Research Article |
Corresponding author: Hiroshi Kajihara ( kajihara@eis.hokudai.ac.jp ) Academic editor: Yasen Mutafchiev
© 2019 Taeseo Park, Sang-Hwa Lee, Shi-Chun Sun, Hiroshi Kajihara.
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:
Park T, Lee S-H, Sun S-C, Kajihara H (2019) Morphological and molecular study on Yininemertes pratensis (Nemertea, Pilidiophora, Heteronemertea) from the Han River Estuary, South Korea, and its phylogenetic position within the family Lineidae. ZooKeys 852: 31-51. https://doi.org/10.3897/zookeys.852.32602
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Outbreaks of ribbon worms observed in 2013, 2015, and 2017–2019 in the Han River Estuary, South Korea, have caused damage to local glass-eel fisheries. The Han River ribbon worms have been identified as Yininemertes pratensis (Sun & Lu, 1998) based on not only morphological characteristics compared with the holotype and paratype specimens, but also DNA sequence comparison with topotypes freshly collected near the Yangtze River mouth, China. Using sequences of six gene markers (18S rRNA, 28S rRNA, histone H3, histone H4, 16S rRNA, and COI), the phylogenetic position of Y. pratensis was inferred among other heteronemerteans based on their sequences obtained from public databases. This analysis firmly placed Y. pratensis as a close relative to Apatronemertes albimaculosa Wilfert & Gibson, 1974, which has been reported from aquarium tanks containing tropical freshwater plants in various parts of the world as well as a wild environment in Panama.
Anguilla japonica, brackish-water invertebrates, freshwater invertebrates, Yellow Sea
An explosive proliferation of unidentified, brackish-water heteronemerteans was observed in the Han River Estuary, South Korea, in the spring of 2013. Our morphological observation of the Han River ribbon worms indicated that they represent Yininemertes pratensis (Sun & Lu, 1998), a brackish-water heteronemertean known only by its original description from the Yangtze (Changjiang) River Estuary, China (for the nomenclature of the genus, see
Facing a plethora of undescribed species with dwindling number of experts, some nemertean taxonomists agreed that taxonomic descriptions of ribbon worms will have to shift from traditional, internal-anatomy-based style to histology-free one with a combination of high-quality external images and molecular phylogeny (
In this paper, we report the identity of Han River nemerteans based on morphological characteristics in comparison to the type material of Y. pratensis as well as DNA barcoding data from the type locality. Also, we infer the phylogenetic position of Y. pratensis among Heteronemertea based on a multi-locus molecular analysis.
Approximately 700 individuals of ribbon worms were collected from local fishermen’s glass-eel nets for Anguilla japonica, set at about 37°36'08"N, 126°48'23"E, in Goyang, South Korea, approximately 40 km upstream of the mouth of the Han River (Figs
List of specimens identified as Yininemertes pratensis (Sun & Lu, 1998) in this study with catalogue numbers at the National Institute of Biological Resources Invertebrate Section, Incheon, Korea (NIBR IV) and the Invertebrate Collection of the Hokkaido University Museum, Sapporo, Japan (ICHUM) as well as their sampling date and locality.
Catalogue number | Sampling date and locality | Remarks |
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NIBR IV 0000409587–0000409590 | 6 April 2015, Goyang, South Korea | > 300 individuals fixed in 10% formalin |
NIBR IV 0000409591–0000409595 | 6 April 2015, Goyang, South Korea | > 300 individuals fixed in 100% EtOH |
NIBR IV 0000409596–0000409617 | 6 April 2015, Goyang, South Korea | 22 voucher specimens used for DNA extraction |
NIBR IV 0000758851 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758852 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758853 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758854 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758855 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758856 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758857 | 13 May 2016, Bailonggang, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000758848 | 14 May 2016, Chongming, China | 1 specimen fixed in 70% EtOH |
NIBR IV 0000754958 | 14 May 2016, Chongming, China | 1 specimen fixed in 70% EtOH |
ICHUM 5259 | 6 April 2015, Goyang, South Korea | 8 specimens fixed in 10% formalin |
ICHUM 5260 | 6 April 2015, Goyang, South Korea | Serial transverse sections of the anterior portion of a specimen, Mallory trichrome, 36 slides. |
Small pieces of tissue taken from 22 specimens collected from the Han River and seven specimens from Yangtze River were used for total genomic DNA extraction using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Partial sequences of six gene markers (nuclear 18S rRNA, 28S rRNA, histone H3, and histone H4; mitochondrial 16S rRNA, and COI) were used for molecular analyses using the same primers published by
GenBank accession numbers of sequences determined in the present study from voucher specimens of Yininemertes pratensis (Sun & Lu, 1998) deposited in the National Institute of Biological Resources Invertebrate Collection, Incheon, Korea (NIBR IV).
To assess phylogenetic affinity of the Han River nemerteans, maximum likelihood (ML) analysis and Bayesian Inference (BI) were carried out with 31 lineid heteronemertean species for which the aforementioned six gene sequences were available in public databases (Table
GenBank accession numbers of sequences used in the present phylogenetic analysis (Histone H4 sequences indicated by asterisks (*) were kindly provided by Dr Sebastian Kvist).
18S rRNA | 28S rRNA | Histone H3 | Histone H4 | 16S rRNA | COI | Reference | |
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Apatronemertes albimaculosa Wilfert & Gibson, 1974a | JF293030 | HQ856860 | JF277733 | JF277666 | JF277587 | HQ848584 |
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Cerebratulus lacteus (Leidy, 1851) | JF293044 | HQ856857 | JF277728 | JF277653 | JF277575 | HQ848576 |
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Cerebratulus marginatus Renier, 1804 | JF293042 | HQ856858 | JF277729 | JF277652 | JF277576 | HQ848575 |
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Gorgonorhynchus albocinctus Kajihara, 2015 | LC010650 | LC010651 | – | – | – | LC010649 |
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Gorgonorhynchus cf. bermudensis Wheeler, 1940b | KF935300 | KF935356 | KF935412 | * | KF935467 | KF935517 |
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Kulikovia alborostrata (Takakura, 1898)c | – | AJ436877 | – | – | AJ436822 | AJ436932 |
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Kulikovia manchenkoi |
JF293035 | HQ856856 | JF277730 | JF277683 | JF277572 | HQ848574 |
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Lineus acutifrons Southern, 1913 | JF304778 | HQ856855 | JF277727 | JF277681 | JF277573 | GU590937 |
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Lineus bilineatus (Renier, 1804) | JF293041 | HQ856844 | JF277731 | JF277682 | JF277571 | – |
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Lineus lacteus (Rathke, 1843)e | JF293065 | HQ856850 | JF277725 | JF277656 | JF277584 | HQ848583 |
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Lineus longissimus (Gunnerus, 1770) | – | AJ436880 | – | – | AJ436825 | AJ436935 |
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Lineus ruber (Müller, 1774)f | JF293040 | HQ856853 | JF277718 | JF277655 | JF277583 | HQ848580 |
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Lineus sanguineus (Rathke, 1799)g | KF935301 | KF935357 | KF935413 | * | KF935468 | KF935518 |
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Maculaura alaskensis (Coe, 1901a)h | – | AJ436882 | AJ436981 | – | AJ436827 | AJ436937 |
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Micrura chlorapardalis Schwartz & Norenburg, 2005 | KF935292 | KF935348 | KF935404 | * | KF935459 | KF935512 |
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Micrura dellechiajei (Hubrecht, 1879) | KF935294 | KF935350 | KF935406 | * | KF935461 | KF935514 |
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Micrura fasciolata Ehrenberg, 1828 | JF293038 | HQ856846 | JF277721 | JF277660 | JF277585 | HQ848577 |
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Micrura ignea Schwartz & Norenburg, 2005 | JF293043 | HQ856859 | JF277734 | JF277664 | JF277588 | HQ848587 |
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Micrura purpurea (Dalyell, 1853) | JF293036 | HQ856845 | JF277726 | JF277663 | JF277577 | HQ848586 |
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Micrura verrilli Coe, 1901a | KF935288 | KF935344 | KF935400 | * | KF935455 | KF935508 |
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Micrura sp.i | KF935293 | KF935349 | KF935405 | * | KF935460 | KF935513 |
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Notospermus geniculatus (Delle Chiaje, 1828) | KF935295 | KF935351 | KF935407 | * | KF935462 | – |
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Notospermus sp. 1 (SK76) | KF935296 | KF935352 | KF935408 | * | KF935463 | KF935515 |
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Notospermus sp. 2 (SK65) | KF935297 | KF935353 | KF935409 | * | KF935464 | – |
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Notospermus sp. 3 (SK50) | KF935298 | KF935354 | KF935410 | * | KF935465 | KF935516 |
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Parborlasia corrugata (McIntosh, 1876) | JF293037 | HQ856851 | JF277732 | JF277662 | JF277578 | – |
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Parvicirrus dubius (Verrill, 1879) | – | AJ436885 | – | – | AJ436830 | AJ436940 |
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Pseudomicrura afzelii Strand & Sundberg, 2011 | GU445924 | GU445919 | – | – | GU445914 | GU392013 |
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Riseriullus occultus |
JF293031 | HQ856848 | JF277724 | JF277679 | JF277581 | HQ848581 |
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Tenuilineus bicolor (Verrill, 1892) | – | AJ436878 | AJ436980 | – | AJ436823 | AJ436933 |
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Zygeupolia rubens (Coe, 1895) | JF293045 | HQ856861 | JF277735 | JF277661 | JF277574 | HQ848585 |
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Yininemertes pratensis (Sun & Lu, 1998) | KY274047 | KY274069 | KY274091 | KY274113 | KY274025 | KY274003 | Present study |
Outgroup | |||||||
Baseodiscus mexicanus (Bürger, 1893) | KF935281 | KF935337 | KF935393 | * | KF935449 | KF935503 |
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Baseodiscus unicolor Stiasny-Wijnhoff, 1925 | KF935284 | KF935340 | KF935396 | * | KF935451 | KF935505 |
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PartitionFinder ver. 1.1 (
Using 29 sequences (22 from Korea, seven from China) of 658-bp partial COI gene, haplotype network analyses were performed with Network ver. 5.0.0.1 (available at http://www.fluxus-engineering.com) using median-joining method (
The external feature of the Han River nemerteans agreed with the original description of Y. pratensis in that these worms were variously dark brown, brick red, and tinged with violet sometimes (Fig.
In specimens collected from the Han River, the proboscis was not branched, and reddish in color (Fig.
Photographs of Yininemertes pratensis (Sun and Lu, 1998) taken in life. A A haul of a glass-eel net at the Han River Estuary, South Korea, on 6 April 2015 B magnification of a swarm of the same worms as in A from the Han River Estuary taken in the laboratory; arrow heads indicating the characteristic transverse narrow rings in the intestinal region C a specimen dug from clayey mud sediment with vegetation at Bailonggang in the Yangtze River Estuary, China, May 13, 2016 D a specimen dug from non-vegetated clay sediment at Chongming Island in the Yangtze River Estuary, China, 14 May 2016 E topotype from the Yangtze River Estuary showing an overview of whole specimen F topotype from China showing magnification of head, ventral view G topotype from China, magnification of intestinal region, showing the characteristic narrow transverse rings, indicated by arrow heads.
Yininemertes pratensis (Sun and Lu, 1998), photograph in life (A) and photomicrographs of transverse sections (B, D, E, G, H ICHUM 5260 C DH005C, paratype F DH005A, holotype). A Anesthetized state with proboscis partially protruded, NIBR IV 0000409596 B, C proboscis; large arrow heads indicating fibers contributing to muscle cross; small arrow heads showing rhabdoids D cerebral region showing type-3 neuron E, F foregut region, arrow heads indicating intra-epithelial somatic muscle fibers G, H cephalic region showing well-developed cephalic lacuna.
Lengths of the six gene markers determined for Korean and Chinese materials were: 16S, 507–508 bp; 18S, 1000–1003 bp; 28S, 1132 bp; COI, 658 bp; H3, 331 bp; and H4, 160 bp. Resulting ML tree (ln L = −51290.378661) and BI tree (harmonic mean of estimated marginal likelihood for two runs = −52096.68) were topologically more or less the same, with Y. pratensis being a sister of Apatronemertes albimaculosa Wilfert & Gibson, 1974 in both trees with 100% bootstrap support value and 1.0 posterior probability (Fig.
Median-joining and statistical parsimony networks were identical in shape, comprising eight haplotypes with a maximal difference of five mutations (Fig.
Maximum likelihood tree (ln L = −51290.378661) for heteronemerteans based on concatenated 18S rRNA, 28S rRNA, histone H3, histone H4, 16S rRNA, and COI dataset showing phylogenetic position of Yininemertes pratensis (Sun and Lu, 1998). Numbers near nodes are bootstrap values for maximum-likelihood analysis and posterior probability for Bayesian inference. Scale bar indicates the number of substitutions per site.
Median-joining network for eight haplotypes detected among 29 Yininemertes pratensis specimens (22 from Han River, Korea; 7 from Yangtze River, China; statistical-parsimony method yielded the same topology). Numbers in each circle (pie chart) indicate sample size which is proportional to the size of each pie diagram.
Number of individuals analysed for population genetic analysis, number of haplotypes, nucleotide diversity, haplotype diversity, Tajima’s D, and Fu’s Fs based on 658-bp partial COI gene sequences from populations of Yininemertes pratensis (Sun and Lu, 1998) in the Han River and Yangtze River Estuaries.
Locality | Number of individuals | Number of haplotypes | Nucleotide diversity (S.D.) | Haplotype diversity (S.D.) | Tajima’s D | Fu’s Fs |
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Han River Estuary, Korea | 22 | 8 | 0.001849 (0.001365) | 0.7316 (0.0897) | −0.80 | −3.87* |
Yangtze River Estuary, China | 7 | 3 | 0.000868 (0.000907) | 0.5238 (0.2086) | −1.23 | −0.92 |
total | 29 | 9 | 0.001632 (0.001234) | 0.6847 (0.0899) | −1.18 | −5.08** |
Because fundamental biological aspects of Y. pratensis such as diet, life duration, breeding season, reproductive strategy (semelparous/iteroparous) and mode (oviparous, viviparous, and ovoviviparous), and larval ecology (if the species produces larvae in the first place) are unknown, the causes for the Y. pratensis outbreaks since 2013 in the Han River Estuary, South Korea, are open to speculation. One of the potential factors conceivable to explain the Yininemertes outbreaks is that the species might be capable of asexual reproduction. Until recently, asexual reproductive capacity among Heteronemertea had been confirmed only in the lineid Lineus sanguineus (Rathke, 1799) and L. pseudolacteus (Gontcharoff, 1951) (cf.
The family Lineidae McIntosh, 1874 currently contains about 90 genera and 370 species of heteronemerteans, which are morphologically characterized by having horizontal lateral cephalic slits and three apical organs. Most are marine, but six species (each in a monotypic genus) have been described from freshwater or brackish-water habitat. These are Planolineus exsul Beauchamp, 1928 from Indonesia; Siolineus turbidus Du Bois-Reymond Marcus, 1948 from Amazon; Hinumanemertes kikuchii Iwata, 1970 from Japan; A. albimaculosa from freshwater tanks in Germany (
We are grateful to Dr Sebastian Kvist for sharing his unpublished histone H4 sequences; to Kwang-Soo Kim, Seul Yi, and Guang Xi for their help in collecting specimens from Shanghai; and to Professor Gonzalo Giribet for the information on a specimen deposited in the Museum of Comparative Zoology (MCZ IZ 135331). Dr Malin Strand (as a reviewer), two anonymous reviewers, and Dr Yasen Mutafchiev (as an editor) gave us thoughtful comments that improved earlier version of the manuscript. The present study was partially supported by a grant (NIBR201601111) from the National Institute of Biological Resources (NIBR) funded by the Ministry of Environment (MOE), Republic of Korea for TP; and Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (grant number 17K07520) for HK.