Research Article |
Corresponding author: Gi-Sik Min ( mingisik@inha.ac.kr ) Academic editor: David Gibson
© 2018 Hee-Min Yang, Ronald Sluys, Masaharu Kawakatsu, Gi-Sik Min.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Yang H-M, Sluys R, Kawakatsu M, Min G-S (2018) 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). ZooKeys 781: 1-17. https://doi.org/10.3897/zookeys.781.26324
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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.
Antarctica, molecular phylogeny, new records, Obrimoposthia , Paucumara , South Korea
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
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.
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 killein 10% glacial acetic acid and, subsequently, fixed for 24 hour 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 μm and 8 μm, mounted on albumen-coated slides, stained with Mallory-Heidenhain/Cason (see
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
List of species taxa of which 18S rDNA gene data was used for constructing the phylogenetic trees, with accession numbers for gene sequences available from GenBank.
Classification | Species | GenBank number |
---|---|---|
Suborder Maricola Hallez, 1892 | ||
Family Cercyridae Böhmig, 1906 | Cercyra hastata | KM200902 |
Sabussowia dioica | JN009785 | |
Sabussowia ronaldi | KM200923 | |
Family Uteriporidae Böhmig, 1906 | Ectoplana limuli | D85088 |
Obrimoposthia wandeli | MH108586 | |
Paucumara trigonocephala | MH108587 | |
Sluysia triapertura | MF383119 | |
Uteriporus sp. | AF013148 | |
Family Bdellouridae Diesing, 1862 | Bdelloura candida | Z99947 |
Palombiella stephensoni | DQ666008 | |
Pentacoelum kazukolinda | KM200905 | |
Family Procerodidae Diesing, 1862 | Procerodes dohrni | JN009783 |
Procerodes littoralis | Z99950 | |
Procerodes plebeius | DQ665997 | |
Incertae sedis | Maricola sp. | KC869825 |
Suborder Cavernicola Sluys, 1990 | ||
Cavernicola sp. | KC869823 | |
Novomitchellia bursaelongata | KU096054 | |
Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguñà & Riutort, 1998 | ||
Family Dugesiidae Ball, 1974 | Dugesia gonocephala | DQ666002 |
Dugesia japonica | D83382 | |
Dugesia ryukyuensis | AF050433 | |
Dugesia subtentaculata | AF013155 | |
Family Planariidae Stimpson, 1857 | Crenobia alpina | M58345 |
Phagocata vitta | DQ665998 | |
Polycelis felina | DQ665996 | |
Order Fecampiida Rohde, Luton & Johnson, 1994 | ||
Kronborgia isopodicola | AJ012513 | |
Piscinquilinus sp. | AJ012512 | |
Urastoma cyprinae | AF167422 |
The 18S ribosomal DNA gene was amplified using Polymerase Chain Reaction (PCR) with four primers: 1F, 4F, 7R, 9R (see
bc bursal canal;
br brain;
ca common atrium;
cb copulatory bursa;
cod common oviduct;
cvd common vas deferens;
ed ejaculatory duct;
el eye lens;
go gonopore;
in intestine;
mo mouth opening;
od oviduct;
pb penis bulb;
pg penis gland;
ph pharynx;
pp penis papilla;
sg shell gland;
spt septum;
ug unicellular gland;
vd vas deferens;
vi vitellaria
The BI and ML phylogenetic trees showed the same topology (Figure
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
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.
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;
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
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
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 (
With respect to their anatomy, the South Korean animals exhibit a distinct lens in each of their eyes (Figure
According to
Previous records of Paucumara trigonocephala from Japan, Australia, the Bismarck Archipelago and probably Hong Kong were summarized in
Although the species was probably observed in Hongkong as early as 1857 (see
In the molecular phylogenetic trees generated by
synonym: Procerodes sanderi [Hauser, 1987]
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;
Holotype Procerodes sanderi: MZU PL. 00290, sagittal sections on 48 slides (nos. A-140/A-188).
MZU PL. 00291, sagittal sections on 30 slides (nos. A788-821) of presumed specimen of Procerodes sanderi from the original collection of J. Hauser.
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 (
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
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 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
Obrimoposthia wandeli. 12 MZUPL 00290-A163, microphotograph of sagittal section of copulatory apparatus of holotype of Procerodes sanderi; anterior to the left 13 MZU PL. 00291(nos. A788-821), sagittal reconstruction of the copulatory apparatus of presumed specimen of Procerodes sanderi.
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.
In an anonymous article in a bulletin, the late Josef Hauser described the presumed new species Procerodes sanderi [Hauser, 1987] (
Nevertheless, examination of our new material collected in 2017, as well as re-examination 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.,
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
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 (