Abstract The head and anterior trunk region of most actinopterygian fishes is stiffened as, uniquely within vertebrates, the pectoral girdles have a direct and often strong connection through the posttemporal to the posterior region of the skull. Members of the mesopelagic fish family Stomiidae have their pectoral girdle separated from the skull. This connection is lost in several teleost groups, but the stomiids have an additional evolutionary novelty—a flexible connection between the occiput and the first vertebra, where only the notochord persists. Several studies suggested that stomiids engulf significantly large prey items and conjectured about the functional role of the anterior part of the vertebral column; however, there has been no precise anatomical description of this complex. Here we describe a unique configuration comprising the occiput and the notochordal sheath in Aristostomias, Eustomias, Malacosteus, Pachystomias, and Photostomias that represents a true functional head joint in teleosts and discuss its potential phylogenetic implications. In these genera, the chordal sheath is folded inward ventrally beneath its connection to the basioccipital and embraces the occipital condyle when in a resting position. In the resting position (wherein the head is not manipulatively elevated), this condyle is completely embraced by the ventral fold of the notochord. A manual manipulative elevation of the head in cleared and stained specimens unfolds the ventral sheath of the notochord. As a consequence, the cranium can be pulled up and back significantly farther than in all other teleost taxa that lack such a functional head joint and thereby can reach mouth gapes up to 120°.

Citation: Schnell NK, Johnson GD (2017) Evolution of a Functional Head Joint in Deep-Sea Fishes (Stomiidae). PLoS ONE 12(2): e0170224. https://doi.org/10.1371/journal.pone.0170224 Editor: Wm. Leo Smith, University of Kansas, UNITED STATES Received: July 6, 2016; Accepted: January 1, 2017; Published: February 1, 2017 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: All relevant data are within the paper. Funding: This study was initiated during a Smithsonian Predoctoral Fellowship of NKS in the Division of Fishes at the National Museum of Natural History and further supported by the Herbert R. and Evelyn Axelrod Endowment Fund for systematic ichthyology in the Division of Fishes (2008/2009). Competing interests: The authors have declared that no competing interests exist.

Materials and Methods Our research employed only ethanol-preserved specimens deposited in museum collections and did not involve animal experimentation or examination of fossil specimens. Examined material is listed in the following section “Material examined” and is deposited in the following institutions: National Museum of Natural History, Smithsonian Institution, USA (USNM); Scripps Institution of Oceanography, USA (SIO); Museum of Comparative Zoology, Harvard University, USA (MCZ); British Museum of Natural History, UK (BMNH); National Museum of Nature and Science, Japan (NSMT-P); Virginia Institute of Marine Science, USA (VIMS). Access to material of those collections was authorized by respective curators and specimens were examined at their original institutions or loaned to the Muséum national d’Histoire naturelle (MNHN). No permits were required for the described study, which complied with all relevant regulations. Preserved specimens were cleared and double stained (c&s) [17,18] and afterwards examined and dissected using a Zeiss Discovery V12/V20 stereomicroscope. One specimen was cleared and triple stained (c&ts) with Sudan Black B, in order to stain nerves, following the alcian blue-alizarin red staining [19]. Photographs were taken with an attached Axiocam microscope camera and processed with the Zeiss AxioVision or ZEN software to obtain composite images with an increased depth of field. The specimens for the histological serial sections (hss) were embedded in paraffin and stained with Azan [20]. All measurements referenced herein are standard length, SL, unless otherwise mentioned. Material examined Aristostomias xenostoma, USNM 296715, 3 specimens, 123 mm, 140 mm, including 1 c&s, 83 mm; Bathophilus sp., USNM 325530, 1 specimen c&s, 73 mm; Bathophilus filifer, SIO 03–189 1 specimen (hss), 80 mm; SIO 76–42, 1 specimen c&s, 75 mm TL; Bathophilus vaillanti, USNM 234150, 1 specimen c&s, 101 mm; Eustomias sp., MCZ 62637, 4 specimens c&s, 26–45 mm (26, 30, 32, 45 mm); USNM 394242, 1 specimen c&s, 59 mm; Eustomias bifilis, SIO 97–89, 2 specimens, 108 mm TL; including 1 c&s, 105 mm TL; Eustomias filifer, BMNH 2007.10.31.64, 1 specimen (hss), 97 mm TL; Eustomias fissibarbis, USNM 270587, 1 specimen, 120 mm; Eustomias macronema, BMNH 2007.10.31.12, 1 specimen c&s, 65 mm TL; Eustomias obscurus, USNM 206711, 5 specimens, 131–199 mm, including 1 c&s, 199 mm, and 1 c&ts, 147 mm; (131, 135, 147, 179, 199 mm); USNM 234416, 1 specimen c&s, 71 mm; USNM 234444, 1 specimen c&s, 59 mm; Flagellostomias boureei, BMNH 2002.8.5.786–788, 1 specimen c&s, 161 mm; Grammatostomias circularis, NSMT-P 99317, 1 specimen (hss), 149 mm; Grammatostomias dentatus, USNM 234036, 1 specimen c&s, 76 mm; VIMS 11846, 2 specimens, 117 mm, including 1 c&s, 111 mm; Idiacanthus antrostomus, SIO 60–459, 2 specimens, 182 mm, including 1 c&s, 320 mm; SIO 70–237, 3 specimens c&s, 57–135 mm (57, 75, 135 mm); SIO 97–85, 1 specimen (hss), 310 mm; Malacosteus australis, USNM 296675, 1 specimen c&s, 110 mm; Malacosteus niger, SIO 73–25, 3 specimens, 135 mm, including hss, 100 mm, and 1 c&s, 130 mm; USNM 296813, 1 specimen c&s, 74 mm; Pachystomias microdon, USNM 297922, 1 specimen c&s, 168 mm; USNM 297923, 1 specimen c&stained with alizarin, 156 mm; Photostomias sp., USNM 296650, 1 specimen c&s, 92 mm; Photostomias guernei, BMNH 2007.10.31.6, 1 specimen c&s, 50 mm; BMNH 2007.10.31.19, 1 specimen c&s, 112 mm.

Discussion Cranial elevation driven by the epaxial musculature is a common feature of feeding in fishes, as it contributes to mouth opening and promotes jaw protrusion [21]. In stomiids, the presence of considerable flexibility in the anterior part of the vertebral column coincident with a functional head joint allows extension of the mouth gape antero-dorsally and enables these taxa to have gapes up to 120° [3, 9]. Although evidence has been provided that the previously recognized six stomiatoid families (Astronesthidae, Chauliodontidae, Idiacanthidae, Melanostomiidae, Malacosteidae, and Stomiidae) should be placed in a single, expanded Stomiidae [15], these families are sometimes still recognized as separate subfamilies [22,23]. The presence of a functional head joint in Aristostomias, Eustomias, Malacosteus, Pachystomias, and Photostomias renders two of those putative subfamilies, Malacosteinae and Melanostomiinae, paraphyletic. The phylogenetic hypothesis inferred by this character corresponds (with one difference) with the latest analysis [16] based on rod opsin and three other nuclear gene fragments (rag1, myh6, enc1). The formation of the occipital condyle by the basioccipital and the exoccipital in Photostomias and Aristostomias suggests a sister relationship between the two taxa, contrary to the relationship proposed in the latest anaylsis [16], wherein Aristostomias is sister to Malacosteus based on the four nuclear loci, their red sensitivity, and photophore emission, exceeding 700 nm. We mapped the presence of the functional head joint on this previous analysis [16] of the family (Fig 3). It’s presence corroborates the placement of Eustomias within the “malacosteine” loosejaw + Pachystomias clade, with Bathophilus + Grammatostomias sister to this clade. Eustomias has traditionally been placed somewhere within the “Melanostomiinae,” and in his extensive morphological treatise on 25 stomiid genera Fink [15] placed it for the first time as sister to Bathophilus + Grammatostomias based on three osteological characters. Our analysis as well as the recent molecular analysis [16] places Eustomias within the “Malacosteinae.” Accordingly, below we discuss the three characters that led to Fink’s [15] alternative placement within the “melanostomiines” (Fig 4). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Illustration of Fink’s [ Illustration of Fink’s [ 15 ] characters placing Eustomias as sister to Bathophilus+Grammatostomias. Cladogram according to Fink [15]; left column, compound hypohyal; middle column, articulation of third epibranchial with fused third and fourth pharyngobranchials of dorsal gill arches; right column, bases of pectoral fin rays. All images show c&s specimens, except (D3) and (G3), which are modified drawings from Fink [14]. (A) Grammatostomias dentatus, USNM 234036. (B1,2,4) Bathophilus vaillanti, USNM 234150. (B3) Bathophilus filifer, SIO 76–42. (C) Eustomias obscurus, USNM 206711. (D) Pachystomias microdon, USNM 297922. (E) Photostomias sp., USNM 296650. Photostomias lacks pectoral fin rays. (F) Malacosteus australis, USNM 296675. (G) Aristostomias xenostoma, USNM 296715. cl, cleithrum; co, coracoid; eb3, third epibranchial, hhd, dorsal hypohyal; hhv, ventral hypohyal; ra, radial; sc, scapula; pb3-4, fused pharyngobranchials 3+4; pfr, pectoral fin ray. Scale bars, 1 mm. https://doi.org/10.1371/journal.pone.0170224.g004 In reference to the hypohyal complex (Fig 4, left column): “In Bathophilus, Eustomias, and Grammatostomias, the hypohyal element is about twice as long anteroposteriorly as it is dorsoventrally, and its longest dorsoventral distance is posterior to the anterior border of the element” (p. 44 [15]). We agree that the compound hypohyals (comprising dorsal and ventral elements) are more elongate in these three genera relative to those of Pachystomias, Malacosteus, Aristostomias, and Photostomias. Those of Bathophilus and Grammatostomias are quite similar in size and shape, but differ from that of Eustomias in which the dorsal hypohyal lies anterior to the extremely elongate ventral element rather than dorsal to it as in Bathophilus and Grammatostomias and all other “Malacosteinae” + Pachystomias (Fig 4, left column). In terms of character information we consider the relative positions of the anterior and posterior hypohyals to one another equally or more relevant than the length to depth ratio of the compound element. In reference to the dorsal gill arches (Fig 4, middle column): “In Bathophilus, Eustomias, and Grammatostomias, the third epibranchial articulates with the third pharyngobranchial at a point anterior to the posterior border of the pharyngobranchial ossification. (…) In other stomiids, the third epibranchial articulates with the third pharyngobranchial adjacent to the posterior border of the pharyngobranchial ossification” (p. 51 [15]). Bathophilus and Grammatostomias are very similar regarding the shape of the fused third and fourth pharyngobranchial (pb3+pb4) [24] and, in fact, in overall configuration of the entire dorsal complex. The third epibranchial (eb3) articulates with the fused pb3+pb4 slightly anterior to the cartilaginous posterior area. Shape of the fused pb3+pb4, articulation of the third epibranchial, and overall configuration of the dorsal complex is similar in Aristostomias. In Eustomias the fused pb3+pb4 element is extremely elongate, and the articulation of the third epibranchial is even farther anterior than in Bathophilus and Grammatostomias. We also note that the point of articulation of the third epibranchial can be intraspecifically variable. In Bathophilus filifer (Fig 4B3) the point of articulation is as far anterior as in Eustomias obscurus (Fig 4C2) and therewith different from the condition in Bathophilus vaillanti (Fig 4B2). We agree that the more anterior articulation of the third epibranchial could be construed as a synapomorphy of the latter three genera, as Fink proposed. In reference to pectoral-fin rays (Fig 4, right column): “In Bathophilus (…), Grammatostomias, and Eustomias (…), the flanges for muscle attachment on the dorsal halves of the non-rod ray fin rays are greatly reduced in breadth (i.e., in the axis perpendicular to the axis of the rays). (…). In other stomiids, such flanges are more developed” (p. 80 [15]). We agree that there are no such flanges in Eustomias and Grammatostomias, although in the latter the first ray has a little broadening at the base. Bathophilus has two separated sets of fin rays. The two smaller ones articulating at the scapula have slightly broadened bases but no obvious flanges. However, the ray attaching to the radial has an obvious triangular base that we would call a flange. Furthermore, the suggested flanges in Pachystomias are no more developed than they are in Grammatostomias. Again, we see no clear character in this complex to support a close relationship between Bathophilus, Grammatostomias, and Eustomias. There is no doubt about the sister group relationship between Bathophilus and Grammatostomias based on these three characters, but we maintain that the hyoid and pectoral fin ray characters are open to alternative interpretations and thus do not unequivocally support a relationship between Eustomias+(Bathophilus+Grammatostomias). Fink’s character of the dorsal gill arches may be valid, but that complex and others need to be more comprehensively investigated to fully understand and support a specific placement of Eustomias within the “Malacosteinae,” particularly in light of our documentation of the distribution of the functional head joint among these taxa. In an anatomical and functional morphology study [3] on the jaw apparatus of two loosejaw genera, published in the 19th century in German, it was noted that the connection between the occiput and the notochord in Photostomias and Malacosteus resembles a ball and socket joint, but the details of this specialized connection could not be fully understood with the rigid, ethanol preserved specimens those authors had available. An occipito-vertebral gap between the first and second vertebrae and intervertebral spaces is documented elsewhere only for the lepidogalaxiid, Lepidogalaxias salamandroides, and here, smaller gaps are present between all succeeding vertebrae [25]. Observations of a live specimen proved that this facilitates movements of the head, e.g. elevating and bending the neck to each side [25]. For barbeled dragonfishes (family Stomiidae), the very few live records do not include feeding activity, and such observations are unlikely to be available as long as specimens cannot be kept alive, but such observations might prove invaluable in delivering a stable hypothesis of the functional significance of this exceptional vertebrate head joint.

Acknowledgments We thank S. Raredon (USNM), C. Klepadlo (SIO), S. Charter (NMFS SWFSC), J. Maclaine (BMNH), G. Shinohara (NSMT), T. Sutton (NSU), and E. Hilton (VIMS) for loan of specimens and radiograph photographs; M. Meinert and C. Nitzsche (University of Tübingen) for the preparation of the serial sections; and E. Hilton, M. L. Habegger, A. Nonaka, A. Ohler, and R. Aurahs for discussion. We are grateful to C. Kenaley, K. Conway, and L. Smith, whose comments highly improved the manuscript.

Author Contributions Conceptualization: NKS GDJ. Funding acquisition: NKS GDJ. Investigation: NKS. Resources: NKS GDJ. Supervision: NKS. Visualization: NKS. Writing – original draft: NKS. Writing – review & editing: NKS GDJ.