New molecular sequences for two genera of marine planarians facilitate determination of their position in the phylogenetic tree, with new records for two species (Platyhelminthes, Tricladida, Maricola)
New molecular sequences for two genera of marine planarians facilitate determination of their position in the phylogenetic tree, with new records for two species (Platyhelminthes, Tricladida, Maricola)
Hee-Min Yang 0
Gi-Sik Min 0
0 Department of Biological Sciences, Inha University , Incheon , Republic of Korea 2 Naturalis Biodiversity Center , P.O. Box 9517, 2300 RA Leiden , The Netherlands 3 9-jo 9-chome 1-8, Shinkotoni, Kita-ku, Sapporo, Hokkaido , Japan
For the first time, molecular sequences of the 18S ribosomal DNA were generated for representatives of the genera Obrimoposthia Sluys & Ball, 1989 and Paucumara Sluys, 1989 of the suborder of the marine triclads, or Maricola, by analyzing the species Obrimoposthia wandeli (Hallez, 1906) and Paucumara trigonocephala (Ijima & Kaburaki, 1916). On the basis of this molecular data the phylogenetic position of these two genera in the phylogenetic tree of the Maricola was determined and compared with their position in the phylogeny based on the analysis of anatomical features. New records for these two species are documented and their taxonomic status is determined on the basis of histological studies.
eol>Antarctica; molecular phylogeny; new records; Obrimoposthia; Paucumara; South Korea
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Although the marine planarians or Maricola Hallez, 1892 form only a small suborder
of triclad flatworms, comprising approximately 80 species, they exhibit a rather great
anatomical diversity, which at times makes it difficult to recognize homological
character states and thus to analyse their phylogenetic relationships. The first comprehensive
study of the phylogenetic relationships among the marine triclads was undertaken by
and was based on anatomical and morphological features. As
morphological support for a monophyletic Maricola was postulated the autapomorphic presence
of adhesive papillae arranged in the ventral annular zone, the latter constituting an
autapomorphy for the entire group of triclads
. More recently,
correctly argued that this marginal band with adhesive papillae is a plesiomorphic
feature and therefore cannot support the presumed monophyly of the Maricola.
Nevertheless, recent phylogenetic studies on the triclads consistently recover the Maricola
as a monophyletic taxon
(see Charbagi-Barbirou et al. 2011, Sluys et al. 2014, Harrath
et al. 2016)
. The same molecular studies reveal relationships within the Maricola that
differ from those hypothesized by Sluys (1989), thus suggesting that eventually major
changes in the current taxonomy of the group may be necessary.
Unfortunately, the number of species incorporated in molecular phylogenetic
studies of marine planarians is rather small, as for only a handful of species gene sequences
are publicly available. Therefore, it is as yet not possible to draw firm conclusions about
the phylogeny, and thus the taxonomy, of maricolans based on such molecular studies.
In this paper we contribute to the solution of this problem by making available gene
sequences of two other species of marine planarian, Paucumara trigonocephala (Ijima
& Kaburaki, 1916) and Obrimoposthia wandeli (Hallez, 1906), which previously have
not been examined. On the basis of this molecular data we analyze the phylogenetic
position of the respective genera in the phylogenetic tree of the Maricola and compare
this result with their position in the phylogeny based on the analysis of anatomical
features. In addition, we document new records for these two species as well as their
taxonomic status, as deduced from histological studies.
Materials and methods
Collected worms were transferred live to the laboratory, where specimens of Paucumara
trigonocephala and Obrimoposthia wandeli were incubated under dark conditions at
temperatures of 18 °C and 4 °C, respectively. For morphological study specimens were killed
in 10% glacial acetic acid and, subsequently, fixed for 24 hours in Bouin’s fluid, and stored
in 70% ethanol. For histological processing specimens were first dehydrated in a graded
ethanol series, cleared in clove oil and then embedded in synthetic wax. Serial sections
were made at intervals of 5 mμ and 8 mμ, mounted on albumen-coated slides, stained
(see Winsor and Sluys 2018)
, and, subsequently, cover
glasses were mounted with DPX. Reconstructions of the copulatory complex were
obtained by using a camera lucida attached to a compound microscope. The material is
deposited in the National Institute of Biological Resources (NIBR), Seoul, Republic of
Korea and Naturalis Biodiversity Center, Leiden, The Netherlands (ZMA collection code).
Before performing the molecular analysis, specimens of P. trigonocephala and O.
wandeli were first starved for more than seven days. Genomic DNA was extracted,
using LaboPassM Tissue Mini Kit (Cosmogenetech, Seoul, South Korea), from either
starved live worms or from 100% ethanol fixed specimens that before fixation had
been starved also for seven days. We obtained two 18S ribosomal DNA sequences
from both species. To infer their position in the phylogenetic tree of the triclads we
constructed Bayesian Inference (BI) and Maximum-likelihood (ML) trees, using 24
planarians as ingroup and three fecampiid species as outgroup taxa (Table 1).
The 18S ribosomal DNA gene was amplified using Polymerase Chain Reaction
(PCR) with four primers: 1F, 4F, 7R, 9R
(see Carranza et al. 1996)
. The PCR
amplifications were conducted in a final volume of 35Lμ under the following conditions: 2 min
at 94 °C, 40 cycles of 20 s at 95 °C, 30 s at 45 °C, and 1min at 72 °C, and 5min at 72 °C
as a final extension. After purifying PCR products, using LaboPassM PCR Purification
Kit (Cosmogenetech, Seoul, Republic of Korea), 18S gene sequences were determined
from both strands by Macrogen Inc. (Seoul, Republic of Korea) by using the 3730xl
DNA analyser with the same primers that were used in the PCR. For constructing
the 18S rDNA phylogenetic tree, we used genomic data of 27 species, including three
outgroup taxa (Table 1). The sequences were aligned using MAFFT version 7 (Katoh
et al. 2017) (using the G-INS-i iterative refinement method and with the other options
set as default) and checked using BioEdit 220.127.116.11
. Regions that could not
be clearly aligned were excluded using Gblocks version 0.91b
(Castresana and Talavera
(with the option of half allowed gap positions and minimum length of a block
being 6). The final length of the alignment was 1,398bp. To find the best-fit
evolutionary model, we used Jmodeltest2
(Darriba et al. 2012)
. GTR+I+G model was selected
by applying the Akaike information criterion. We used Mr Bayes v 3.2
(Ronquist et al.
and RaxML 8.2.10
to infer phylogenies with the Bayesian
Inference (BI) and Maximum-likelihood (ML) method, respectively. For BI method, two
runs for 5 million generations and 25% burn-in was used under the GTR+I+G model.
For the ML analysis, we performed 10,000 replicates with the same model. BI and ML
trees were visualized using Figtree v1.4.3 and edited by using AdobeR PhotoshopR CS5.
Abbreviations used in the figures
common vas deferens;
The BI and ML phylogenetic trees showed the same topology (Figure 1). The suborders
Cavernicola Sluys, 1990, Continenticola Carranza et al., 1998, and several of their
inclusive lower taxa, form monophyletic groups. The Maricola Hallez, 1892 is also a
monophyletic group, although the relationships within this group do not always reflect
current taxonomy. For example, only the family Procerodidae Diesing, 1862 forms a
monophylum, while current families Bdellouridae Diesing, 1862, Uteriporidae
Böhmig, 1906 and Cercyridae Böhmig, 1906 form polyphyletic groups in our tree.
In the phylogenetic tree, the inferred positions of species belonging to the current
families Bdellouridae, Uteriporidae and Cercyridae are supported only by BI, as the
bootstrap supports for ML are < 75. The species Cercyra hastata Schmidt, 1861,
Sabussowia dioica (Claparède, 1863), Pentacoelum kazukolinda (Kawakatsu & Mitchell,
1984), Paucumara trigonocephala and Ectoplana limuli (Ijima & Kaburaki, 1916) form
an exception, in that their positions in our tree are supported by both ML and BI.
Paucumara trigonocephala and Obrimoposthia wandeli are currently classified as
belonging to the Uteriporidae. In our phylogenetic tree P. trigonocephala forms a
highly supported clade with Ectoplana limuli, currently also classified as an
Uteriporidae species. The position of O. wandeli is also inferred with high posterior
probability value in the BI tree and this species forms a clade with the family
Procerodidae, here represented by three species included in our analysis. However, in the ML
tree (not shown but with the same topology as the BI tree), the clade formed by the
three species of Procerodes and Obrimoposthia (Figure 1) had only a bootstrap
support value of 67.
The unidentified species Maricola sp. shows a very long branch that differs strongly
from the branch lengths of other maricolans. Together with the low support values
(bootstrap: 38; posterior probability: 0.63) this suggests that the molecular sequence
of Maricola sp. may be corrupted.
Systematic and integrative account
Order TRICLADIDA Lang, 1884
Suborder MARICOLA Hallez, 1892
Family UTERIPORIDAE Böhmig, 1906
Genus Paucumara Sluys, 1989
Paucumara trigonocephala (Ijima & Kaburaki, 1916)
Material examined. NIBRIV0000821277, Sacheon-si, Gyeongsangnam-do,
Republic of Korea (35°05'05"N 128°03'14"E), 7 June 2017, coll. H-M. Yang, sagittal
sections on 2 slide; ZMA V.Pl. 7279.1, ibid., sagittal sections on 3 slides; V.Pl. 7279.2,
ibid., horizontal sections on 1 slide; V.Pl. 7279.3, ibid., transverse sections on 6 slides.
ZMA V.Pl. 6807, shore of Lake Hi-numa, near the Park, Ibaraki-machi,
HigashiIbaraki-gun, Ibaraki Prefecture, Kantô Region, Honshû, Japan, 13 August 2007, coll.
S. Chinone, preserved specimens. ZMA V.Pl. 6810, ibid., 31 August 2007, coll. S.
Chinone, preserved specimens.
Comparative description and discussion. The external features and anatomy of
the specimens from South Korea correspond in all essential details to the
descriptions of this species published earlier
(see Sluys 1989, Sluys and Ball 1990, Sluys and
. Preserved specimens measured up to approx. 3 mm in length and
1 mm in width. In particular, the shape of the body and the position of the eyes in
living specimens (Figure 2) are very similar to the situation in Australian specimens,
as documented in
Sluys and Kawakatsu (2000)
. The two eyes are set
close to the mid-line of the body and positioned at a considerable distance posterior to
the anterior body margin.
The shape of the front end of the body is very characteristic: anterior to the eyes the
body first narrows to give rise to a kind of “neck” and then widens to form a triangular,
obtusely pointed head with broadly rounded auricles (Figure 2). At the level of the
auricles there is a broad, creamy-white patch that extends across the body. A similar kind
of patch is located immediately behind the eyes, albeit that it does not extend from one
lateral body margin to the other but is confined to mid-dorsum. Additional
creamywhite spots may be located directly in front of and behind the pharyngeal pocket and
at the very tip of the tail. In point of fact, each of the pharyngeal patches may actually
consist of two spots situated close together. These pharyngeal spots as well as the one at
the tip of the tail were not present in every specimen examined and neither were they
reported earlier in the available literature.
In the specimens from South Korea the entire dorsal surface is provided with a
brownish pigmentation. The pigment granules are more or less evenly distributed, but
accumulations occur in front of the eyes, where there is a broad, transverse band, and
in the form of a brown stripe on either side of the pharyngeal pocket and a band of
brown pigment running between the eyes. A brownish colouration, on both dorsal and
ventral body surface, was described also for specimens from northern Australia
and Kawakatsu 2000)
With respect to their anatomy, the South Korean animals exhibit a distinct lens
in each of their eyes (Figure 3) and a copulatory apparatus (Figs 4, 5) similar to that
documented for animals from other parts of the range of the species
(see Sluys and Ball
1990, Sluys and Kawakatsu 2000, and references therein)
. The penis papilla is a stubby
cone. Immediately after having penetrated the penis bulb, the vasa deferentia unite
to form a common vas deferens, which communicates with a much broader
ejaculatory duct. At its distal, ventral section the latter receives the openings of erythrophilic
penis glands. The sac-shaped copulatory bursa fills the entire dorso-ventral space; it is
connected with the common atrium by means of a bursal canal that is lined with an
infranucleated epithelium. The major portion of the bursal canal is rather wide and
irregularly shaped, but the part near its opening into the common atrium is narrow.
This lower, proximal portion of the bursal canal receives the separate openings of the
oviducts. Unfortunately, in specimens NIBRIV0000821277, ZMA V.Pl. 7279.1, and
ZMA V.Pl. 7279.2 we were unable to trace the oviducts and only in the transversally
sectioned specimen ZMA V.Pl. 7279.3 did we observe the oviducts separately opening
into the bursal canal. The entire bursal canal is covered with a well-developed,
subepithelial layer of circular muscle, followed by a thin layer of longitudinal muscle.
Sluys and Ball (1990)
Sluys and Kawakatsu
, the bursal canal receives along its entire length numerous openings of
erythrophilic glands, the cell bodies of which are located ectally to the surrounding coat of
muscle. These glands are different from the shell glands, which open into the bursal
canal ectally to the oviducal openings. In the present material we observed that indeed
at places a granular, erythrophilic secretion is discharged into the lumen of the bursal
canal. However, this secretion is not easy to detect and apparently in our specimens
this situation is much less developed than as described in the literature for other
specimens of Paucumara trigonocephala. We were unable to detect shell glands in our
animals from South Korea.
Previous records of Paucumara trigonocephala from Japan, Australia, the Bismarck
Archipelago and probably Hong Kong were summarized in
Sluys and Ball (1988)
Sluys and Kawakatsu (2000)
. We do here document a new locality for
Japan, viz., Lake Hi-numa; this is a lake that is connected with the sea and, thus, has
a low level of salinity. Histological sections have not been prepared of the preserved
specimens available from this locality (see Material examined) but a picture of a live
specimen (Figure 6) leaves little doubt about its specific identity. It is noteworthy that
this animal also exhibits the whitish patches in the pharyngeal region and at the tip of
the tail (see above).
Although the species was probably observed in Hongkong as early as 1857
our present animals from South Korea represent the first substantiated
record of P. trigonocephala from continental Asia. Here, the animals were collected
from brackish water with a low salinity level, the bottom consisting of mud with small
stones and being devoid of any aquatic vegetation. This habitat is in agreement with
the fact that all over its range the animals show the same characteristic ecology, in that
they live in low-salinity biotopes, which during ebb tide even may be entirely fresh
(Kaburaki 1922, Sluys and Kawakatsu 2000)
In the molecular phylogenetic trees generated by
Souza et al. (2018)
genus and species Sluysia triapertura Leal-Zanchet & Souza, 2018 is the sister-group
of the ectocommensal species Ectoplana limuli (Ijima & Kaburaki, 1916), albeit that
this relationship only has low support in their trees. In our tree (Figure1) the topology
has changed slightly in that P. trigonocephala has become the sister-species of E. limuli,
while S. triapertura forms part of a group of five species that constitutes the sister-taxon
of E. limuli plus P. trigonocephala. That E. limuli and P. trigonocephala share a
sistergroup relationship conforms with the current taxonomy of the Maricola, in which
both species are classified amongst members of the subfamily Ectoplaninae Bresslau,
Genus Obrimoposthia Sluys & Ball, 1989
Obrimoposthia wandeli (Hallez, 1906)
synonym: Procerodes sanderi [Hauser, 1987]
Material examined. NIBRIV0000813547, King George Island, South Shetland
Islands, 62°12'31"S – 58°47'42"W, 2 February 2017, coll. Hee-Min Yang, sagittal
sections on 11 slides; ZMA V.Pl. 7280.1, ibid., sagittal sections on 14 slides. The animals
were collected from a pebble beach; temperature of the sea surface water was 0~1°C;
at a depth of 10m water temperature was 1~0°C. Many worms were observed to be
attached to the seaweed Kelp.
ZMA V.Pl. 951.1, King George Island, South Shetland Islands, 1983, sagittal
sections on 15 slides; V.Pl. 951.2, ibid., whole mount on 1 slide; V.Pl. 951.3, ibid.,
sagittal sections on 13 slides; V.Pl. 951.4, ibid., sagittal sections on 13 slides; V.Pl. 951.5,
ibid., transverse sections on 19 slides; V.Pl. 951.6, ibid., horizontal sections on 8 slides.
Holotype Procerodes sanderi: MZU PL. 00290, sagittal sections on 48 slides (nos.
MZU PL. 00291, sagittal sections on 30 slides (nos. A788-821) of presumed
specimen of Procerodes sanderi from the original collection of J. Hauser.
Comparative description. Preserved specimens, collected in 2017, up to approx. 11 ×
3.5 mm, thus being somewhat larger than reported for preserved animals of Obrimoposthia
wandeli (Hallez, 1906), which measured 4–8 mm in length and 2.5–4 mm in width
. However, much of this variation in size may be due to different fixatives used in
the field, resulting in different states of contraction of the animals. Nevertheless, preserved
specimens of the sample ZMA V.Pl. 951 also were quite large, measuring 8-10 × 3–5 mm.
Dorsal surface of our animals from 2017 mottled blackish or dark brown, with a
pale mid-dorsal stripe, which is only weakly developed on the middle portion of the
body (Figure 7). At the front end of the body the dark pigmentation gives way to a
broad, pale patch on either side of the body, extending from the eyes towards the
antero-lateral body margins; further there is a pale patch on the mid-frontal body margin.
In this way the dark pigmentation on the head forms a kind of V-shaped pattern; the
same pattern was described for other specimens of O. wandeli
external appearance and colouration of our 2017 specimens fully agree with that of
specimens of P. sanderi as originally described in 1987
(see figure in Anonymous 1987)
In the specimens from the 2017 sample the small, rounded testes are situated
ventrally and extend from immediately behind the ovaries to somewhat posteriorly to the
copulatory apparatus, as may be the case also in other specimens of O. wandeli
. The anatomy of the penis papilla of these specimens from the 2017 sample is
precisely the same as that documented for O. wandeli
(see Sluys and Ball 1988, Sluys
and De Vries 1988, Sluys 1989)
The several specimens available from the population of King George Island revealed
the presence of intraspecific variability in the female reproductive system. Generally,
O. wandeli has been described as having a bursal canal that shows a distinct T-junction,
with the posterior branch of the T forming a kind of diverticulum that receives the
opening of the common oviduct
(see Sluys 1989, fig. 141)
. In some animals from King
George Island such a T-junction is indeed present (ZMA V.Pl. 951.1, V.Pl. 951.4, V.Pl.
951.6). However, in others, the situation is different in that in these animals the
common oviduct opens, via a constriction, into the bursal canal, which from there on runs
antero-dorsad and then gently curves rather abruptly ventrad to open into the common
atrium (Figs 9, 10). This portion of the bursal canal shows a clear, lateral bend during
its course towards the common atrium (Figure 11), as reported earlier for O. wandeli
. From the side of this obliquely, antero-dorsally running part of the bursal
canal arises a branch that runs more or less straight forward to communicate with the
copulatory bursa (Figure 9). Precisely the same situation is present in the holotype of P.
sanderi (Figure 12). In some presumed specimens of P. sanderi the branch that runs to
the bursa may even originate very close to the point where the common oviduct
communicates with the bursal canal (Figure 13).
The entire bursal canal, including its side branch, is lined with an infranucleated
epithelium and is surrounded by a thick, subepithelial layer of circular muscle,
bounded by a much thinner layer of longitudinal muscle. Oviducts and common oviduct
are lined with a nucleated epithelium and are surrounded by a thin layer of circular
muscles. The entire bursal canal is surrounded by a broad zone of unicellular glands,
which discharge their erythrophilic secretion into the canal. Erythrophilic shell glands
discharge their secretion into the ventral portion of the bursal canal, near its
communication with the common atrium.
Discussion. In an anonymous article in a bulletin, the late Josef Hauser described
the presumed new species Procerodes sanderi [Hauser, 1987]
Hauser was indeed the author of this article was apparent, for example, from the fact that
in 1988 and 1989 he corresponded on this subject with both Masaharu Kawakatsu and
Ronald Sluys and that he forwarded to these workers photocopies of the article.
Furthermore, in the article the new species is attributed to Hauser. In his article Hauser claimed
that the anatomy of P. sanderi was different from congeneric species, including species
currently assigned to the genus Obrimoposthia. Unfortunately, the article did not provide
a reconstruction drawing of the copulatory apparatus, while the short description of the
reproductive apparatus in the Portuguese language neither did make clear the anatomical
differences between the new species and its congeners. Furthermore, the material that
Hauser made available to both Sluys and Kawakatsu, consisting of printed photographs,
histological slides, and reconstruction drawings, at the time did not convince these two
workers that indeed the specimens represented a new species. As a result, in his
synonymized Procerodes sanderi with Obrimoposthia wandeli. In their
Sluys and Kawakatsu (2005)
reiterated their conclusion, as expressed
also in correspondence with Hauser, that the species P. sanderi is synonymous with O.
Nevertheless, examination of our new material collected in 2017, as well as
reexamination of specimens from King George Island that were collected in 1983 and
were part of Hauser’s samples, including a specimen that he had designated as the
holotype specimen of P. sanderi, revealed that at least within this population there is
clear intraspecific variation in the construction of the female copulatory apparatus.
In earlier studies
(e.g., Sluys and Kawakatsu 2005)
the deviant course of the bursal
canal in some specimens of O. wandeli from King George Island, i.e. absence of the
T-junction and origination of a duct from the side of the bursal canal, was not clearly
observed as no reconstruction drawings were made of the various specimens. The
present series of material that is available undeniably shows that the intraspecific variability
of this population is exhibited by animals collected both in 1983 and 2017. Therefore,
we do here consider this variability in the course of the bursal canal, as described above,
to be a constant, stable feature of at least the population from King George Island and
probably for other populations of O. wandeli as well.
One might contemplate an alternative explanation for the deviant course of the
bursal canal. As the specimens from King George Island were somewhat larger than
generally reported for O. wandeli (see above), one may view their copulatory apparatus
as having reached the final stage of maturation. However, although we can envision
structures becoming larger during maturation, we believe it to be unlikely for
anatomical organs to become structurally different. In other words, we consider it unlikely that
upon maturation a T-junction in the bursal canal will re-assemble in such a way that
it develops into a duct with a distinct loop from which originates a side-branch that
runs to the copulatory bursa. Therefore, we consider these different expressions of the
course of the bursal canal and its connection with the copulatory bursa to be the result
of intraspecific variation, independent of the stage of maturation.
In our phylogenetic tree (Figure 1) O. wandeli is the sister-group of the genus
Procerodes Girard, 1850. This reflects the taxonomic history of the current members of the
genus Obrimoposthia, most of which were formerly assigned to the genus Procerodes.
However, it became increasingly clear that in the past the genus Procerodes constituted
an unnatural assemblage of species that belonged to different natural groups
, one such group being formed by the present members of the genus
(Sluys and Ball 1988, Sluys 1989)
. This has resulted in the situation that in the
most recent taxonomy of the Maricola the genera Procerodes and Obrimoposthia are
even classified in different families, viz. Procerodidae Diesing, 1862 and Uteriporidae
Böhmig, 1906, respectively
(Sluys 1989, Sluys et al. 2009)
. In the phylogenetic tree of
the Uteriporidae based on morphological characters, the genus Obrimoposthia is closely
related to Paucumara Sluys, 1989 and Ectoplana Kaburaki, 1917
(Sluys 1989, fig. 302)
both belonging to the subfamily Ectoplaninae Bresslau, 1933. It is clear that in our
present tree (Figure 1) Obrimoposthia is rather far removed from Ectoplana and Paucumara.
We are grateful to Dr. S. Chinone for making available preserved animals and a
photo of a live specimen of Paucumara trigonocephala from Japan. Dr. A. Leal-Zanchet
(UNISINOS, São Leopoldo, Brazil) is thanked for making available the type specimen
of Procerodes sanderi. M. Hermsen (Naturalis Biodiversity Center) is thanked for the
digital rendering of the figures. The study was supported by a grant from the National
Institute of Biological Resources (NIBR), funded by the Ministry of Environment
(MOE) of the Republic of Korea (NIBR201801202, NIBR201722202).
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