not extend anteriorly beyond the cloaca on the ventral

383 side. The midline finfold in Myxinikela is more extensive and complex than those in modern hagfishes,

384 which develop a simple caudal fin at approximately the posterior 10–20% of the body (Fig. 5J).

385 Myxinikela is similar to lampreys for having a distinct dorsal peak and marked dorsoventral

386 asymmetry, but differs from them in having a continuous finfold instead of developing discrete fins

387 (Fig. 5K).

388

389 Discussion

390

391 Myxinikela (Fig. 8A) shows a mosaic of anatomical traits shared with modern hagfishes, those

392 reminiscent of lampreys, and those unique among cyclostomes. The hagfish traits include: a terminal

393 nasohypophyseal aperture; skeletal structures supporting the nasohypophyseal canal (including

394 subnasal cartilage); first branchial arch positioned substantially more posterior than otic capsule; a

395 single large gallbladder; and the implied presence of barbels, massive velar cavity, and cardinal heart. 396 Myxinikela provides the hard-minimum dateDraft for these characters (Fig. 8B). 397 The following morphological characters in Myxinikela are more similar to lampreys than to

398 hagfishes: branchial basket; the separation between feeding passage (esophageal portion of pharynx)

399 and branchial passage; and midline finfold extending anteriorly on dorsal side and having a distinct

400 peak. In addition to these characters, Myxinikela has general features of the cyclostome crown:

401 anguilliform profile; (probable) keratinous tooth plates in oral cavity; and massive parachordal

402 cartilage. Except for the keratinous tooth plates (synapomorphy of the cyclostome crown), many of

403 these traits occur outside the crown group (branchial basket in Euphanerops; midline finfold

404 morphology in anaspids in general; body profile similar to euconodonts) (Janvier and Arsenault 2007;

405 Miyashita et al. 2019a). Therefore, these characters are likely symplesiomorphies of the cyclostome

406 crown (Figs. 7, 8B). The separation between feeding and respiratory passages or the presence of

407 parachrodal cartilage cannot be evaluated in the stem group (the osteostracan condition for the former

408 character discussed in the following section). As such, Myxinikela serves as a useful data point with

409 which to set polarities for these characters.

410 Finally, the characters unique to Myxinikela among cyclostomes include: eyes elliptic,

411 pigmented, small (approximately the size of otic capsules), and positioned dorsally and close to

412 midline; proximity between eyes and otic capsules; and branchial apparatus short (shorter than preoptic

413 head length) and not separated from the rest of the head by a lingual apparatus. Except about position

414 of the eyes, these traits fall in the middle of the continuum from a generalized vertebrate toward the

415 extreme morphology of modern hagfishes. Therefore, they are best interpreted to reflect the

416 ‘intermediate’ nature of Myxinikela in the evolution of hagfishes (Fig. 8).

417

418 Phylogenetic affinity and internal consistency of character reconstruction

419 My revision of Myxinikela leaves little ambiguity about the myxinoid affinity of the taxon, and is 420 consistent with the recent cladistic analysesDraft that placed Myxinikela on the myxinoid stem (Gabbott et 421 al. 2016; Miyashita et al. 2019a, in review) (Fig. 8B). Re-interpretation of the anatomy of Myxinikela

422 does not challenge any character coding in the most recent update to this taxon (Miyashita et al.

423 2019a). The tree presented here (Fig. 8B) reflects this consensus. This phylogenetic position — and the

424 set of morphological characters preserved in the two specimens — depicts Myxinikela as a curious

425 hybrid of hagfish- and lamprey-like features: a rather squat hagfish with branchial baskets, well-

426 differentiated midline fin, and squinting eyes of a lamprey (Fig. 8A).

427 The combination of characters as interpreted here might raise a question of internal consistency.

428 Myxinikela is hagfish-like in having a terminal nasohypophyseal aperture, velum, and posteriorly

429 placed branchial apparatus (Figs. 5, 7). These characters may appear inconsistent with the apparent

430 esophageal-branchial separation and branchial baskets present in the same animal, which are

431 reminiscent of modern adult lampreys. Here, I argue that they are not functionally incompatible with

432 each other. In hagfish, the nasohypophyseal canal is flow-through and the velum acts as both a pump

433 and a valve (Figs. 5C, F, 7). By contrast, the ventilation of an adult lamprey is generally considered a

434 specialization toward sucking for feeding and attachment. To have negative pressure within the oral

435 cavity, the branchial constriction provides high-volume pumping, and the velum acts solely as a valve

436 to close the branchial passage (Kawasaki and Rovainen 1988; Rovainen 1996). Thus, the branchial

437 passage needs to be separated for adult lampreys to generate strong suction force without creating flow

438 reversal (Rovainen and Schieber 1975; Hardisty 1979).

439 However, this functional prerequisite in lampreys does not exclude alternative forms of

440 ventilation with separate feeding and respiratory passages in other lineages. For example, osteostracans

441 (stem gnathostomes) have been consistently reconstructed with both a velum and separation between

442 the branchial and feeding passages, each supported by multiple lines of osteological correlates (Fig. 7)

443 (Janvier 1981, 1985a, 1985b, 1996; Miyashita 2016). Modern hagfishes have a substantial velum but

444 do not have a common afferent branchial passage separate from the esophageal pharynx (Fig. 5C). 445 Instead, each afferent branchial duct branchesDraft off the main pharyngeal passage individually (Fig. 5I). 446 This is likely a consequence of the posterior shift of the branchial apparatus because otherwise a

447 common branchial passage would be prohibitively long to maintain pressure for the respiratory flow.

448 Therefore, a pumping velum and a separate branchial passage are not mutually exclusive. Both are

449 correlates of the anatomical configuration for ventilation and feeding. These functional considerations

450 even suggest that the lamprey and osteostracan conditions may be more primitive in vertebrate

451 evolution than the pattern seen in modern hagfishes (Fig. 7).

452 Myxinikela may be most comparable to the osteostracan condition, except about using the

453 nasohypophyseal system for water intake and having a much smaller and posteriorly placed branchial

454 apparatus (Fig. 7). In this stem hagfish, the aperture implies the nasohypophyseal canal was flow-

455 through. Corollaries of this reconstruction are: a) the mouth was subterminal and closed as in hagfish;

456 and b) the feeding apparatus was probably fully eversible, consistent with the apparent absence of an

457 oral funnel or perioral keratinous teeth. The branchial basket implies pumping via constriction, but this

458 does not exclude the velum from providing a valve and an additional pumping device at the split of the

459 feeding and respiratory passages. Admittedly, this reconstruction is at odds with character

460 interpretation. In particular, the feeding passage and/or the velar cavity identified in the specimens of

461 Myxinikela may invite alternative interpretations, which will have different implications for the

462 morphological structures used for ventilation in this taxon.

463 Similarly, the oral feeding apparatus (‘orofacial skeleton’) identified in FMNH PF15373 (Figs.

464 1, 2) might appear inconsistent with an apparently small space available for a lingual apparatus — a

465 complex of protractors, retractors, and their skeletal bases to move the feeding apparatus in

466 cyclostomes (Fig. 5A). The lingual apparatus is either housed within the branchial basket in lampreys

467 or set in the space between the posteriorly displaced branchial region and the rest of the head in

468 hagfishes (Hardisty 1979, 1981; Hardisty and Rovainen 1982; Yalden 1985). Neither could be the case

469 for Myxinikela. The branchial basket only covers impressions of the branchial pouches, and the 470 branchial region is not posteriorly displacedDraft as much as in modern hagfishes. Therefore, the lingual 471 apparatus — which is assumed present in Myxinikela based on the presence of the orofacial skeleton, or

472 keratinous tooth plates — may have occupied the space below the branchial pouches and basket.

473 Indeed, there is an amorphous tissue stain in this area in both FMNH PF15373 and PF8472 (Figs. 1–4),

474 but its shape or composition remains unclear. Given all evidence available at this point, Myxinikela has

475 both the oral and lingual apparatti, with the latter occupying the hypobranchial position and being

476 comparatively small — and likely symplesiomorphic (retention of a primitive state) — with respect to

477 the hagfish crown. The presumed hypobranchial position may be the state from which the crown

478 hagfish and lamprey conditions were derived, but there is no other taxon with which to root this

479 condition except for the elusive euconodonts (Goudemand et al. 2011).

480 My reconstruction of barbels in Myxinikela may also appear somewhat inconsistent with the

481 presence of pigmented eyes in the same animal. The barbels in modern hagfishes are typically

482 considered sensory adaptations in the absence of image-forming vision (Ronan 1988; Andres and von

483 Düring 1993; von Düring and Andres 1998). However, conspicuous barbels and well-developed eyes

484 are not mutually exclusive, as many actinopterygian fishes have both. Thus, the presence of pigmented

485 eyes does not rule out that of barbels in Myxinikela.

486 Additionally, I interpreted that the ‘cheek’ infilling in FMNH PF8472 includes a cardinal heart,

487 implying that Myxinikela had a circulatory system similar to modern hagfishes. Modern hagfishes

488 represent a curious departure from the closed, generally high-pressure circulatory systems of

489 vertebrates (Farrell 1991). They have a massive subcutaneous sinus between the body and loose skin,

490 and the blood pressure is among the lowest known among living vertebrates (Johansen 1960; Hol and

491 Johansen 1960; Davie et al. 1987; Forster et al. 1991; Forster 1997; Lomholt and Franko-Dossar 1998).

492 They have accessory, pumping blood sinuses of which the cardinal heart is one (Greene 1900; Cole

493 1926; Forster 1997, 1998). Sitting between the base of the velar skeleton and the facial cartilage, the 494 cardinal heart drains into the anterior cardinalDraft vein as the velar skeleton flexes and extends to pump 495 water, thereby also contracting and relaxing the blood sinus (Cole 1926; Johansen 1960; Forster 1997).

496 Unfortunately, lines of inferences are limited in the specimens of Myxinikela. The cardinal heart in

497 FMNH PF8472 is identified on the basis of its position, size, and consistency with the preservation of

498 the heart and other large, fluid-filled cavities. Alternatively, the stain is entirely a velar cavity. But such

499 a large velum would still require a cardinal heart at its base, as it functions in an analogous manner to a

500 synovial joint (Strahan 1958; Miyashita 2016). Regardless of the precise identity of the cheek stain, the

501 peculiar circulatory anatomy of modern hagfishes likely predated the crown group and, at least in part,

502 has a deep evolutionary root in the Paleozoic times.

503

504 Taphonomic contexts and taxonomic comparison

505 Taphonomic insights are essential to consideration of morphological characters preserved and those not

506 preserved in Myxinikela (Purnell et al. 2018). In this respect, the two specimens (FMNH PF15373 and

507 PF8472) can complement each other with different sets of the morphological structures, thereby

508 representing two different taphonomic pathways (Table 1). Interestingly, the features preserved as

509 putrefied infilling in FMNH PF 8427 (oral, nasohypophyseal, velar, branchial spaces; cardinal heart)

510 have low preservation potentials as a tissue in a modern decay experiment (Sansom et al. 2011, 2013).

511 This is also true for a gut trace (Sansom et al. 2011, 2013), which is clearly preserved in both

512 specimens. Despite differential preservation of the soft tissues between the two specimens, there is little

513 ambiguity about their taxonomic identity. Many of the diagnostic features are common between the

514 two (elliptic pigmented eyes close to midline and otic capsules; branchial region entirely in postotic

515 position; tapering snout). These same features also exclude all other putative cyclostomes of the Mazon

516 Creek fauna.

517 Among them, Gilpichthys greenei (Bardack and Richardson 1977) (Fig. 6A–D) is considered a

518 hagfish on the basis of the paired, longitudinal feeding apparatus in the oral cavity (Janvier 1993, 1996, 519 2008). In addition, the potential ‘gonads’ reportedDraft in some of the specimens (Bardack and Richardson 520 1977; Miyashita et al. in review) are consistent with the condition observed among modern hagfishes,

521 which is characterized by small number, large relative size, and thick egg capsules (Conel 1933;

522 Gudger and Smith 1933; Martini 1998; Patzner 1998; Powell et al. 2005; Martini and Beulig 2013).

523 Gilpichthys is distinguished from Myxinikela for the small, pigmented eyespots, round anterior

524 terminus of head, and paired, longitudinal feeding apparatus in the oral cavity. These structures are

525 among the most resistant to decay (Sansom et al. 2011, 2013) and differ substantially from those of

526 Myxinikela in position, shape, and size. Myxinikela is rare in comparison to other Mazon Creek

527 cyclostomes, whereas the specimens of Gilpicithys massively outnumber those of Myxinikela in the

528 collections (60 specimens were examined by the author at FMNH and ROM). Therefore, it is unlikely

529 that the specimens of Gilpichthys represent different stages of the same decay series with either of the

530 specimens of Myxinikela.

531 Fossilization results in information loss (Purnell et al. 2018), but the loss is non-random and can

532 affect phylogenetic interpretations (Sansom et al. 2010; Sansom and Wills 2013, 2017; Sansom 2015).

533 For Myxinikela, an anticipated outcome of non-random decay is the ‘stem-ward slippage’ where

534 derived characters tend to be less resistant to decay, thus making the taxon appear more primitive

535 (Sansom et al. 2010; Sansom and Wills 2013). This particular problem likely does not apply to

536 Myxinikela for two unique biological conditions. First, the taxon is a lone stable branch in the hagfish

537 stem (Fig. 8B). It is set apart from the hagfish crown by a ghost lineage that extends more than 200

538 million years (Miyashita et al. 2019a). Where only topological relationships are concerned, the tree has

539 no other reasonable node from which Myxinikela could have slipped stem-ward. Second, the hagfish

540 crown group — the next node after Myxinikela — is characterized more by the loss of vertebrate

541 synapomorphies than by the acquisition of novel characters (Miyashita et al. 2019a). Therefore,

542 hagfishes seem to present an interesting exception to the general observation of stem-ward slippage.

543 With plesiomorphic conditions predominantly being presence — and apomorphic predominantly being 544 absence or reduction — taphonomic loss ofDraft character information does not directly pull the stem taxon 545 down the tree.

546 Consequently, Myxinikela might be predisposed to over-interpretation. Bardack (1991, 1998)

547 identified structures preserved in FMNH PF15373 closely after modern hagfishes. The branchial

548 structures considered as branchial baskets in this paper were posited as blood vessels, and the barbels

549 were fully reconstructed where the outline is unclear (Table 1). In this re-description, too, many

550 structures are postulated after modern hagfishes. The presence of barbels, nasohypophyseal aperture,

551 cardinal heart, velum, heart, and gallbladder in Myxinikela is all at odds with correspondence with the

552 anatomy of modern hagfishes (Fig. 5). For many of these structures, few lines of evidence exist except

553 anatomical consistency.

554 Conversely, a number of traits of modern hagfishes may have been present but not preserved in

555 Myxinikela. These include other accessory hearts (branchial, caudal), subcutaneous sinus, slime glands,

556 single semicircular canal, spatially restricted lateral line, and more. A decay experiment has shown that

557 the accessory hearts and slime glands have low preservation potentials, whereas decay process of other

558 traits remains undocumented (Sansom et al. 2011, 2013). Among these traits, the slime glands are

559 preserved as infilling of the glands — decomposed keratin fibers — in a fossil hagfish from the

560 Cretaceous of Lebanon, Tethymyxine (Miyashita et al. 2019a). Given the high preservation potentials of

561 keratin in fossils from the Mazon Creek localities, it is reasonable to assume that the slime glands were

562 absent in Myxnikela, at least in the form expected from modern hagfishes. Generally, however, the

563 absence of soft-tissue traits is difficult to test.

564 Aside from anatomical inferences, the habitat of Myxinikela siroka implies ecology of the taxon

565 quite unlike that of modern hagfishes. The two specimens of Myxinikela come from the nearshore

566 marine facies (‘Essex fauna’) of the Francis Creek Shale. Also home to other cyclostomes of Mazon

567 Creek (Gilpichthys, Mayomazon, Pipiscius; Fig. 6) and Tullimonstrum, this estuarine habitat

568 experienced fluctuating salinity and turbidity (Baird et al. 1986; Baird 1997a, 1997b; Sallan and Coates 569 2014). Tethymyxine from the Late CretaceousDraft of Lebanon is the other fossil hagfish and a branch 570 within the crown group. Its only specimen comes from a low-latitude shallow habitat, which is an inter-

571 reef basin on a carbonate platform (Miyashita et al. 2019a). In comparison, modern hagfishes are

572 exclusively marine, sensitive to salinity changes, and mostly confined to deep benthos (Martini 1998).

573 They occasionally and transiently migrate through shallow waters, but do not tolerate high-temperature

574 and low-salinity nearshore habitats (Martini 1998). As such, fossil hagfishes occupied habitats

575 unconventional to modern hagfishes. Like Tethymyxine, Myxinikela is rare in the extensively sampled

576 type locality (Baird and Anderson 1997). Thus, Myxinikela may have been a low-abundance permanent

577 resident, a seasonal migrant, or a transient member from peripheral habitats.

578

579 Conclusion

580

581 The discovery of a second specimen of Myxinikela siroka (FMNH PF8472) provided an opportunity to

582 revisit the original descriptions of this stem hagfish and recalibrate hard minima of morphological traits

583 that distinguish modern hagfishes. Bardack (1991, 1998) reconstructed Myxinikela essentially as a

584 morphologically unequivocal hagfish. In this re-description, I reinterpreted some of these traits in the

585 holotype FMNH PF15373 (Bardack’s ‘branchial vessels’ as branchial baskets; ‘liver’ as gallbladder;

586 outlines questioned for barbels, mouth, cloaca, and precloacal fin; nasal skeleton and feeding apparatus

587 now not delineated into individual elements; esophageal and intestinal portions of digestive tract

588 identified). FMNH PF8472 added new information on the cavities through their infillings

589 (nasohypophyseal, oral, velar, esophageal, and branchial space; cardinal heart) and three-dimensional

590 profile of the animal. FMNH PF8472 and PF15373 showed different modes of preservation. Cartilages

591 were taphonomically lost in FMNH PF8472, whereas the body was compressed and cavity infilling not

592 preserved in FMNH PF15373. Nevertheless, the characters preserved in common (sensory capsules, 593 branchial region, body outlines) clearly indicatedDraft their taxonomic identity, and distinction from any 594 other cyclostomes of the Mazon Creek fauna. Based on these insights, I presented an alternative

595 reconstruction of Myxinikela as a stem myxinoid that retains general features of cyclostomes, which

596 were lost among more derived hagfishes (e.g., pigmented eyes, cartilaginous branchial basket).

597 A suite of characters present in Myxinikela showed that many distinctive features of modern

598 hagfishes long preceded the origin of the crown group. These included structures for ventilation and

599 feeding: nasohypophyseal system and its skeletal support, incipient posterior displacement of branchial

600 region, massive velum, and cardinal heart. Myxinikela also retained many morphological characters

601 that now appear to be symplesiomorphic and synapomorphic conditions of the cyclostome crown.

602 These traits again included anatomical correlates of ventilation and feeding, such as the separation of

603 feeding and respiratory passages and feeding apparatus housed within an oral cavity. With the

604 pigmented eyes, branchial region not decoupled from the head, and extensive midline finfold,

605 Myxinikela indicated that transitions of these symplesiomorphic characters toward the crown group

606 condition occurred later than its divergence from the hagfish stem. Therefore, character changes in the

607 sensory structures and fin likely followed ventilation and feeding adaptations in the evolution of

608 hagfishes.

609

610 Acknowledgments

611

612 As if to climb a tree to seek a fish…

613 Mencius, Liang Hui Wang (volume 1).

614

615 This ancient idiom — which could be interpreted in the context of systematic research on fishes — is

616 an analogy for a wrong method to achieve a desired outcome. The irony for this paper aside, Mencius is 617 here attempting to persuade King Liang Hui,Draft who was waging a conquest war, to win the hearts of the 618 people with benevolence instead. With this quote I reflect on the teaching of Dr. A. Richard Palmer.

619 Rich is a benevolent and patient mentor, and a role model who pushed his students to be curious, brave,

620 and maybe a little obsessed. He is a staunch guardian of scientific integrity, and has a keen sense with

621 which to detect both clever setups and potential flaws in experimental and analytical designs. He also

622 has an inexhaustible supply of curiosity for all things “cool” and a streak of playfulness — or perhaps

623 mischievousness — with which he could catch others completely off-guard. In addition to all, the leap

624 of analogy seen in the quote, and in the Menciusian school in general, shows a broad vision and an

625 ability to connect disparate observations, both Rich’s quintessential traits as an evolutionary biologist.

626 Rich changed the course of my professional career. A second-year undergrad and a

627 stereotypical dinosaur fan boy who never grew up, my eyes were literally wide open as he walked his

628 Survey of Invertebrates class through sponges that “sneeze,” echinoderm larvae that clone themselves,

629 and flatworms that engage in penis fencing. I followed him to Bamfield Marine Sciences Centre in the

630 following summer, and again, and ended up spending four field seasons in the west coast station. This

631 experience solidified the direction of my thesis research. Naively, I picked hagfish. I was intrigued by

632 all their peculiarities, but they also seemed to fall somewhere in the middle of my interests —

633 vertebrates (dinosaurs) and invertebrates (all the other interesting critters). In the decade that followed,

634 Rich guided me patiently as I became a more broadly trained evolutionary biologist. It is my great

635 pleasure to return to one of my academic roots for this Festschrift and dedicate this paper in his honour.

636 I thank W. Simpson (FMNH) and K. Seymour (ROM) for access to the collections. I examined

637 FMNH PF15373 first when the specimen was on loan to S. Gabbott and M. Purnell (University of

638 Leicester). I thank them both for hosting my visit and having discussion on Myxinikela. I acknowledge

639 discussion on cyclostomes in anatomical, systematic, and even cultural details with M. Coates

640 (University of Chicago) and his lab (A. Caron, B. Otoo, S. Sang, K. Tietjen, and V. Venkat). 641 Comments from an anonymous referee improvedDraft this manuscript. This work was supported by the 642 University of Chicago Fellow Program, Vanier Canada Graduate Scholarship, and I.W. Killam

643 Scholarship.

644

645 References

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872

873 Figure Captions

874

875 Fig. 1. Holotype of Myxinikela siroka Bardack, 1991 (FMNH PF15373), a stem myxinoid from the

876 Moscovian (Middle Pennsylvanian) Mazon Creek fauna of Illinois. (A, B) Part in photograph (A) and

877 interpretive drawing (B). (C, D) Counterpart in photograph (C) and interpretive drawing (D). Grey

878 shade indicates tissue remains; solid line delineates changes in mineral compositions; and stippling

879 shows specific tissues or their outlines.

880

881 Fig. 2. Detailed anatomy of the head region in the holotype of Myxinikela siroka Bardack, 1991

882 (FMNH PF15373). (A, B) Part in photograph (A) and interpretive drawing (B). (C, D) Counterpart in 883 photograph (C) and interpretive drawing (D).Draft Grey shade indicates tissue remains; solid line delineates 884 changes in mineral compositions; and stippling shows specific tissues or their outlines. Not to scale.

885

886 Fig. 3. Paratype of Myxinikela siroka Bardack, 1991 (FMNH PF8472) from the same locality. Unlike

887 holotype, FMNH PF8472 is preserved with putrefied remains trapped within cavities but without any

888 definitive cartilaginous structure. The specimen is more three-dimensional than the holotype. (A, B)

889 Part in photograph (A) and interpretive drawing (B). (C, D) Counterpart in photograph (C) and

890 interpretive drawing (D). Grey shade indicates tissue remains; solid line delineates changes in mineral

891 compositions; and stippling shows specific tissues or their outlines.

892

893 Fig. 4. Detailed anatomy of the head region in the paratype of Myxinikela siroka Bardack, 1991

894 (FMNH PF8472). (A, B) Part in photographs in different light angles, showing mode of preservation.

895 The high light angle (A) reveals no internal structures to the oral, velar, and branchial areas unlike in

896 the holotype, indicating chemical preservation of cavity infillings. The low light angle (B) shows three-

897 dimensional preservation of the specimen through shades cast by cavity infilings, otic capsules, and

898 outline of the animal. (C, D) Part in photographs in different focal depths to capture three-dimensional

899 preservation of the head. Focus on the snout (C) reveals details of the oral and nasohypophyseal areas,

900 whereas focus on the cheek (D) shows sensory capsules overlapping the velar cavity and its peripheral

901 structures. Panels A+B and C+D are not to scale against one another.

902

903 Fig. 5. Anatomy of living cyclostomes — the hagfishes (Myxine glutinosa Linnaeus, 1758) (A–C, F)

904 and Eptatretus stoutii Lockington, 1878 (G, H), and the lamprey (Lampetra fluviatilis Linnaeus, 1758)

905 (D, E, K) — as comparative models for Myxinikela. Structures considered present in Myxinikela are

906 shaded in red.

907 (A) Myxine glutinosa Linnaeus, 1758, dissected, in left lateral view, showing the barbels, 908 lingual apparatus, posteriorly displaced branchialDraft region, heart, and visceral structures. Proportions and 909 positions of these structures differ in Myxinikela.

910 (B–E) Comparison of chondrocrania (B, D) and sagittal sections (C, E) of the heads between

911 Myxine (B, C) and Lampetra (D, E). In the snout and face, the chondrocranial elements preserved in

912 Myxinikela have their counterparts in modern hagfishes (B). Myxinikela also has parachordal cartilage

913 (structure in common with hagfishes and lampreys) and branchial basket (present in lampreys but

914 absent in modern hagfishes) (D). Myxinikela has evidence for the hagfish-like nasohypophyseal canal

915 and velum (C), and for the lamprey-like separation between the feeding and respiratory passages (E).

916 (F–H) Anatomy and function of the velum and its peripheral structures in modern hagfishes.

917 (F) Schematics of the velum in maximum flexion in Myxine. (G, H) Transverse sections of Eptatretus,

918 showing the velar cavity, velar skeleton, cardinal heart, and peripheral structures. The cardinal heart

919 sits between the velar skeleton and the main chondrocranium, and acts as a functional analogue of a

920 synovial joint as the velar movement pumps blood draining into the anterior cardinal vein (Miyashita

921 2016).

922 (I) Dissection of the visceral organs in a female of Myxine, showing organization, spatial

923 relationships, and proportions of the organs. Note that the gallbladder is a single large organ posterior

924 to the heart, whereas the liver consists of two separate lobes. (J, K) Comparison of caudal anatomy

925 between Myxine (J) and Lampetra (K). Myxinikela has a well-differentiated midline finfold similar to

926 lampreys than to hagfishes. A, C, I, J: modified after Marinelli and Strenger (1956). B, D: modified

927 after Miyashita et al. (2019b). E, K: modified after Marinelli and Strenger (1954). F: modified after

928 Strahan (1958). G, H: adapted from Miyashita (2012). Not to scale.

929

930 Fig. 6. Cyclostomes from the Mazon Creek fauna for comparison with Myxinikela: (A–D) enigmatic

931 taxon Gilpichthys greenei Bardack & Richardson, 1977, which is potentially a stem myxinoid; (E, F) 932 putative petromyzontiform Pipiscius zangerliDraft Bardack & Richardson, 1977; (G) stem 933 petromyzontiform Mayomyzon piecoensis Bardack and Zangerl, 1968. (A) FMNH PF8411 shows the

934 commonly identified soft tissues in well-preserved specimens of Gilpicithys (eyespot, feeding

935 apparatus, intestine, myomeres). (B) FMNH PF8348 is preserved with multiple cranial structures that

936 are not commonly found in specimens of Gilpichthys (otic capsules, snout and branchial skeletons). (C)

937 FMNH PF8464 has eleven circular structures organized longitudinally (arrowheads), which are

938 interpreted as gonads (Bardack and Richardson 1977) or eggs (Miyashita et al. in review; this paper).

939 (D) ROM 56861 is a head region preserved with the sensory capsules and branchial skeleton. (E)

940 FMNH PF16082 is interpreted as a yolk-sac carrying hatchling (Miyashita et al. in review), showing

941 many anatomical details such as the sensory capsules and branchial basket. (F) FMNH PF8346 is rare

942 among large specimens of Pipiscius for being preserved in lateral compression, whereas most adults of

943 this taxon are preserved in dorsoventral compression. (G) ROM 56806 is preserved with many soft

944 tissue structures shared in common between Mayomyzon and other stem/crown lampreys, such as the

945 separation between branchial and esophageal portions of the pharynx. All scale bars equal 5 mm.

946

947 Fig. 7. Comparison of oropharyngeal anatomy among jawless vertebrate lineages. At the crown

948 vertebrate node, the feeding and respiratory passages were likely separate, and the velum probably

949 functioned as a pump. Schematic drawings (not to scale) are presented with a table comparing character

950 states. See main text for discussion of character optimization for each branch. Color codes: light cyan =

951 oropharyngeal cavity; blue = branchial pouches; purple = nasohypophyseal canal; light brown = body;

952 dark brown = velum; pink = cartilages.

953

954 Fig. 8. Myxinikela is a stem myxinoid that retains general features of cyclostomes. Using Myxinikela as

955 a calibration point for character evolution within cyclostomes, modifications to the anatomical

956 correlates of feeding and ventilation occurred first in the myxinoid total group, followed by the sensory 957 structures, physiological adaptations (e.g., Draftslime), and elaboration of the feeding and ventilation 958 correlates toward the myxinoid crown. (A) Reconstruction of Myxinikela, showing the mosaic of

959 features seen in modern hagfishes and lampreys (illustration by the author). (B) A consensus summary

960 tree of the cyclostome crown, accompanied by chronological occurrences of the likely outgroups (stem

961 cyclostomes) anaspids and euconodonts. Position of Gilpichthys remains uncertain. Topology and node

962 age estimates follows Miyashita et al. (2019a, in review). Stem-ward arrowhead indicates potential

963 plesiomorphies, whereas crown-ward arrowhead represents potential apomorphies. For each, character

964 states are listed under DELTRAN (delayed transformation) optimization for maximum parsimony.

965 Annotations: *These characters have low preservation potentials and potentially have earlier origins

966 and/or broader distributions. † Velar sinus seen in ammocoete larvae of modern lampreys may be a

967 homologue of the cardinal heart in hagfishes. If this is the case, the cardinal heart may have a deeper

968 evolutionary origin, at least to the crown cyclostome node.

969

Draft

Fig. 1. Holotype of Myxinikela siroka (FMNH PF15373), a stem myxinoid from the Moscovian (Middle Pennsylvanian) Mazon Creek fauna of Illinois. (A, B) Part in photograph (A) and interpretive drawing (B). (C, D) Counterpart in photograph (C) and interpretive drawing (D). Grey shade indicates tissue remains; solid line delineates changes in mineral compositions; and stippling shows specific tissues or their outlines.

180x164mm (300 x 300 DPI)

Draft

Fig. 2. Detailed anatomy of the head region in the holotype of Myxinikela siroka (FMNH PF15373). (A, B) Part in photograph (A) and interpretive drawing (B). (C, D) Counterpart in photograph (C) and interpretive drawing (D). Grey shade indicates tissue remains; solid line delineates changes in mineral compositions; and stippling shows specific tissues or their outlines. Not to scale.

86x203mm (300 x 300 DPI)

Draft

Fig. 3. Paratype of Myxinikela siroka (FMNH PF8472) from the same locality. Unlike holotype, FMNH PF8472 is preserved with putrefied remains trapped within cavities but without any definitive cartilaginous structure. The specimen is more three-dimensional than the holotype. (A, B) Part in photograph (A) and interpretive drawing (B). (C, D) Counterpart in photograph (C) and interpretive drawing (D). Grey shade indicates tissue remains; solid line delineates changes in mineral compositions; and stippling shows specific tissues or their outlines.

180x214mm (300 x 300 DPI)

Draft

Fig. 4. Detailed anatomy of the head region in the paratype of Myxinikela siroka (FMNH PF8472). (A, B) Part in photographs in different light angles, showing mode of preservation. The high light angle (A) reveals no internal structures to the oral, velar, and branchial areas unlike in the holotype, indicating chemical preservation of cavity infillings. The low light angle (B) shows three-dimensional preservation of the specimen through shades cast by cavity infilings, otic capsules, and outline of the animal. (C, D) Part in photographs in different focal depths to capture three-dimensional preservation of the head. Focus on the snout (C) reveals details of the oral and nasohypophyseal areas, whereas focus on the cheek (D) shows sensory capsules overlapping the velar cavity and its peripheral structures. Panels A+B and C+D are not to scale against one another.

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Fig. 5. Anatomy of living cyclostomes — the hagfishes (Myxine glutinosa) (A–C, F) and Eptatretus stoutii (G, H), and the lamprey (Lampetra fluviatilis) (D, E, K) — as comparative models for Myxinikela. Structures considered present in Myxinikela are shaded in red. (A) Myxine glutinosa, dissected, in left lateral view, showing the barbels, lingual apparatus, posteriorly displaced branchial region, heart, and visceral structures. Proportions and positions of these structures differ in Myxinikela. (B–E) Comparison of chondrocrania (B, D) and sagittal sections (C, E) of the heads between Myxine (B, C) and Lampetra (D, E). In the snout and face, the chondrocranial elements preserved in Myxinikela have their counterparts in modern hagfishes (B). Myxinikela also has parachordal cartilage (structure in common with hagfishes and lampreys) and branchial basket (present in lampreys but absent in modern hagfishes) (D). Myxinikela has evidence for the hagfish-like nasohypophyseal canal and velum (C), and for the lamprey-like separation between the feeding and respiratory passages (E). (F–H) Anatomy and function of the velum and its peripheral structures in modern hagfishes. (F) Schematics of the velum in maximum flexion in Myxine. (G, H) Transverse sections of Eptatretus, showing the velar cavity, velar skeleton, cardinal heart, and peripheral structures. The cardinal heart sits between the velar skeleton and the main chondrocranium, and acts as a functional analogue of a synovial joint as the velar movement pumps blood draining into the anterior cardinal vein (Miyashita 2016). (I) Dissection of the visceral organs in a female of Myxine, showing organization, spatial relationships, and proportions of the organs. Note that the gallbladder is a single large organ posterior to the heart, whereas the liver consists of two separate lobes. (J, K) Comparison of caudal anatomy between Myxine (J) and Lampetra (K). Myxinikela has a well-differentiated midline finfold similar to lampreys than to hagfishes. A, C, I, J: modified after Marinelli and Strenger (1956). B, D: modified after Miyashita et al. (2019b). E, K: modified after Marinelli and Strenger (1954). F: modified after Strahan (1958). G, H: adapted from Miyashita (2012). Not to scale.

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Fig. 6. Cyclostomes from the Mazon Creek fauna for comparison with Myxinikela: (A–D) enigmatic taxon Gilpichthys greenei, which is potentially a stem myxinoid; (E, F) putative petromyzontiform Pipiscius zangerli; (G) stem petromyzontiform Mayomyzon piecoensis. (A) FMNH PF8411 shows the commonly identified soft tissues in well-preserved specimens of Gilpicithys (eyespot, feeding apparatus, intestine, myomeres). (B) FMNH PF8348 is preserved with multiple cranial structures that are not commonly found in specimens of Gilpichthys (otic capsules, snout and branchial skeletons). (C) FMNH PF8464 has eleven circular structures organized longitudinally (arrowheads), which are interpreted as gonads (Bardack and Richardson 1977) or eggs (Miyashita et al. in review; this paper). (D) ROM 56861 is a head region preserved with the sensory capsules and branchial skeleton. (E) FMNH PF16082 is interpreted as a yolk-sac carrying hatchling (Miyashita et al. in review), showing many anatomical details such as the sensory capsules and branchial basket. (F) FMNH PF8346 is rare among large specimens of Pipiscius for being preserved in lateral compression, whereas most adults of this taxon are preserved in dorsoventral compression. (G) ROM 56806 is preserved with many soft tissue structures shared in common between Mayomyzon and other stem/crown lampreys, such as the separation between branchial and esophageal portions of the pharynx. All scale bars equal 5 mm.

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Fig. 7. Comparison of oropharyngeal anatomy among jawless vertebrate lineages. At the crown vertebrate node, the feeding and respiratory passages were likely separate, and the velum probably functioned as a pump. Schematic drawings (not to scale) are presented with a table comparing character states. See main text for discussion of character optimization for each branch. Color codes: light cyan = oropharyngeal cavity; blue = branchial pouches; purple = nasohypophyseal canal; light brown = body; dark brown = velum; pink = cartilages.

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Fig. 8. Myxinikela is a stem myxinoid that retains general features of cyclostomes. Using Myxinikela as a calibration point for character evolution within cyclostomes, modifications to the anatomical correlates of feeding and ventilation occurred first in the myxinoid total group, followed by the sensory structures, physiological adaptations (e.g., slime), and elaboration of the feeding and ventilation correlates toward the myxinoid crown. (A) Reconstruction of Myxinikela, showing the mosaic of features seen in modern hagfishes and lampreys (illustration by the author). (B) A consensus summary tree of the cyclostome crown, accompanied by chronological occurrencesDraft of the likely outgroups (stem cyclostomes) anaspids and euconodonts. Position of Gilpichthys remains uncertain. Topology and node age estimates follows Miyashita et al. (2019a, in review). Stem-ward arrowhead indicates potential plesiomorphies, whereas crown-ward arrowhead represents potential apomorphies. For each, character states are listed under DELTRAN (delayed transformation) optimization for maximum parsimony. Annotations: *These characters have low preservation potentials and potentially have earlier origins and/or broader distributions. † Velar sinus seen in ammocoete larvae of modern lampreys may be a homologue of the cardinal heart in hagfishes. If this is the case, the cardinal heart may have a deeper evolutionary origin, at least to the crown cyclostome node.

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Table

Table 1. Comparison of interpretations on the soft tissues preserved in the specimens of Myxinikela

siroka. Grey shade indicates a group of elements interpreted collectively in either Bardack (1991, 1998)

or this study. Interpretations proposed in this paper are highlighted in bold typeface. Asterisk (*)

denotes structures identified on the basis of putrefied remains either trapped within a cavity or pushed

out from an opening.

FMNH PF15373 FMNH PF8472 Bardack (1991, 1998) DraftThis study This study Mouth — (Oral cavity?)*

Perioral barbels (Partially present?) —

Nasohypophyseal barbels (Partially present?) —

Subnasal cartilage Subnasal cartilage —

Nasohypophyseal aperture Nasohypophyseal canal and canal*

Nasal arches — “Nasal skeleton” Nasal capsule basket —

Palatal cartilage —

Eyes Eyes Eyes

Otic capsules Otic capsules Otic capsules

— Parachordal cartilage —

Keratinous tooth plates — “Orofacial skeleton”* Anterior lingual cartilages —

FMNH PF15373 FMNH PF8472

Bardack (1991, 1998) This study This study

Velar space and cardinal — — heart*

Branchial blood vessels Branchial baskets —

Branchial pouches* Branchial pouches*

Branchial pouches Digestive tract (esophageal Digestive tract (esophageal portion) portion)*

Heart Heart Heart

Digestive organ (liver) Gallbladder Gallbladder

— Intestine Intestine

— Myomeres —

Precloacal median fin — Draft —

Cloaca — —

Median fin Median fin —