Gross anatomy of the Pacific hagfish, Eptatretus burgeri, with special reference to the coelomic viscera

Hagfish (Myxinoidea) are a deep‐sea taxon of cyclostomes, the extant jawless vertebrates. Many researchers have examined the anatomy and embryology of hagfish to shed light on the early evolution of vertebrates; however, the diversity within hagfish is often overlooked. Hagfish have three lineages, Myxininae, Eptatretinae, and Rubicundinae. Usually, textbook illustrations of hagfish anatomy reflect the morphology of the Myxininae lineage, especially Myxine glutinosa, with its single pair of external branchial pores. Here, we instead report the gross anatomy of an Eptatretinae, Eptatretus burgeri, which has six pairs of branchial pores, especially focusing on the coelomic organs. Dissections were performed on fixed and unfixed specimens to provide a guide for those doing organ‐ or tissue‐specific molecular experiments. Our dissections revealed that the ventral aorta is Y‐branched in E. burgeri, which differs from the unbranched morphology of Myxine. Otherwise, there were no differences in the morphology of the lingual apparatus or heart in the pharyngeal domain. The thyroid follicles were scattered around the ventral aorta, as has been reported for adult lampreys. The hepatobiliary system more closely resembled those of jawed vertebrates than those of adult lampreys, with the liver having two lobes and a bile duct connecting the gallbladder to each lobe. Overall, the visceral morphology of E. burgeri does not differ significantly from that of the known Myxine at the level of gross anatomy, although the branchial morphology is phylogenetically ancestral compared to Myxine.


| INTRODUCTION
One key to understanding the early evolution of vertebrates is to examine the morphology of our jawless sister group, the cyclostomes.This group and the gnathostomes (jawed vertebrate lineage) diverged about 500 million years ago (Kuraku & Kuratani, 2006;Benton et al., 2015).The cyclostome lineage has two subgroups: the lampreys (Petromyzontiformes) and hagfishes (Myxinoidea).Historically, these taxa have sometimes been considered paraphyletic, but today their monophyly is supported by molecular phylogeny (Heimberg et al., 2010;Kuraku, 2008;Kuraku et al., 1999;Mallatt & Sullivan, 1998;Takezaki et al., 2003) and comparative morphology (Oisi et al., 2013;Ota et al., 2007;Yalden, 1985).The absence of many characteristics of jawed vertebrates (e.g., jaws, paired nostrils, limbs, cucullaris muscles, and vertebral centra) makes cyclostomes essential organisms for studying vertebrate evolution (Janvier, 1996;Ota et al., 2011).In addition, they have many cyclostome-specific features (e.g., lingual apparatus, velum, and mucous cartilage) not found in jawed vertebrates (Janvier, 1996;Oisi et al., 2013), and whether these features are inherited from the common ancestors of vertebrates or are synapomorphies of cyclostomes remains controversial (Yokoyama et al., 2021; also see Sugahara, 2021).The morphology of the hagfishes is regarded to be highly derived, including the degeneration of eyes because of adaptation to the deep sea (Dong & Allison, 2021;Gabbott et al., 2016), and many studies have instead used lampreys as a model for cyclostomes.However, recent studies have suggested that some lamprey characteristics that had been regarded as ancestral to the cyclostomes, such as the larval-type oral apparatus (Miyashita et al., 2021) and the transformation of the larval endostyle into the thyroid after metamorphosis (Takagi et al., 2022), are in fact the derived state from the acquisition of the ammocoete larval stage (still, careful discussion is necessary; see Mallatt, 2023).Lampreys and hagfish split around 470-390 million years ago (Kuraku & Kuratani, 2006; also see Miyashita et al., 2019), and their morphology has highly diverged.Thus, there is a strong need to gain a better understanding of the anatomy of hagfishes.
Although the morphology of the hagfishes is often thought to be similar among species, they are, in fact, quite diverse.Hagfish are comprised of three subfamilies (Myxininae, Eptatretinae, and Rubicundinae), six genera, and 88 species worldwide (Fricke et al., 2022).Differences in their morphology are most apparent in their branchial pores and horny teeth, which are often used to identify species (Jørgensen et al., 1998;Nelson et al., 2016; Figure 1).The external branchial pores are located in the middle part of the body (Figure 1a).The number and morphology of these pores differ among hagfish species and are essential traits for taxonomy (Dean, 1904; Figure 1b).(Nelson et al., 2016).The morphological diagrams are after Nakab o (2013).
In the present article, we focus on the anatomy of an Eptatretinae hagfish, the inshore hagfish Eptatretus burgeri.In East Asia, E. burgeri is commonly found in relatively shallow habitats.It is traditionally consumed and served as cuisine mainly in Korea and in some parts of Japan, where the species is easy to obtain (Gorbman et al., 1990;Honma, 1998).Recent embryological studies have been conducted on E. burgeri, and some knowledge on its comparative embryology is now available (Oisi et al., 2013(Oisi et al., , 2015;;Ota et al., 2007).Gross anatomical description of a Japanese hagfish, probably this species, has been done in the past in Japanese (Yamatsuta, 1903), but it is brief and not sufficient to discuss detailed comparative anatomy.Furthermore, we dissected unfixed specimens of hagfish, because we expect that further work will be conducted to examine the detailed molecular biology of this animal with techniques such as single-cell RNA sequencing.For this purpose, the tissue or organ of interest must be isolated appropriately through dissection; however, since fixation with formalin affects the preservation of RNA (Denisenko et al., 2020;Li et al., 2014), sample collection should be performed on unfixed specimens whenever possible.Because fixed tissues differ in appearance from unfixed ones, anatomical descriptions of fixed specimens may not be suitable for researchers who aim to conduct such studies.Thus, in this study, we compare E. burgeri, which has six pairs of branchial pores, to M. glutinosa to advance a comprehensive understanding of hagfish morphology and provide information on hagfish tissues using both unfixed and fixed specimens.

| Animals
E. burgeri specimens were collected from Sagami Bay, Kanagawa Prefecture, Japan, in February (40-45 m depth) and May (80-100 m depth) 2020.The animals from the former location were transported to the Center for Education and Research in Field Sciences, Faculty of Agriculture, Shizuoka University, Japan, and the latter were sent to the laboratory at the University of Tsukuba, Japan.Brown hagfish (Eptatretus atami; syn.Paramyxine atami) were obtained from Suruga Bay, Yaizu, Shizuoka Prefecture, Japan, in February 2021 (320 m depth), and transferred to the Field Science Center, Shizuoka University.The hagfishes were kept in seawater at 10 C before dissection.We identified these species based on external morphology, including the branchial pores and dental cusps (after Dean, 1904; see Figure 1).All animal experiments were performed in accordance with the Guides for Care and Use of Laboratory Animals of Shizuoka University and the University of Tsukuba.

| Anesthesia, fixation, and dissection
Hagfish were anesthetized with a mixture of 0.1% ethyl m-aminobenzoate (Nacalai tesque, Kyoto, Japan) and 0.1% sodium hydrogen carbonate (Wako, Osaka, Japan) in seawater.We then euthanized them by immersing them in a fixative solution under deep anesthesia.For unfixed specimens, we humanely euthanized deeply anesthetized animals by severing the medulla or performing decapitation.For fixation, we soaked the hagfish specimens in 20% formalin for 2 days and transferred them into 70% ethanol.We mainly followed Marinelli and Strenger (1956) for terminology.

| External morphology
First, we describe the external morphology of E. burgeri (Figure 2).E. burgeri is ochre to brownish red on its dorsal and lateral sides, with a pale dorsal midline skin fold, and white to ecru on its ventrum.Total lengths of our specimens were 39.5-58.0cm.They have six pairs of external branchial pores (Figure 2).The distance between each branchial pore is two or three times the branchialpore diameter in E. burgeri.The caudal-most branchial pore on the left side is larger than the others because it is fused with the apertura ductus oesophageocutanei, as in other hagfish species (see also Dean, 1904).
Mucous pores open at regular intervals on the ventral side of the body in a pair of rows as with the external branchial pores.The rostral end of these rows is at about the same anterior-posterior position as the eyes, which is more anterior than the external branchial pores.These rows are also present on the tail, caudal to the cloaca, although they are interrupted around the cloaca (Figure 2a).Rows of white and prolate-shaped mucous glands (glandular mucosa) are found under the mucous pores (Figure 3).
On the dental plate, E. burgeri has three fused cusps in the anterior row and two fused cusps in the posterior row (Figure 2).The number of unicusp teeth varies among species, with some within-species differences (Jørgensen et al., 1998;Kase et al., 2017).Three pairs of tentacles are present on either side of the snout.
The above features are not so different in the other species in the same genus, E. atami (Figure 2).E. atami is dark brown or blackish purple; the total lengths were 43.6-55.3cm.Comparing E. atami with E. burgeri, there are notable differences in the number of dental cusps and the arrangements of the branchial pores.The number of cusps on the innermost tooth in the posterior row is two in E. burgeri, while it is three in E. atami; the distance between the branchial pores is narrower in E. atami in comparison with E. burgeri (Figure 2b).However, these two species have identical topographical relationships, including the internal organs.Thus, although there are minor differences in coloration and teeth, the number and topography of anatomical structures, such as branchial pores and tentacles, are highly conserved in the same Eptatretus genus.

| Lingual apparatus
Underneath the skin, the body trunk of hagfishes is broadly covered with a thin layer of muscles, the m.parietalis and m. decussatus (Figure 3a).For unfixed E. burgeri specimens, we pinched the ventral skin rostral to the branchial region and just ventral to the lingual apparatus with tweezers and opened the abdomen along the midline (Figure 3b,c).
The lingual apparatus is unique to cyclostomes, and differs from the tongue (hypobranchial musculature) of jawed vertebrates in that it is derived from the mandibular arch (i.e., the first pharyngeal arch; Oisi et al., 2015, and references within).The lingual apparatus is a prominent structure in the hagfish pharynx and provides mobility to the dental plate (Clark & Summers, 2007).It consists of the protractor muscles: m. protractor cartilaginis, m. protractor dentium superficialis, and m. protractor dentium profundus (Figure 3c,d) that originate in the medial cartilaginous keel (cartilago linguae basalis) of the lingual apparatus and terminate in the dental plate.The main body of the thick rod-shaped lingual apparatus is dorsal to these protractor muscles.The rod-shaped structure is entirely covered by m. tubulatus, with the muscle fibers oriented horizontal to the body axis and encircling the rod-shaped structure.The longitudinal muscles (m.longitudinalis linguae and m. perpendicularis) are found medial to the m.tubulatus.These originate in the cartilago musculi perpendicularis and terminate at the posteromedial part of the dental plate with the stiff tendon (tendo musculi longitudinalis linguae).The muscles that make up the lingual apparatus were stiffened in our fixed specimens, but white and soft in unfixed samples with a texture that was clearly distinguishable from that of the other trunk muscles (Figure 3c).

| Branchial apparatus, heart, and thyroids
The gills and heart are located caudal to the lingual apparatus in hagfish.These pharyngeal elements initially develop anterior to the head in the hagfish embryo, as in other vertebrates.During development, the anterior half of the head becomes elongated, and the gills and heart are shifted posteriorly (Oisi et al., 2013).
The branchial apparatus of hagfish has a unique morphology: the branchial pouch forms a bladder-shaped structure (bursa branchialis) that connects with the pharynx and external body through ducts (Marinelli & Strenger, 1956).In E. burgeri, we observed six pairs of hemispherical bulsa branchialis lined up on each side (Figure 4a).The inner gill duct (ductus branchialis afferens) connects the pharynx and bulsa branchialis.The outer gill duct (ductus branchialis efferens) emerges from the ventral side of the bulsa branchialis and is connected to the branchial pore (Figure 4a).
On the left side of the body, caudal to the caudal-most ductus branchialis efferens, we identified the ductus oesophageocutanei, a structure unique to hagfish (Figure 4b).This duct directly connects the alimentary canal to the exterior of the body at the boundary between the pharynx and intestine and is located only on the left side.The alimentary canal is slightly constricted at the branching point of this duct.
The heart is located along the midline, caudal to the caudal-most bulsa branchialis and rostral to the liver (Figure 4b).The heart consists of a ventricle and an atrium.The atrium is located caudal to and on the left side of the ventricle.As in other vertebrates, the ventral aorta extends anteriorly from the ventricle and leads to the branchial arteries (arteria branchialis afferens) in each bursa branchialis.The ventral aorta bifurcates around the branching point of the fourth branchial arteries (Figure 4b-d), as already reported in Bdellostoma cirrhatum (syn.E. burgeri) in Yamatsuta (1903).This differs from the description of M. glutinosa, in which the ventral aorta does not bifurcate and remains along the midline (Marinelli & Strenger, 1956), and from that of E. cirrhatus, in which the ventral aorta bifurcates close to the ventricle (Icardo et al., 2016a;Icardo et al., 2016b).The surface of the heart and ventral aorta lacks the vascular system (e.g., coronary or hypobranchial arteries) seen in many jawed vertebrates (for coronary arteries of jawed vertebrates, see Mizukami et al., 2022).
In unfixed samples (Figure 4c), the bulsa branchialis was easily identifiable as a flexible gill structure containing small blood vessels.White fat is found around this branchial region, and the thyroid follicles are embedded in it (Figure 4d).The thyroid consists of yellow, 100-500 μm long oval thyroid follicles scattered around the ventral aorta from the anterior part of the ventricle to the posterior end of the lingual muscle (Figure 4d).

| Hepatobiliary and urogenital organs
The hepatobiliary organ is found posterior to the heart.The liver is divided into anterior and posterior lobes, with the gallbladder located between them (Figure 5a).The bile duct, hepatic ducts, blood vessels, and connective tissues are also found between the two lobes and connect the hepatic organs with the gastrointestinal tract.In unfixed specimens, the gallbladder was a thin bladder filled with green bile (Figure 5b).The surface of this bladder is covered with a network of small arteries.Apart from some large arteries, no vascular networks were visible on the gallblader in fixed specimens (Figure 5c).Although a previous study has suggested the presence of a pancreatic principal islet at the junction of the extrahepatic bile duct and intestine in hagfishes (Youson & Al-Mahrouki, 1999), we could not find this structure in the present gross dissection.Further histological investigation will be necessary to observe this pancreatic structure.Spleens were not found.
The common bile duct (ductus choledochus) is located between the two liver lobes, and transports bile from the gallbladder to the intestine (Figure 5c).From the interface between the gallbladder and the common bile duct, two hepatic ducts branch off and enter the anterior and posterior lobes of the liver.These ducts are short, giving the appearance that the gallbladder is attached directly to the liver.The blood vessels on the gallbladder are the peripheral arteries branched from the coeliac artery (arteria coeliaca) that runs longitudinally along the intestinal tract.The portal vein (vena portae), a major vein in the liver, bifurcates proximal to the gallbladder and enters the anterior and posterior lobes of the liver from the same position as the anterior and posterior hepatic ducts.The posterior branch (v.portae hepatis posterior) passes near the ventral side of the posterior lobe of the liver.This vein exits the liver at the caudal edge of the posterior lobe and extends on the surface of the intestine, where it is termed the v. subintestinalis.We could not determine the peripheral pattern of v. portae hepatis anterior in the fixed specimens.In the unfixed specimens, we observed that this vessel branches all over the medial side (i.e., gallbladder side) of the anterior lobe of the liver (Figure 5b).
The alimentary canal consists of a large simple intestine extending in a straight line (Figure 6a).Unlike most jawed vertebrates, hagfish have no stomach.The intestinal tract of E. burgeri is a linear structure rich in blood vessels, as has been reported for other hagfishes.The intestinal wall is thick, elastic, and made up of longitudinally aligned folds on the inner surface (Figure 6a).
The kidneys are paired on the roof of the body cavity, as in other vertebrates, which are represented by the pronephros (see Romagnani et al., 2013).The gonads of hagfish are found in the peritoneal cavity and are attached to the intestinal tract.The testis consists of white-to creamcolored, translucent tissue (Figure 6d), and the ovary is yellowish and opaque (Figure 6b,c).In the waters around Japan, E. burgeri ovarian maturity is reported to occur in September, and the testes are at their maximum size around July (Nozaki et al., 2000;Patzner, 1978aPatzner, , 1978b)).However, although we collected the hagfish in February, the gonads were in various stages of growth (Figure 6b,c).The membranous ovary contains eggs at various stages of maturity.Immature eggs appear as small white prolate spheroids that turn yellow and oval as they mature.Sexual maturity can be confirmed in females by touching the abdomen from the outside because we can touch the mature eggs in the abdominal cavity through the thin abdominal wall.

| Head and brain
The m. parietalis continues serially from the trunk, covering the caudal half of the head, with the anterior end of this muscle reaching the posterior edge of the corbiculum nasale (Figure 7a).The eyes are semi-transparent and easy to observe in the unfixed samples (Figure 7a); in the fixed samples, their texture is different, and the eyes are more difficult to identify (Figure 3a).Since the cranial roof is covered by m. parietalis, we first remove this muscle.Then, we removed the thin cartilage of the cranial roof with scissors for observation of the brain.There is little space between the brain and the brain capsule (Figure 7a).The brain consists of the telencephalon, diencephalon, mesencephalon, and rhombencephalon (Figure 7b; also see Dupret et al., 2014;Sugahara et al., 2017;Suzuki, 2021), as in other vertebrates.There is no overt epiphysis.The habenular ganglion (ganglion habenulae) is located on the midline between the telencephalon and the diencephalon.On each side of the rhombencephalon is a cartilaginous auditory capsule (capsula auditiva) with a single-canaled inner ear.This single canal is a derived condition among the cyclostomes (Higuchi et al., 2019).At the rostral end of the brain, the fila olfactoria enter the nasal cavity and is closely attached to the corbiculum nasale, making it difficult to detach without damaging it.Brain texture changed significantly after fixation.Because the brain loses flexibility and becomes brittle after fixation, isolation of the brain should be performed in the unfixed condition.

| Hagfish have unique diversities in the branchial region
Myxininae, Eptatretinae, and Rubicundinae differ considerably in their external morphology in that the first group has a single pair of branchial pore apertures, and the rest groups have multiple pairs.In Myxininae, multiple ductus branchialis efferens confluently open into a single branchial pore, although each ductus tube remains separate in Eptatretinae (Figure 4b) and Rubicundinae (Kuo et al., 2010).Phylogenetically, this suggests that the Myxininae-type branchial morphology is a derived condition among hagfishes (Figure 8).Except for the branchial apparatus, the other visceral regions of E. burgeri were almost identical to those of known Myxine.
The branchial region is known to be highly diverged among hagfishes, especially in the number of the bursa branchialis.In Eptatretus, bulsa branchialis has been reported to range from 5 to 14 pairs (Dean, 1904;Mincarone & McCosker, 2004).Furthermore, some species even have intraspecific variations, such as in E. stoutii (syn.Homea stouti), the number of gill pairs is usually 12 but can range from 10 to 15 (Dean, 1904).In Myxine, the number of bulsa branchialis does not vary as much as in Eptatretinae, and ranges from five to seven pairs (Jørgensen et al., 1998).In the third sub-family, Rubicundinae, there are five pairs of bulsa branchialis in Rubicundus spp.(Rubicundinae hagfishes are known by only a few individuals worldwide, and their phylogenetic position is controversial.It was often included in Eptatretinae, but in recent years is considered an outgroup of Eptatretinae and Myxininae; Fernholm et al., 2013;Miyashita et al., 2019).The Cretaceous †Tethymyxine tapirostrum, suggested to be a sister group to Rubicundus, has eight pairs of bulsa branchialis (Miyashita et al., 2019).The Rubicundinae and Mixininae hagfishes are as diverse as Elasmoranchii, with The evolutionary history of the hagfishes and lampreys, with special reference to the pharynx and hepatobiliary organs.The schemes of the pharynx are left lateral views, and those of hepatobiliary organs are ventral views.The evolutionary origin of many structures, such as the lingual apparatus, is still a subject of debate.Some fossil records suggest that the separation of the esophageal and respiratory pharynx, a characteristic found only in adult lampreys among extant vertebrates, was established in the common ancestor of vertebrates and then lost independently in the hagfish and gnathostome lineages, although this hypothesis remains controversial (Mallatt, 2023).
intraspecific differences ranging from five to seven gill pairs (Shirai, 1992;Wegner, 2015).However, no group should exhibit a greater increase in gill number or intraspecific variation among all known vertebrates than Eptatretinae hagfishes.
There is also diversity in the branching pattern of the ventral aorta among hagfishes.In the E. burgeri specimens dissected in the present study, the ventral aorta branches in a "Y" pattern about a third of the way from the ventricle.In contrast, in M. glutinosa, the ventral aorta is unbranched and "I-shaped," as in extant chondrichthyans (Marinelli & Strenger, 1956).This bifurcating structure in E. burgeri is identical to that of E. stoutii (Müller, 1834), but different from E. cirrhatus in which the ventral aorta bifurcates near the ventricle (Icardo et al., 2016a; see also Dean, 1904).
In contrast to the diversity in the branchial apparatus among the extant hagfish, all extant lampreys have seven bulsa branchialis and seven corresponding branchial pores, with less morphological diversity within the extant species than the hagfish.Although the morphology is uncertain in some extinct species, such as †Hardistiella montanensis, the Carboniferous lamprey †Mayomyzon pieckoensis has seven pairs of gills (Bardack, 1991;Miyashita et al., 2021, and references therein), leading that the number of gills in lampreys is likely roughly fixed from the Carboniferous to the present.
It is challenging to determine the number of gills in the common ancestor of cyclostomes, whether it was nearly fixed as in lampreys or diverse as in hagfishes.However, evidence from the fossil record (such as stem hagfish, †Myxinikela siroka) suggests that hagfishes exhibit a high degree of divergence in their branchial structures, such as the loss of the cartilaginous branchial basket and the posterior shift of the branchial region (Miyashita, 2020; see the below section).The branchial structure of the ancestral hagfish was probably similar in form to that of lampreys.Still, we do not know what caused the number of gills in hagfishes to increase even to have intraspecific variation.Perhaps, the developmental process and time may have redundancy in response to the extreme environment of the deep sea, and the number of gills might be affected by developmental adaptations to different marine environments.In fact, the circulatory system of the hagfish is in some respects considered to have changed as a result of adaptation to the deep-sea environment.For example, in addition to their true heart (sometimes called the branchial heart), hagfish have multiple "accessory hearts" (Johansen, 1963;Nishiguchi et al., 2016).The significance and function of these accessory hearts are unknown, although some authors have suggested that they enable the storage of more than 30% of their blood in the sinus system to keep blood pressure low and ensure gill function (Forster, 1997).The number and locations of these accessory hearts are also known to vary among species.For example, M. glutinosa has two cardinal hearts, one branchial heart, one portal heart, and one pair of caudal hearts, but E. okinoseanus does not have caudal hearts (Nishiguchi et al., 2016).In addition, hagfish maintain a lower blood pressure than other vertebrates, especially among the Myxine (Farrell, 2007).Thus, hagfishes exhibit morphological variations in their circulatory system and gills, which their unique living environments may have partially influenced.Furthermore, the posterior shift of the branchial apparatus discussed below should contribute to their distinctive pharyngeal anatomy.

| Caudal shift of cardiobranchial structures and enlargement of the lingual apparatus
The division of the head and trunk appears to be ambiguous in adult hagfish.Underneath the skin, their bodies are covered with segmented m. parietalis up to the area surrounding the brain, and the branchial pores open at about the middle of the body (Figures 2 and 3).This should be related to the developmental processes: the caudal shift of the pharyngeal structures and the rostral shift of the trunk somite derivatives, as observed in the development of E. burgeri (see Oisi et al., 2015).
The vertebrate heart is originally a structure closely associated with the pharyngeal arch, and the head mesoderm and cranial neural crest cells contribute to it during the pharyngula stage (Meilhac & Buckingham, 2018;Tzahor & Evans, 2011).The location of the heart during the pharyngeal embryonic stage in jawed vertebrates is ventral to the pharyngeal arches.In lampreys, the heart is consistently positioned behind the caudalmost pharyngeal arch, leading to differences in the embryonic environment around the caudal part of the heart compared to jawed vertebrates (Higashiyama et al., 2016a).Despite this, the branchial region of lampreys, including the heart, is located anteriorly throughout the body, signifying its association with the head even in the adult.During embryonic development in hagfishes, the heart and gills undergo posterior translocation, leading to a configuration in which the gills are situated in the middle of the trunk.This unique process results in the peculiar morphology of hagfishes, such as the alteration of the distribution of hypoglossal nerves, which typically run along the head-trunk boundary in most vertebrates, into segmented occipitospinal nerves.This change allows the nerves to innervate the hypoglossal musculature located anterior to the heart and gill region from lateral rather than circumventing the pharynx caudally (Oisi et al., 2015).
The amniotes are the other typical example of a heart migrating to the trunk through development.Their heart shifts caudally to the thorax during development, away from the cranium, to establish an elongated movable neck (Hirasawa et al., 2016).This translocation of the heart during the establishment of an amniote neck results in the heart being surrounded by trunk derivatives, such as the rib cage.
The caudal shift of the branchial and heart region in hagfishes should differ from the translocation of the heart in amniotes.In tetrapods, the mandibular arch differentiates into the jaw and the hyoid arch into the hyoid apparatus and cutaneous muscles of the neck.They are rarely remodeled, even in the evolutionary acquisition of an elongated amniote neck.Remodeling occurs instead in the caudal-most part of the pharyngeal arch series, resulting in the extension of the esophagus behind the caudalmost pharynx, giving rise to an amniote neck.By contrast, in the hagfishes, elongation occurs in the rostral pharynx.Our present observations reveal that the posterior branchial arteries are located near the heart as a series of undifferentiated gill arches (Figure 4).A previous embryonic study demonstrates that the fourth pharyngeal pouches as well as more caudal ones differentiate as simple branchial pores and shift caudally with the heart following the remodeling of the first through third pharyngeal arches (Oisi et al., 2015).Thus, the anterior part of the pharynx, not the esophagus, is elongated in hagfishes without establishing a neck.Simultaneously, the trunk somites shift rostrally to cover the head surface (Oisi et al., 2015).This is also an important difference from jawed vertebrates, whose necks are characterized by cucullaris muscle and its derivatives (Oisi et al., 2015 and references therein).
This rostral extension of the somites is also found in lampreys (Kuratani et al., 1999); however, the translocation of the cardiobranchial region does not occur.In the fossil stem hagfish, †M.siroka, the position of the gills is more cephalic and closer to that of lampreys than in the extant hagfishes (Bardack, 1991;Miyashita, 2020), suggesting that the caudal translocation of the cardiobranchial region is an autapomorphy for the extant hagfishes.The underlying cause of this cardiobranchial translocation remains unknown.However, differences in the size and position of the lingual apparatus between lampreys and hagfishes might play a role, with a large lingual apparatus occupies the space between the cranium and the branchial region in hagfishes (Figure 3), as opposed to being smaller and located on the ventral side of the branchial region in lampreys (Marinelli & Strenger, 1954;Yalden, 1985).Thus, the shift in cardiobranchial position could be related to the enlargement of the lingual apparatus, although the causal relationship remains elusive.Perhaps, the specialized branchial position of hagfishes may be linked to their feeding behavior, which entails puncturing prey carcasses with their enlarged lingual apparatus.The pharyngeal and branchial regions of hagfishes are in a specialized condition among cyclostomes, possibly reflecting their unique habitat in the deep sea.

| Thyroid gland of E. burgeri represents the common form of the cyclostomes
The thyroid gland of the cyclostomes is not a glandular organ with clusters of follicles, but rather, the thyroid follicles are scattered throughout the pharynx (Matsumoto & Ishii, 1992).Hagfish thyroid follicles are similar in early development and morphology to those of gnathostomes and represent the ancestral condition among extant vertebrates (Takagi et al., 2022).Electron microscopic analyses have revealed the presence of dense granules in the thyroid follicular epithelial cells of hagfishes, which are thought to contain iodine (Fujita, 1975;Fujita & Shinkawa, 1975;Henderson & Gorbman, 1971;Suzuki, 1985).Also, numerous microvilli are present on the apical membrane on the medial side of the follicular structure of thyroid follicular epithelial cells (Suzuki & Kawabata, 1988).These observations indicate that the thyroid follicles of hagfish do not differ significantly from those of other vertebrates in terms of histology.
In lampreys, the thyroid gland takes on the function of an exocrine organ, known as an endostyle during the larval period.This was long considered to be an ancestral condition, but recently it has been suggested to be a secondary condition (Takagi et al., 2022).Although there is some difference in the arrangement of blood vessels, both lamprey and hagfish have the scattered distribution of thyroid follicles along the bifurcated ventral aorta (for lamprey, see Takagi et al., 2022).This suggests that the ancestral thyroid gland was composed of scattered follicles, at least in the common ancestor of the cyclostomes, and that the thyroid as a glandular organ arose for the first time in the gnathostome lineage.A similar trend is seen in the pancreas, as discussed below.

| Hagfishes possess an ancestral-type hepatobiliary system
Morphological evolutionary studies of the hepatobiliary organs are less well-established than those of other body systems, such as the musculoskeletal system.Because of this, evolutionary studies of the hepatobiliary system are poorly developed, even though it is a characteristic structure of vertebrates.
The hepatobiliary system of the lamprey is distinct from that of jawed vertebrates, for example, in having a single liver lobe (Marinelli & Strenger, 1954).Some older studies have claimed that the gallbladder is completely absent in lampreys, but this is incorrect; the gallbladder is present during the ammocoetes larval period and is lost in the adult stage (Morii et al., 2010;Scammon, 1916;Youson, 1993;Youson & Sidon, 1978).This degeneration of the gallbladder during metamorphosis is unique to lampreys.In this respect, adult lampreys are more specialized than their ammocoetes larvae.
In contrast to lampreys, the hepatobiliary organs of hagfish resemble those of jawed vertebrates at the gross anatomical level.For example, hagfish maintain two liver lobes and a large gallbladder throughout their lives (Figure 5).Because of their elongated shape, the liver lobes are generally called anterior and posterior lobes, which correspond to the right and left lobes, respectively, of other vertebrates.The gallbladder is supplied by vessels branching from the artery of the body cavity (a.hepatica).The major veins of the liver gather in the vena portaea and enter the heart through a short route through the sinus venosus.The topographical relationships among the major veins are identical to those of mammals (Higashiyama, Sumitomo, et al., 2016), birds (Higashiyama & Kanai, 2021), and probably chondrichthyans (Hochstetter, 1888), but distinct from those of lampreys.Thus, the hepatobiliary morphology is highly conserved among the vertebrates, and the lamprey case is likely to be a derived form.
Histologically, the hepatobiliary system of hagfish resembles that of jawed vertebrates, although with some important differences.Namely, in both hagfish and jawed vertebrates, the intrahepatic bile ducts and hepatic artery run parallel to the portal vein to form portal triads, and smooth muscle is distributed around the hepatic artery and gallbladder (Ota et al., 2021;Shiojiri et al., 2019;Umezu et al., 2012).However, it has recently been reported that in hagfish, both the intrahepatic and extrahepatic bile ducts and the portal vein lack smooth muscle layers.These characteristics have not been found in jawed vertebrates as far as is known (Ota et al., 2021).
Whereas lamprey and hagfish differ in the structure of their liver and bile ducts, both taxa are characterized by their lack of a compact pancreas (pancreatic organ).In extant jawed vertebrates, the pancreas develops from the subdivision of a single embryonic primordium (the hepatic diverticulum) (Higashiyama et al., 2018, and references within), but this primordium is probably not present in cyclostomes.The exo-and endocrine cells exist sparsely in the intestinal wall (Youson & Al-Mahrouki, 1999;Yui et al., 1988), and could not be identified in our examination of gross anatomy.Thus, as with the thyroid, the pancreas as an organ as well as the pancreatic duct likely arose in the common ancestor of the jawed vertebrates, although we cannot exclude the possibility that the compact pancreas was lost secondarily in the common ancestor of the cyclostomes.Detailed embryological data is needed to provide further insight into this question.

| CONCLUSION
We dissected specimens of E. burgeri and compared its gross morphology, mainly around the pharynx and coelomic organs, with that of other species.We identified several morphological differences between E. burgeri and other species in the pharyngeal region and the cardiovascular system but were not able to do so for other organs.E. burgeri is indeed a typical form to use as a model for hagfishes.Our discussion showed that the branchial structures of hagfishes represent a highly derived condition in the cyclostome.At the same time, however, the hepatobiliary system is more derived in lampreys.This highlights the difficulty in determining the morphology of the early cyclostomes and the need for a thorough discussion, including consideration of fossil evidence (Figure 8).Note that we did not conduct a detailed dissection of the musculoskeletal or nervous systems in the present study, but it will need to be considered in the future.Some differences in these systems could be tied to differences in ecology among hagfish species.For example, the eyes of Myxine are almost completely buried beneath the cutaneous layer (Marinelli & Strenger, 1956), whereas the position of the eyes can be identified externally in Eptatretus.It is plausible that there are differences in the shape of the brain and peripheral nerves associated with these sensory-organ differences.Comparisons between the subfamilies would provide a better understanding of the evolution and development of hagfishes, which in turn would lead to a better understanding of cyclostomes and early vertebrates.

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I G U R E 1 Morphological variation among hagfishes and their phylogenetic relationships.(a) Diagram of a hagfish, showing a left lateral view of the entire body, a ventral view of the head, and the tooth arrangement.(b) Phylogenetic relationships among the chordates.The Myxiniformes contain two lineages: Eptatretinae and Myxininae.The Eptatretinae have several pairs of external branchial pores, and the Myxininae have a single pair of branchial pores

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I G U R E 2 The external morphology of two hagfish species, Eptatretus burgeri and E. atami.(a) Photographs of each species, showing an enlarged view of the caudal part of E. burgeri.(b) Differences between the two species in the arrangement of tooth rows and external branchial pores.Scale bars = 1 cm.

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I G U R E 3 Positions of the organs and lingual apparatus of Eptatretus burgeri.(a) Fixed specimen with skin removed.The trunk is covered by two muscles with different fiber directions.(b) We cut and opened the body wall muscle using an unfixed specimen to show the lingual apparatus and internal organs.The photograph shows a ventral view.(c) Pharynx and lingual apparatus.(d) The lingual apparatus of a fixed specimen.Scale bars = 1 cm.F I G U R E 4 Cardiac anatomy and thyroid follicles of Eptatretus burgeri.(a) Left lateral view of a fixed specimen.We removed the lateral body wall and exposed the left lateral branchial structure.(b) Ventral view of the branchial organs of the same specimen as shown in panel (a).The left lateral body wall was removed, exposing the left-sided rows of the ductus branchialis efferens.(c) Ventral view of the branchial structure in an unfixed specimen.(d) Ventral view of the heart in an unfixed specimen.The thyroid follicles are in the white adipose tissue.The distribution of the thyroid follicles is shown in the diagram.Scale bars = 1 cm.

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I G U R E 5 The hepatobiliary organs of Eptatretus burgeri.(a) Ventral views of an unfixed specimen.The liver in the left-hand panel is shown almost in its natural position.In the right-hand panel, we have shifted the liver to the left side of the body, showing the junction of the hepatobiliary system with the intestinal tract.(b) The hepatobiliary system of an unfixed specimen of E. atami, where the liver and gallbladder were severed from the intestinal tract and are shown from the intestinal side.(c) The hepatobiliary system of a fixed specimen shown from the ventral side.The liver in the left-hand panel is in its natural position.The middle panel shows the liver flipped over to the right side of the body, showing the extrahepatic biliary tract and vascular system.Scale bars = 1 cm.

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I G U R E 6 The hindgut and urogenital organs of unfixed specimens of Eptatretus burgeri.(a) The intestinal tract is shown from the ventral side.Enlarged views are shown of the kidneys and the inner wall of the intestinal tract.In this specimen, the gonads are not mature, and the sexes are not distinguishable.(b) The female body cavity with mature ovaries.(c) A female with immature ovaries.(d) A mature male specimen.Scale bars = 1 cm.

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I G U R E 7 The head of Eptatretus burgeri, with the skin removed.(a) The unfixed specimen.(b) The fixed specimen.Scale bars = 1 cm.