Rediscovery of the abyssal species Peniagone leander Pawson and Foell, 1986 (Holothuroidea: Elasipodida: Elpidiidae): the first record from the Mariana Trench area

Abstract

The elpidiid holothurian Peniagone leander Pawson and Foell, 1986 is recorded for the first time from the Mariana Trench area at a depth of 5 571 m. The type description of this species was based on only in-situ photographs. P. leander is re-described and illustrated based on a preserved material and in-situ photographs taken on the seabed. The 16S rRNA was also obtained from the specimen and submitted to GenBank. This is the fourth discovery of this species, extending its bathymetric range in the western Pacific Ocean.

Full text

Journal of Oceanology and Limnology
https://doi.org/10.1007/s00343-020-0067-9
Rediscovery of the abyssal species Peniagone leander Pawson
and Foell, 1986 (Holothuroidea: Elasipodida: Elpidiidae):
the fi rst record from the Mariana Trench area*
GONG Lin
1, 2, 3 , LI Xinzheng
1, 2, 3, 4 , XIAO Ning
1, 2, 3, ** , HE Lisheng
5 , ZHANG Haibin
5 ,
WANG Yong
5
1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao
266200, China.
3 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
4 University of Chinese Academy of Sciences, Beijing 100049, China
5 Department of Life Science, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya 572000, China
Received Jan. 27, 2020; accepted in principle Mar. 21, 2020; accepted for publication May 12, 2020
© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract The elpidiid holothurian Peniagone leander Pawson and Foell, 1986 is recorded for the fi rst
time from the Mariana Trench area at a depth of 5 571 m. The type description of this species was based on
only in-situ photographs. P . leander is re-described and illustrated based on a preserved material and in-situ
photographs taken on the seabed. The 16S rRNA was also obtained from the specimen and submitted to
GenBank. This is the fourth discovery of this species, extending its bathymetric range in the western Pacifi c
Ocean.
Keyword : Elpidiidae; abyssal zone; deep-sea holothurian; benthopelagic species; new record
1 INTRODUCTION
The genus Peniagone Théel, 1882 with 32 known
species (WoRMS, 2019) is the most species-rich in
the Elpidiidae family. The species of Peniagone are
widely distributed in the Atlantic, Pacifi c, Indian and
Antarctic Oceans. They have a rather wide vertical
distribution, from 400 m ( Peniagone vignoni
Hérouard, 1901, recorded in the Antarctic Ocean) to
8 300 m ( Peniagone azorica Marenzeller von, 1892,
recorded in the Kermadec Trench), and many are
benthopelagic (Pawson and Foell, 1986; Gebruk,
2008; Cross et al., 2012; Rogacheva et al., 2012,
2013). The expanded mouth tentacles and fused
posterior tube feet are adaptations to the benthopelagic
lifestyle (primarily for swimming) of the genus
(Gebruk, 2008; Rogacheva et al., 2013).
The large swimming holothurian Peniagone
leander Pawson and Foell, 1986 was fi rst described
based on in-situ photographs taken in the Clarion-
Clipperton Fracture Zone (14°45′N–13°50′N,
127°39′W–125°49′W), eastern central Pacifi c, at a
depth of approximately 4 400–5 000 m. The
observation was used to describe a new species on the
basis of the photographs alone, without details from
ossicles since no specimens were obtained. P . leander
is known from the abyssal zone, has rarely been
encountered and is very fragile similar to most
swimming holothurians. P . leander was observed and
sampled during the French Nodinaut cruise in 2005
(Fifi s and Scolan, 2005). Although a specimen of
P . leander was sampled during the cruise, its
description was not published. Fujikura et al. (2008)
reported the species from the Japan Trench and
* Supported by the National Key R&D Program of China (No.
2018YFC0309804), the China Ocean Mineral Resources Research and
Development Association Program (No. DY135-E2-3-04), the Strategic
Priority Research Program of the Chinese Academy of Sciences (No.
XDB42000000), and the Biological Resources Program, Chinese Academy
of Sciences (No. KFJ-BRP-017-38)
** Corresponding author: xiaoning@qdio.ac.cn

2 J. OCEANOL. LIMNOL.,
Nankai Trough areas at a depth of 3 764–4 836 m.
This was the third discovery of this species, and the
fi rst record of it in the western Pacifi c.
In June 2016, a biodiversity survey cruise of the
Mariana Trench was organized by the Institute of
Deep-sea Science and Engineering, Chinese Academy
of Sciences (IDSSE), using the R/V Xiangyanghong 9
and manned submersible Jiaolong . During the cruise,
three individuals of P . leander were observed. A
whitish specimen (individual #1) was collected by the
mechanical arm of the submersible Jiaolong . Another
whitish specimen (individual #2) and a light violet
specimen (individual #3) were only observed on the
video. In this study, we re-describe P . leander , giving
details of ossicles which were lacking in the original
description. This is the fi rst record of the species from
the Mariana Trench area.
2 MATERIAL AND METHOD
2.1 Specimen preservation
The specimen was collected by the mechanical arm
of manned submersible Jiaolong at a depth of 5 571 m
from the Mariana Trench area (11°48′N, 142°07′E).
After collection, the specimen was preserved in 95%
ethanol and deposited at the Institute of Deep-sea
Science and Engineering, Chinese Academy of Sciences.
2.2 Morphological observation
For scanning electron microscope (SEM)
examinations of ossicles, small pieces of tissue from
dorsal body wall, ventral body wall and tentacles of
the holothurian were macerated in sodium
hypochlorite solution, and thereafter rinsed several
times in distilled water before being transferred to
95% ethanol and air dried. The ossicles were then
transferred to an aluminum stub, coated with gold,
and observed under SEM (Hitachi S-3400N) at the
Institute of Oceanology, Chinese Academy of
Sciences (IOCAS).
2.3 Molecular analysis
Total genomic DNA was extracted from a small
piece of tissue using a Tissue DNA Kit (OMEGA
Qingdao, China) according to the manufacturer’s
instructions. Partial sequences of 16S rRNA genes
was amplifi ed with primers16S-AR/BR (Kessing et
al., 1989) with the following program: initial
denaturation for 5 min at 94°C, followed by 30 cycles
of denaturation for 30 s at 94°C, 30 s at 48°C, 1 min at
72°C, and a fi nal extension for 5 min at 72°C.
Polymerase chain reaction (PCR) amplifi cation was
carried out with a total reaction volume of 25 μL,
containing 12.5-μL Premix TaqTM (TaKaRa, Japan),
8.5-μL DNase free ddH2O, 1-μL each primer, 2-μL
template DNA. The 16S rRNA sequence was submitted
to GenBank with an accession number of MN567717.
Considering that only three species of Peniagone
have 16S rRNA sequences in GenBank and two more
species were classifi ed only to the genus level, we
added COI sequences to give a more comprehensive
phylogenetic tree to determine the taxonomic status
of P . leander . 16SrRNA and COI sequences of all
species in the Elpidiidae, including 5 species from
Peniagone and 5 additional species from fi ve genera
in the family Elpidiidae, were downloaded from
GenBank for phylogenetic analysis. Mitochondrial
sequence data of Peniagone sp. YYH-2013 and
Scotoplanes sp. TT-2017 were included here and
Psychropotes longicauda was used as the out-group
(Table 1).
The homologous sequences, including 9 sequences
of 16S rRNA and 8 sequences of COI genes, were
aligned using MUSCLE 3.8 (Edgar, 2004) under the
default parameters. The best-fi t model of DNA
substitution for each partitioned dataset were assessed
by ModelTest v3.7 (Posada and Crandall, 1998).
Finally, the concatenated dataset consisted of 1 044 bp
(16S/COI=459/585 bp), the alignment gaps were
represented as ‘-’ and the missing data were marked
with ‘?’.
RAxMLGUI v1.5 (Silvestro and Michalak, 2012)
Table 1 Details of specimens and GenBank accession
number used in this study
Family Species
GenBank accession number
16S COI
Psychropotidae Psychropotes longicauda KU987549.1 KU987478.1
Elpidiidae
Amperima robusta KX856728.1 KX874381.1
Elpidia glacialis HM196429.1
Peniagone diaphana KX856725.1 KX874384.1
Peniagone sp. YYH-2013 KF915304.1
Peniagone sp. AKM-2016 KX856726.1 KX874385.1
Peniagone incerta HM196399.1
Peniagone vignoni HM196397.1
Peniagone leander MN567717
Protelpidia murrayi KX856727.1 KX874382.1
Rhipidothuria racovitzai KX856729.1
Scotoplanes sp. TT-2017 LC416624.1

3GONG et al.: Rediscovery of the abyssal species P . leander
was used to conduct the maximum likelihood (ML)
analysis with the GTRGAMMA substitution model
for all partitions in the concatenated dataset. Mrbayes
3.2 (Huelsenbeck and Ronquist, 2001) was used to
conduct the Bayesian inference (BI) analysis, Markov
Chains were run for 2 million generations, with
sampling every 100 generations. The fi rst 25% of
trees were discarded as burn-in, and the remaining
trees were summarized in 50% majority rule
consensus tree to estimate the posterior probabilities.
Finally, we used Tracer v1.7 (Rambaut et al., 2018) to
diagnose the eff ective sample size (ESS) values for all
sampled parameters to make sure convergence was
reached.
3 TAXONOMY
Order Elasipodida Théel, 1882
Suborder Psychropotina Hansen, 1975
Family Elpidiidae Théel, 1879
Genus Peniagone Théel, 1882
Peniagone leander Pawson and Foell,
1986 (Figs.1–3)
Peniagone leander Pawson and Foell, 1986: 293–
299, Figs. 1–8, Clarion-Clipperton Fracture Zone,
14°45′N–13°50′N, 127°39′W–125°49′W, 4 400–
5 000 m; Fifi s and Scolan, 2005: 72–74, Fig.
Peniagone sp.1, Clarion-Clipperton Fracture Zone,
13°55.63′N, 130°12.20′W, 4 800 m; Fujikura et al,
2008: 288, Fig. 22.60, Japan Trench, 4 836 m, Nankai
Trough, 3764 m, coordinates unknown.
3.1 Type locality
Eastern central Pacifi c, 4 400–5 000 m.
3.2 Material examined
IDSSE Lab-2016-0629, 1 adult (length 230 mm),
Mariana Trench area, fi ne sand, St. JL-Dive121,
11°48′N, 142°07′E, 5 571 m, 29 June 2016.
3.3 Diagnosis
Body ovoid, not fl attened. Colour in life light
violet, pink to reddish-brown. Anus dorsal. Ten
tentacles. Velum broad, composed of two pairs of
dorsal papillae; the central pair fused; the outer pair
very short. Two pairs of short, pointed dorsal papillae
posterior to the velum. Tube feet, 2 short free in the
posterior part of the body and 4 posteriormost fused
forming a lobe. Dorsal ossicles with straight or
slightly curved arms up to 0.5 mm in length and
slightly curved apohyses 0.15–0.3 mm in length; both
arms and apophyses spinous. Ventral ossicles primary
crosses, with four arms 0.16–0.4 mm long, slightly
bent; short apophysies on each arm; occasionally
irregular ossicles are present on ventrum.
3.4 Description of the material from the Mariana
Trench area
Body cylindrical to ovoid, 230 mm in length
(observed on fresh material on board), body width
approximately 110 mm. Dorsal side with conspicuous
transverse wrinkles, ventral side convex, bulbous
posteriorly. Colour in vivo whitish to light violet;
colour in alcohol whitish. Skin from transparent to
semi-transparent.
Tube feet 2 pairs of small free in the posterior part
of the body and 4 pairs in the posteriormost portion of
the ventral sole, decreasing slightly in length towards
the mid-ventral line (Fig.2a & b). Posteriormost tube
feet fused forming a swimming lobe.
Velum short, broad, consisting of two pairs of
almost completely fused dorsal papillae. The central
pair of papillae longer than the outer pair. Another
two pairs of small, free and pointed papillae posterior
to the velum. These papillae inconspicuous.
Tentacles Ten. Tentacle stalks cylindrical, fairly
long (58 mm in a 230-mm-long individual). The
terminal discs bilobed on the aboral margin.
Ossicles Ossicles of dorsal skin: Peniagone -type,
consisting of a central stem 0.14–0.36 mm long with
four downwardly straight or slightly curved arms and
four slightly curved apophyses (Fig.3a–c); both arms
and apophyses spinous. Some variations occurred in
the arm angle of curvature (in some ossicles arms
slightly more curved than others). Maximum height
of Peniagone -type ossicle 0.8 mm.
Ventral ossicles fl attened crosses with a short stem
0.1–0.15 mm long and highly spinous arms bearing
four, short blunt spinous apophyses; arms 0.16–
0.4 mm long, apophyses 0.03–0.1 mm long (Fig.3d–e).
Side-branches common on the arms of ventral ossicles
(Fig.3f).
Tentacles containing curved spinous rods as well
as large spinous primary crosses (Fig.3g–k),
measuring up to 0.6 mm.
3.5 Remarks
Based on the body shape, velum morphology,
number and position of dorsal papillae, number and
arrangement of tube feet, we can confi rm that the
individuals from the Mariana Trench area belong to
Peniagone leander . However, the specimens from the
type locality diff er from those from the Mariana

4 J. OCEANOL. LIMNOL.,
Trench area by having a brownish color with a less
transparent body wall and a much more conspicuous
lateral ridge. In the Mariana Trench area individuals,
the body color in life ranges from whitish to light
violet, and the body wall is translucent or transparent,
with the internal organs visible through the body wall.
An important characteristic missing in the original
description is one additional pair of free tube feet
developed close to the central ventral ambulacrum.
These tube feet can be seen on the in-situ images in
Pawson and Foell (1986) and Fujikura et al. (2008).
Descriptions of ossicles of P . leander are given for
the fi rst time.
3.6 GenBank accession number
MN567717 (16S rRNA).
3.7 Molecular data
The phylogenetic trees constructed using BI (Fig.4)
and ML (Fig.5) analyses were generally congruent
however the genera Rhipidothuria and Elpidia
showed diff erent topological structure. The phylogeny
shows that Peniagone leander and other Peniagone
species form a separate clade within the family
suggesting the monophyly of the genus, with support
values >0.7 for both the ML and BI analyses.
3.8 Distribution
Eastern central Pacifi c, 4 400–5 000 m; Nankai
Trough and Japan Trench areas, 3 700–4 840 m;
Mariana Trench area, 5 571 m.
3.9 Relationship
Peniagone leander most closely resembles the
other swimming sea cucumber, P . diaphana (Théel,
1882), in the morphology of its velum and number
and arrangement of tube feet. Peniagone leander
diff ers from P . diaphana by having a more infl ated
body, two pairs of dorsal papillae posterior to the
velum, more or less conspicuous transverse wrinkles
on the dorsal surface, and an extra pair of free tube
feet close to the medioventral line. However, the
ossicles in P . diaphana are slightly smaller, with arm
lengths usually 0.2–0.4 mm (Hansen, 1975; Gebruk,
1990).
4 DISCUSSION
4.1 In-situ observation of Peniagone leander
Pawson and Foell, 1986
Three individuals were observed in the video. The
body of individual #1 was approximately parallel to
the seafl oor with its postanal fan touching the sediment
surface and mouth tube above the seabed (Fig.1l). A
total of 4 minutes elapsed from the time when we fi rst
found the specimen to when we were able to collect it
using the mechanical arm of the submersible. Within
those 4 minutes, the specimen had moved its body
slightly.
Individual #2 and individual #3 were hanging
above the substrate (Fig.1k), and their bodies were
undulating on the current as if they were at “rest”.
Approximately 15 seconds later, individual #3 began
to bend towards the seabed and expanded tentacles
with bilobed aboral margins to ingest material from
the seabed (Fig.1n–o). We tried to obtain individual
#3 using a sediment push-core but failed.
As we operated a sediment push-core near
individual #2, the sediments were stirred into the
seawater, and individual #2 began to take off from the
seabed approximately 4 minutes later. After leaving
the seabed, it swam in an elegant pattern. The
propulsion was accomplished by the stroke of both
the mouth tube and posterior tube feet. When the
mouth tube looked upward, the postanal fan fl exed to
the dorsal and the tentacles expanded, showing an “S”
curve (Fig.1a). Then, the mouth tube and postanal fan
fl exed to the ventral, and the tentacles began to
gradually fold (Fig.1b–e). Over approximately
5 seconds, the oral tentacles shifted position, from
horizontal to vertical. Next, the mouth tube and
postanal fan began to fl ex to dorsal and the tentacles
began to gradually expand (Fig.1f–j). The oral
tentacles shifted position from vertical to horizontal
over approximately 5 seconds. Then, these movements
were repeated in slow, rhythmic strokes.
Peniagone leander was not sensitive to the
submersible’s lights, its disruption of the sediments or
the sounds caused by operating the mechanical arm.
They were not advanced swimmers and in the
presence of danger, they need some time before they
are able to take off from the seabed. Although we
were unable to consistently observe the swimming
posture of Peniagone leander , we consider it a
frequent swimmer, unlike Peniagone diaphana ,
which is known as a preferential swimmer and can
spend more than 12 h in seawater without contacting
the substrate (Barnes et al., 1976).
4.2 Systematic status of Peniagone leander Pawson
and Foell, 1986
The phylogeny of the Elpidiidae, which is based on

5GONG et al.: Rediscovery of the abyssal species P . leander
abcd
ef g h
ijkl
onm
Fig.1 Peniagone leander Pawson and Foell, 1986
a–k. underwater photographs of individual #2: a–j. diff erent phases of swimming; k. “hang” in the water; l–m. individual #1, catalog number IDSSE Lab-
2016-0629: l. in situ on the seabed; m. ventral view when unfi xed on deck; n–o. individual #3 in situ on the seabed.

6 J. OCEANOL. LIMNOL.,
morphology, has been addressed repeatedly. Théel
(1882) established the original classifi cation of
Elpidiidae based on the external characteristics.
Perrier (1902) primarily based his system on specimen
ossicles. Hérouard (1923) regarded the genera of
Elpidiidae as belonging to two evolutionary lineages:
the fi rst branch was based on a three-radial ossicle
type, whereas a four-radial (cross-shaped) ossicle
type served as the basis for the second branch. Ekman
(1926) divided the Elpidiidae into two subfamilies,
Elpidiinae and Peniagoninae. Although based on
completely diff erent theories, the two subfamilies
were identical to the two evolutionary lineages
assumed by Hérouard (1923). Hansen (1975) rejected
the views of Hérouard and Ekman. He concluded that
the ossicles do not justify a division of the family into
two subfamilies. Gebruk (1983) proposed a new view
of the phylogenetic relationships within the Elpidiidae.
The genera of the Elpidiidae may be divided into
three evolutionary branches characterised by the
formation of specifi c types of ossicles and the
structure of the peripharyngeal calcareous ring. One
of the evolutionary branches included Psychrelpidia
and Rhipidothuria , which have typical cross-shaped
ossicles only and paired gonads, lack a rectal caecum
and have up to 8 pairs of arms on the segments of the
peripharyngeal calcareous ring (in Psyhrelpidia ). The
second branch included Peniagone and Achlyonice ,
which have up to 12–15 pairs of thin long arms on the
segments of the peripharyngeal ring (Hansen, 1975).
The third branch included Scotoplanes , Protelpidia ,
Ellipinion , Amperima , Kolga , Irpa , and Elpidia ,
which have C-shaped spicules or very unusual
ossicles and up to 4 or 5 pairs of arms on the segments
of the peripharyngeal calcareous ring.
There are few works on the molecular phylogeny
of the Elpidiidae, and this group has only recently
been explored simply through molecular approaches
recently (Miller et al., 2017). In our tree, we increased
the family Elpidiidae by 11 species and 6 genera, and
the tree was generally consistent with previous results.
All the species of Peniagone clustered together into a
strongly supported monophyletic clade (Bayesian
posterior probabilities is 0.99, bootstrap value is
71%). Peniagone diaphana was a sister group to
Peniagone leander and both of them have an ovoid or
fl atten body shape, almost reduced tube feet and
weakly developed velum. Peniagone incerta was a
sister group to Peniagone vignoni and both of them
have an elongate or somewhat fl attened body shape.
In another clade, Scotoplanes was a sister group to
Protelpidia , which was consistent with the foundings
of Gebruk (1983) that Scotoplanes is most closely
related to Protelpidia . The characteristics shared by
the two genera included the presence of straight rod-
shaped ossicles with complex structures, number and
localisation of dorsal papillae, and numbers of tube
feet pairs. Amperima was a sister group to Scotoplanes
+ Protelpidia , which was also consistent with the
fi ndings of Gebruk (1983), who found that Amperima
has a close relationship with Scotoplanes and
Protelpidia . All of these genera have ossicles with
numerous small C-shaped elements. In the Bayesian
tree, Rhipidothuria was a sister group to Elpidia ,
while in the ML tree, Rhipidothuria was a sister group
to Scotoplanes + Protelpidia + Amperima . Gebruk
(1983) suggested that Elpidia was located on the third
evolutionary branch, but Elpidia was very distantly
related to the other genera within the third branch
based on molecular data. Rhipidothuria was most
closely related to Psychrelpidia according to Gebruk
(1983), and Psychrelpidia was not included in our
analysis. We cannot know whether the formation of a
clade containing Rhipidothuria and Psychrelpidia is
supported by molecular data. Since the status of
Elpidia is not consistent between molecular data and
morphological characteristics and some of the closely
related genera (based on morphology) were not
included in the present analysis, more extensive taxon
coverage and more molecular markers are required in
further studies to elucidate the relationships among
the genera of Elpidiidae. However, the phylogeny
based on molecular data was roughly consistent with
the morphology-based tree suggested by Gebruk
(1983). Thus, specifi c morphological details of
ab
3 mm
Fig.2 Peniagone leander Pawson and Foell, 1986, IDSSE
Lab-2016-0629
a. dorsal view; b. ventral view.

7GONG et al.: Rediscovery of the abyssal species P . leander
ab
c
d
e
f
g
h
ij k
h
0.4 mm
a–c
0.3 mm
d–h
100 µm 100 µm 100 µm
Fig.3 Images of ossicles from Peniagone leander Pawson and Foell, 1986
a–c. dorsal body wall; d–f. ventral body wall; g–k. tentacle.

8 J. OCEANOL. LIMNOL.,
ossicles and peripharyngeal calcareous rings can be
reliable synapomorphies for the elpidiid genera.
5 DATA AVAILABILITY STATEMENT
The authors declare that the data supporting the
fi ndings of this study are available within the article.
The original data of measurements will be available
on request from the fi rst author.
6 ACKNOWLEDGMENT
We are grateful to the crews of the R/V
Xiangyanghong 9 for their support in collecting the
deep-sea specimens during the cruises.
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0.04
Peniagone vignoni
Rhipidothuria racovitzai
Amperima robusta
Psychropotes longicauda
Elpidia glacialis
Peniagone leander
Peniagone diaphana
Peniagone incerta
Protelpidia murrayi
Scotoplanes sp. TT-2017
Peniagone sp. AKM-2016
Peniagone sp. YYH-2013
1
-
0.99
0.93 1
0.75
0.99
0.97 1
Elpidiidae
Fig.4 Phylogenetic tree obtained of Bayesian inference
based on the combined data set of 16S rRNA and
COI gene sequences
Numbers at each node are Bayesian posterior probabilities. Only
values ≥ 0.5 are shown.
Peniagone vignoni
Peniagone leander
Peniagone diaphana
Amperima robusta
Scotoplanes sp. TT-2017
Peniagone sp. AKM-2016
Psychropotes longicauda
Rhipidothuria racovitzai
Peniagone sp. YYH-2013
Protelpidia murrayi
Elpidia glacialis
Peniagone incerta
-
0.03
82
51
71
94
84 82
56 96
Elpidiidae
Fig.5 Phylogenetic tree obtained of ML based on the
combined data set of 16S rRNA and COI gene
sequences
Numbers at each node are ML analysis bootstrap values. Only
values ≥ 50% are shown.

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