Table Of ContentMorphology and molecular phylogenetic relationships
of Barathronus multidens (Ophidiiformes: Bythitidae)
by
Sergei A. EvSEEnko* (1), natalia v. GordEEvA (2),
Yana Yu. BolShAkovA (1) & Stas h. koBYliAnSkY (1)
Abstract. – deep-water Barathronus multidens (ophidiiformes: Bythitidae) was previously known from the two
type specimens from off the coast of Florida and Morocco. in this paper, we characterize morphologically and
molecularly a juvenile (45 mm Sl) caught by a Sigsbee trawl at the Mid-Atlantic ridge (16º50’n) between the
surface and 3,227 m. dnA sequences of B. multidens are obtained for the first time. They are determined from
the sequences of the mitochondrial genes ND4 and 16S, and the nuclear gene RAG 1. Within Bythitidae, the
genera Barathronus and Sciadonus form a monophyletic clade. Sciadonus is nested within Barathronus with the
B. multidens lineage being the earliest branching of the clade.
Résumé. – Sur la morphologie et les relations phylogénétiques de Barathronus multidens (ophidiiformes :
Bythitidae).
© SFI
Received: 18 Apr. 2017 l’espèce profonde Barathronus multidens (ophidiiformes: Bythitidae) était précédemment connue par les
Accepted: 24 Nov. 2017
deux spécimens-type du large de la Floride et du Maroc. Un juvénile (Sl 45 mm), capturé par un chalut Sigsbee
Editor: G. Duhamel
sur la dorsale médio-Atlantique (16º50’n) entre la surface et 3 227 m, permet de compléter les données mor-
phologiques et moléculaires. les séquences Adn sont ici obtenues pour la première fois. Elles proviennent des
gènes mitochondriaux ND4 et 16S et du gène nucléaire RAG 1. Au sein des Bythitidae, les genres Barathronus et
Sciadonus forment un clade. Sciadonus se place au sein de Barathronus et la lignée de B. multidens est la bran-
Key words
che la plus précoce du clade.
Bythitidae
Barathronus multidens
Aphyonids
Mid-Atlantic ridge
Molecular phylogeny
According to Møller et al. (2016), morphological description of the specimen and a molecular
Mariana Trench
deep-sea diversity the Aphyonidae is now considered phylogenetic analysis of its relationships with other species
a member of the Bythitidae, which of the Bythitidae.
involves reassignment of Barathro-
nus and the remaining six aphyonid genera (nielsen, 2015),
forming a monophyletic clade within the Bythitidae. The MATERIALS AND METHODS
cosmopolitan deep-sea genus Barathronus includes 11 valid
species, most of which are known from just a few specimens. A single specimen of B. multidens was caught during
Five species of Barathronus are reported from the Atlantic the first ecological survey in the Russian Claim Area on the
ocean: B. bicolor Goode & Bean, 1886 from off the coast of Mid-Atlantic ridge (MAr). it has been conducted between
South Caroline to Brazil; B. linsi nielsen, Mincarone & di 17°14’n and 16°40’n in February-March 2015, during the
dario, 2015 from the continental slope of northeastern Bra- 37th cruise of the r/v ‘Professor logachev’. Trawling oper-
zil; B. parfaiti (vaillant, 1888) from between the Azores and ation was done using a Sigsbee trawl on the terrace of the
France; B. unicolor nielsen, 1984 from the western north western slope of the rift valley of the Mid-Atlantic ridge,
Atlantic; and B. multidens nielsen, 1984 from off the coast covered with pteropod sand and ooze. it was carried out on
of Florida and Morocco. 4 March 2015 at station number 158. The trawl was on the
Barathronus multidens is previously known from the two bottom at 18.00 at the depth 3227 m, at a site with coordi-
type specimens from the central West and East Atlantic, in nates 16°50.052’N; 46°33.045’W. The profile distance was
which the eyes were not discernible (nielsen, 1984). in this 1800 m; the profile end was at a depth of 3283 m after 2.5
vein, the finding of the 3rd specimen of B. multidens, with hours of trawling, at a site with coordinates 16°51.021’n;
noticeably developed eyes, from a new locality at the Mid- 46°32.767’W. Taking into account the possibility of by-catch
Atlantic ridge extends the known range and morphological during lifting of the trawl to the surface, the horizon of catch
states of the species. This study consists of a comparative of this specimen was located between the surface and the
(1) P.P.Shirshov institute of oceanology, russian Academy of Sciences, 36 nakhimovskyi
Prospekt, 117997 Moscow, russia. [[email protected]] [[email protected]]
(2) vavilov institute of General Genetics, russian Academy of Sciences, 3 Gubkina ul., 119991 Moscow, russia. [[email protected]]
* Corresponding author [[email protected]]
Cybium 2018, 42(2): 137-141.
Morphology and relationships of Barathronus multidens EvsEEnko et al.
depth of 3227 m. Specimen was found between the inner and
outer nets of the trawl, with mesh sizes 5 and 45 mm, respec-
tively. The catch contained a large amount of pteropod sand
and ooze, remains of which are seen in the unpaired fins of
the fish (Fig. 1). Specimen was fixed in 4% formalin solution
prepared with seawater; two months later, it was transferred
to a storage in a 70% ethanol solution. It was cleared in KOH
and stained with alizarin, and stored in glycerol to allow us to
count the number of vertebrae and rays in the fins. Besides,
radiograph of the specimen has been taken also. Currently, it
is deposited in the fish collection at the Institute of Oceanol-
ogy, russian Academy of Sciences (iorAS 03293). Figure 1. – Barathronus multidens, 45 mm Sl. Photo taken just
A small piece of muscle tissue was preserved in 96% after the capture of the fish. Cat. No. IORAS 03293.
ethanol and stored at –20oC. Extraction of dnA was per-
was included as the outgroup taxon (nC_002081 (both
formed with the diatomPrep kit (“laboratory iZoGEn”,
mitochondrial markers), AF369064). Best fitting models
russia). Following Møller et al. (2016), we amplified two
of sequence evolution were determined in ModElGEn-
mitochondrial (mt) fragments and one nuclear gene: a 600
ErATor with using the Akaike information criterion (AiC)
base pairs (bp) region of the mitochondrial 16S ribosomal
(keane et al., 2006). GTr+i+G (Tavaré, 1986; Yang, 1994)
rnA (mtdnA-16S) gene (Palumbi, 1996), a 900 bp region
for each of the mtdnA markers, and Trn+G (Tamura and
of the mitochondrial NADH dehydrogenase subunit 4 (mtd-
nA-ND4) gene (Miya and nishida, 1999), and the partial nei, 1993) for the RAG1 were identified as the most appro-
nuclear recombination activating gene 1 (ndnA-RAG1; li priate nucleotide substitution models. Phylogenetic analy-
et al., 2007). PCr products were tested for quality via run- sis was performed using Bayesian inference implemented
ning on a 1% agarose gel and then were purified by ethanol in MrBayes 3.2.6 (ronquist et al., 2012) and Maximum
precipitation. The sequencing was performed in both direc- likelihood (Ml) using rAxMl 8.0 (Stamatakis, 2014).
tions using the same primers as were used for the amplifica- due to incomplete data, molecular relationship of the genus
tion in an ABi Prizm 3130xl Thermal Cycler with Bigdye Barathronus was inferred from mtdnA-16S and ndnA-
v. 3.1 sequencing kit (Applied Biosystems inc.). Sequence RAG1 separately. After the alignment, the total length of the
reads were assembled and aligned in Geneious v. r7 (kearse sequences was 558 bp for 16S and 522 bp for RAG1. The
et al., 2012). Sequences for species of the Bythitidae and Bayesian analysis was run for four million generations, sam-
closely related families dinematichtyidae and ophidiidae pling every 400 generations, with four runs each distributed
available on nCBi GenBank were used for the representa- across six chains. Stability in the analysis was reached after
tion of phylogenetic relationship of B. multidens: Bara- 400,000 generations. Posterior probabilities and Ml boot-
thronus diaphanus (nCBi GenBank Accession numbers: strap values (after resampling with 1000 replicates) were
kX120066, kX150901, kX150881), Sciadonus galatheae plotted on Bayesian consensus trees.
(kX120089, kX150924, kX150880), Aphyonus gelatino-
sus (kX120065, kX150900, kX150877), Bythitidae: Bel-
lotia galatheae (kX120068, kX150903, kX150882), Cata- RESULTS
etyx messieri (kX120070, kX150905, kX150883), Cata-
etyx niki (kX120071, kX150906, kX150879), Diplacan- Description of juvenile
thopoma brachysoma (kX120079, kX150914, kX150871), Juvenile, 45 mm SL (Fig. 1). Dorsal-fin rays 73, anal-fin
Tuamotuichthys schwarzhansi (kX120090, kX150925, rays 62, pectoral-fin rays 23, one ventral-fin ray, caudal-fin
kX150884), Bidenichthys capensis (kX120069, kX150904, rays 10, total vertebrae 77 (34 precaudal + 43 caudal verte-
kX150874), dinematichtyidae: Dinematichthys iluocoete- brae). Anterior gill arch with 25 gill rakers. origin of dorsal
oides (kX120078, kX150913, kX15087), Ogilbia cayorum fin above vertebra number 29, anterior anal-fin ray below
(kX120085, kX150920, kX150878), ophidiidae: Acan- vertebra number 36 and anterior anal ray below dorsal-fin
thonus armatus (kX120063, kX150898, kX150885) and ray number 11-12.
Chilara taylori (kX120072, kX150907, kX150895). Two Head rather small (head length 18.0% SL), nearly as wide
available sequences of 16S and RAG1 fragments (kY509530 as deep and markedly higher and broader than body. Snout
and kY509537, respectively) for Parabrotula tanseimaru blunt. nostrils with low rim. Body elongate, tapering gradu-
(Parabrotulidae) were also included in the analysis because ally to caudal fin. Skin loose, gelatinous and translucent.
this family was recently determined to presumably be within Scales absent. Anterior part of body prior to dorsal-fin ori-
the bythitid clade (Campbell et al., 2017). Gadus morhua gin and anus surrounded by a transparent sheath, subdermal
138 Cybium 2018, 42(2)
EvsEEnko et al. Morphology and relationships of Barathronus multidens
space behind head rather wide. Dorsal, anal and caudal fins of small melanophores mediolaterally and along dorsal and
united. Dorsal fin origin somewhat anterior to midpoint of postanal margins of body.
fish (predorsal length 43.0% SL), anal fin origin just behind Juvenile weakly ossified. Ossification took place in skull
midpoint of fish (preanal length 51.0% SL). Pelvic fins each and splanchocranium; vertebral ossification progressively
with a single ray, pelvic fin base below posterior margin of decreases posteriorly. vertebral centra narrow, not fully
gill cover. Pectoral peduncle broader than long. formed, rectangular-shaped in a lateral view, markedly sepa-
Eyes small (eye diameter 11.6 % head length), pale yel- rated from each other by a narrow space.
low, hidden under transparent skin; directed forward and a Fin-rays in median fins well ossified.
little sideways so that longitudinal axis of eye lies at an angle
of about 70 degrees to longitudinal axis of body, providing Genetic analysis
almost binocular vision; each eye located in silvery reflec- Sequences obtained for RAG 1 (646 bp), 16S (557 bp)
tor, covering posterior part of eyeball; lens absent. Eye with and ND4 (854 bp) were registered in nCBi Genbank under
centrally placed, small black pigment spot about 0.25 mm in nos. kY120957, kY110996 and kY110997, respectively.
diameter. A narrow black ring (0.65 mm in diameter) forms no stop codons, frame shifts or abrupt indels were found in
reduced iris, surrounding central black spot. any of the mtdnA and nuclear sequences. The topology of
Mouth large, oblique, length of upper jaw two times the phylogenetic trees constructed separately for mtdnA and
head length. Major part of upper jaw with numerous, small, ndnA markers (Fig. 2) revealed a similar pattern of rela-
close-set teeth. Anterior third of each dentary with eight rath- tionship among the bythitin species. According to both
er small teeth in outer row and three larger teeth in inner row; topologies, B. multidens together with other Barathro-
posterior part of dentary with six recurved fangs. vomer with nus species (B. diaphanus Brauer 1906 and B. maculatus
five large, recurved fangs (three fangs and one small tooth Shcherbachev, 1976) and Sciadonus galatheae (nielsen,
on left and two fangs on right half of vomer). Palatines eden- 1969) represents a derived monophyletic clade, which has
tate. otolith oval, its greatest width 1 mm. operculum with a high values of Bayesian posterior probability and Ml
a weak bifurcate spine covered by skin. The 25 gill rakers on bootstrap support. Within this clade, B. multidens is located
first gill arch rather long and thin: 5 rakers on epibranchial, on the basal position, with other mentioned species more
15 on ceratobranchial and 5 on hypobranchial. Peritoneum closely related to each other. Mean estimate of inter-specific
transparent, with dark brown pigment cells laterally: a row nucleotide distances obtained for more informative RAG 1
Figure 2. – Phylogenetic relationships of Barathronus multidens: Bayesian consensus trees of ndnA-RAG1 (left) and mtdnA-16S (right)
sequences. numbers above branches indicate posterior probabilities and Ml bootstrap values out of 1000 replicates (only if greater than
0.5 and 50%, respectively).
Cybium 2018, 42(2) 139
Morphology and relationships of Barathronus multidens EvsEEnko et al.
of the group comprising B. diaphanus, B. maculatus and specimen of B. multidens described above. Common to them
S. galatheae (1.1 ± 0.4%), is almost three times less than the were also same placement of the eyes, providing binocular
distance between this group and B. multidens (2.7 ± 0.7%); vision, absence of the lens, and pale yellow colour of eyes.
on average, all other examined bythitin species differ by The similarity was reinforced by the presence of a single
3.4 ± 0.5%. large dark spot on the lateral surface of the caudal part of
the body on the left side in the compared specimens, which
was probably formed by a luminous tissue. however, these
DISCUSSION fishes differ markedly in the relative size of the eyes (11.6%
of the head length in our specimen vs. about 18% of the head
Morphologic analysis length in specimen from the Marianas). in this regard, the
Among the five known Atlantic species of Barathro- question about genus or species of the Marianas specimen is
nus (nielsen et al., 2015), the studied specimen differs still open. Probably it belongs to the genus Barathronus.
from B. bicolor in having the transparent peritoneum, from
B. parfaiti by the number of precaudal vertebrae (34 vs. 37), Phylogenetic position of Barathronus multidens
total number of vertebrae (77 vs. 84) and dorsal-fin rays (73 The unique data on genetic variation of B. multidens and
vs. 80-85), and by the total number of gill rakers on first gill its phylogenetic position within the family tree may lead to
arch (25 vs. 29-30), from B. linsi by the number of dorsal- an assumption that this species may be close to a common
fin rays (73 vs. 78) and anal-fin rays (62 vs. 69), and from ancestral form of Barathronus and sister a genus Sciadonus.
B. unicolor by the ventral flexure of the anterior vertebrae Although Barathronus and Sciadonus form a single mono-
(no flexure in present specimen). phyletic clade, B. multidens are located at the base of this
our specimen is most similar to B. multidens in meristic clade and markedly distant from other examined species of
characters and by the presence of a transparent peritoneum. Barathronus and Sciadonus, and it is likely that the latter
At the same time, it differs from B. multidens by the number species diverged recently considering the relatively small
of fangs on the vomer (five vs. 11), which could be caused inter-specific differences in comparison to other bythitids. It
by a delay in the appearance and late development of teeth in is possible to establish a complete internal phylogeny of the
our juvenile specimen, observed also in young of Barathro- group of former Aphyonidae (Barathronus, Sciadonus and
nus (nielsen et al., 2015). Aphyonus genera) in the future, when more dnA sequences
Comparing the published data and our materials on of unexamined and new species become available.
aphyonids (nielsen, 1969, 1984, 2015; nielsen et al., 2015),
one can note a large similarity in the morphology of the eyes Distribution
of Nybelinella and Barathronus, namely the presence of a Barathronus multidens is known from both the west-
small centrally placed pigmented part of the eye, and a nar- ern and eastern north Atlantic at depths between 3610 and
row black ring, surrounding the central spot. According to 4279 m (nielsen, 1984) and from the Mid-Atlantic ridge
Munk (1966), the eyes of N. erikssoni (nybelin, 1957) and (around 17°n) (Fig. 3) at depths between the surface and
B. affinis Brauer, 1906 are very much alike. They much 3227 m.
resemble B. bruuni nielsen, 1969 and B. parfaiti (nielsen,
1969). The eyes of the newly caught B. multidens speci-
men (Fig. 1) are yellow as in Ipnops murrayi Günther, 1878
(pers. obs.). herring (2002) mentioned that the retinal plate
of Ipnops and Nybelinella may be able to detect infrared
light. Another common feature concerns the position of the
eyes. This is clearly seen when comparing the holotype of
B. bruuni 39 mm Sl (nielsen, 1969), represented by juve-
nile, newly caught adult B. bicolor 117 mm Sl (nielsen et
al., 2015) and our juvenile specimen of B. multidens 45 mm
Sl (Fig. 1). Their eyes are directed forward, which probably
provides binocular vision.
it is of interest to note the recent information about the
first underwater video of an aphyonid fish, approximately 10
cm in length, in the area of the Mariana Trench at a depth
of about 2500 m, made during the noAA Ship Okeanos
Explorer cruise on the 1 July 2016 (Mundy, 2016). Judg-
Figure 3. – Sites of occurrences of Barathronus multidens: l: type
ing from the video, its general appearance is similar to the specimens, : juvenile 45 mm Sl.
140 Cybium 2018, 42(2)
EvsEEnko et al. Morphology and relationships of Barathronus multidens
Acknowledgements. – We thank the Scientific Leader of the 37th MUndY B., 2016. – http://oceanexplorer.noaa.gov/okeanos/explo-
cruise of ‘Professor logachev’, v.E. Beltenev, and the crew and rations/ex1605/dailyupdates/media/video/0701-fish/0701-fish.
scientific team for their help in obtaining the specimen used in our html
study. The cruise was organised by the Polar Marine Geosurvey MUnk o., 1966. – ocular Anatomy of Some deep-Sea Teleosts.
Expedition and financed through the Federal Subsoil resources dana report 13(70), 62 p. Copenhagen: A.F. host & Son.
Management Agency of the Ministry of natural resources of the
niElSEn J.G., 1969. – Systematics and biology of the Aphyoni-
russian Federation. The authors are thankful to Tina Molodtsova
dae (Pisces, ophidioidea). Galathea Rep., 10: 7-88.
for photographs of the specimen. This research was funded in
part by the RFBR grant № 16-04-00365 and RSF grant № 14-50- niElSEn J.G., 1984. – Two new, abyssal Barathronus spp. from
00095. the north Atlantic (Pisces, Aphyonidae). Copeia, 3: 579-584.
niElSEn J.G., 2015. – revision of the aphyonid genus Aphyonus
(Teleostei, ophidiiformes) with a new genus and two new spe-
cies. Zootaxa, 4039(2): 323–344.
REFERENCES
niElSEn J.G., MinCAronE M.M. & di dArio F., 2015. – A
CAMPBEll M.A., niElSEn J.G., SAdo T., ShinZATo l, new deep-sea species of Barathronus Goode & Bean from Bra-
kAndA M, SAToh T.P. & MiYA M., 2017. – Evolutionary zil, with notes on Barathronus bicolor Goode & Bean (ophidii-
affinities of the unfathomable Parabrotulidae: Molecular data formes: Aphyonidae). Neotrop. Ichthyol., 13(1): 53-60.
indicate placement of Parabrotula within the family Bythiti- PAlUMBi S.r., 1996. – nucleic Acids ii: the polymerase chain
dae, ophidiiformes. Mol. Phylogenet. Evol., 109: 337-342. reaction. In: Molecular Systematics (hillis d.M., Moritz C. &
hErrinG P., 2002. – The Biology of the deep ocean. 314 p. Mabl B.k., eds), 2nd edit., pp. 205-247. Sinauer Ass. inc, Mas-
oxford Univ. Press, oxford. sachusetts, USA.
kEAnE T.M., CrEEvEY C.J., PEnTonY M.M., nAUGhTon ronQUiST F., TESlEnko M., vAn dEr MArk P., AYrES
T.J. & MClnErnEY J., 2006. – Assessment of methods for d.l., dArlinG A., hÖhnA S., lArGET B., liU l.,
amino acid matrix selection and their use on empirical data SUChArd M.A. & hUElSEnBECk J.P., 2012. – MrBayes
shows that ad hoc assumptions for choice of matrix are not jus- 3.2: efficient Bayesian phylogenetic inference and model
tified. BMC Evol. Biol., 6: 29. choice across a large model space. Syst. Biol., 61: 1-4.
kEArSE M., Moir r., WilSon A. et al. [14 authors], 2012. – STAMATAkiS A., 2014. – rAxMl version 8: A tool for phyloge-
Geneious basic: an integrated and extendable desktop software netic analysis and post-analysis of large phylogenies. Bioinfor-
platform for the organization and analysis of sequence data. matics, 30(9): 1312-1313.
Bioinformatics, 28(12): 1647-1649. TAMUrA k. & nEi M., 1993. – Estimation of the number of
li C.h., orTÍ G., ZhAnG G. & lU G.Q., 2007. – A practical nucleotide substitutions in the control region of mitochondrial
approach to phylogenomics: the phylogeny of ray-finned fish dnA in humans and chimpanzees. Mol. Biol. Evol., 10: 512-
(Actinopterygii) as a case study. BMC Evol. Biol., 7: 44. 526.
MiYA M. & niShidA M., 1999. – organization of the mitochon- TAvAré S., 1986. – Some probabilistic and statistical problems in
drial genome of a deepsea fish, Gonostoma gracile (Teleostei: the analysis of dnA sequences, in some mathematical ques-
Stomiiformes): first example of transfer RNA gene rearrange- tions in biology. In: dnA Sequence Analysis (Miura r.M., ed.),
ments in bony fishes. Mar. Biotechnol., 5: 416-426. pp. 57-86. Providence, ri: Am. Math. Soc.
MøllEr P.r., knUdSEn S.W., SChWArZhAnS W. & YAnG Z., 1994. – Maximum likelihood phylogenetic estimation
niElSEn J.G., 2016. – A new classification of viviparous bro- from dnA sequences with variable rates over sites: approxi-
tulas (Bythitidae) – with family status for dinematichthyidae – mate methods. J. Mol. Evol., 39: 306-314.
based on molecular, morphological and fossil data. Mol. Phylo-
genet. Evol., 126: 391-408.
Cybium 2018, 42(2) 141
