Pleuronema marinum Dujardin, 1841

Yeo, Jeong Hyeon, Choi, Ji Hye & Jung, Atef Omar and Jae-Ho, 2022, New record of Pleuronema marinum Dujardin, 1841 (Protozoa, Ciliophora) from South Korea, Journal of Species Research 11 (4), pp. 278-286 : 279-284

Pleuronema marinum Dujardin, 1841 ( Figs. 1, 2 )

1841 Pleuronema marina - Dujardin, Histoire Naturelle zoophytes, p. 475, Planche XIV, Fig. 3 (original description).

1858 Pleuronema chrysalis Perty - Claparède & Lachmann, Mémoires de l’Institut National Genevois, 5: 274, Planche XIV, Fig. 8 (revision).

1866 Pleuronema chrysalis Perty - Diesing, Sitzungsberichte Akademie der Wissenschaften in Wien, Mathematisch-Naturwissenschaftliche Klasse Abteilung I, 5: 85 (revision).

1881 Pleuronema marina Dujardin - Kent, A manual of the infusoria: including a description of all known flagellate, ciliate, and tentaculiferous protozoa, British and foreign, and an account of the organization and affinities of the sponges. Volume II, p. 543 (revision).

1876 Pleuronema chrysalis - Fromentel, Études sur les microzoaires ou infusoires proprement dits comprenant de nouvelles recherches sur leur organisation, leur classification et la description des espèces nouvelles ou peu connues, p. 301, Planche XXI, Fig. 10, XXII, Fig. 16 (revision).

1885 Pleuronema marina (Dujardin) - Fabre-Domergue, Journal of Physiology, Paris, 21: 558, Planche XXIV Figs. 4, 5 (redescription, misidentification).

1931 Pleuronema marinum Dujardin, 1841 - Kahl, Tierwelt Deutschlands, 21: 389, Fig. 6526 (redescription).

1933 Pleuronema marinum Dujardin, 1841 - Kahl, Tierwelt der Nord- und Ostsee, 23: 84, Fig. 1041 (redescription, guide to marine ciliates).

1937 Pleuronema marinum Duj. - Noland, Transactions of the American Microscopical Society, 56: 169, Fig. 5D (redescription, misidentification).

1960 Pleuronema marinum Dujardin - Dragesco, Travaux de la Station Biologique de Roscoff, 12: 271, Figs. 140A, 142a (redescription, misidentification).

1963 Pleuronema marina Dujardin, 1841 - Borror, Ar-

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chiv für Protistenkunde, 106: 496, Figs. 82, 83, 86 (redescription, misidentification).

1964 Pleuronema marinum Dujardin, 1841 - Small, Dissertation, p. 3 (revision).

1968 Pleuronema marinum Dujardin, 1841 - Dragesco, Protistologica, 4: 86, Figs. 1A, 2 , 4A (redescription, misidentification).

1970 Pleuronema marinum Dujardin, 1841 - Burkovsky, Acta Protozoologica, 7: 483, Fig. 9 (redescription, misidentification).

1971 Pleuronema marinum Dujardin, 1841 - Agamaliev, Acta Protozoologica, 8: 386, Figs. 2 , 3A- C (redescription, misidentification).

1985 Pleuronema marinum Dujardin, 1841 - Aladro Lubel, Anales del Instituto de BiologÍa , Universidad Nacional Autónoma de México. Serie ZoologÍa, 55 (year 1984): 18, Lámina 9, Fig. 1 (redescription, misidentification) .

1986 Pleuronema marinum Dujardin, 1836 - Dragesco & Dragesco-Kernéis, Faune tropicale (Éditions de l’ ORSTOM), 26: 360, 95E- H (redescription, misidentification).

1992 Pleuronema marinum Dujardin, 1841 - Carey, Marine interstitial ciliates. An illustrated key, p. 144, Fig. 531 (misidentification, guide to marine interstitial ciliates).

1999 Pleuronema marinum Dujardin, 1836 - Alekperov & Asadullayeva, Turkish Journal of Zoology, 23: 218, Fig. 4 (redescription, misidentification).

2005 Pleuronema marinum Dujardin, 1836 - Alekperov,

An atlas of the free living ciliates (classes Kinetofragminophora, Colpodea, Oligohymenophora, Polyhymenophora), p. 162, Drawing 49.4, Fig. 15.2 (redescription, misidentification).

2014 Pleuronema marinum Dujardin, 1836 - Kadhim & Mahmood, Iraqi Journal of Science, 55: 661, Fig. 12 (misidentification).

2016 Pleuronema marinum Dujardin, 1841 - Pan, Hu,

Jiang, Wang & Hu, Journal of Eukaryotic Microbiology, 63: 290, Figs. 3 , 4, Table 1 (redescription based on stained specimens with 18S rRNA gene).

2021 Pleuronema marinum Dujardin, 1841 - Alekperov & TahirovА, Amurian Zoological Journal, 13: 492, Figs. 5, 6 (no description, but a micrograph of stained specimen available; misidentification).

Material examined. Estuarine water sample (12.6‰, 11.4℃) collected from Gyeonso-dong , Gangneung-si, Gangwon-do, Republic of Korea (37°46′12.8″N 128°56′ 57.2″E) on 24 January 2022 .

Diagnosis (n = 7). Cell size 100-145 × 30-45 μm in vivo (127-172 × 61-80 μm after wet silver nitrate impregnation), outline narrowly elliptical (obovate in wet silver nitrate preparations), posterior body end slightly narrower than anterior end, dorsoventrally flattened at posterior portion of body. One globular to irregularly-shaped macronucleus (irregularity more distinct in Bouin-fixed cells), 20.4-27.3× 13.5-24.4 μm in size; two to four globular to elliptical micronuclei attached to macronucleus, 3.3-4.8 × 2.6-3.6 μm in size; one or two preoral kineties; one contractile vacuole at right margin slightly below mid-body with one excretory pore; extrusomes filiform, straight or distinctly curved, forming cortical seam because perpendicularly arranged to pellicle; 57-64 somatic kineties, 10- 31 prolonged caudal cilia; buccal area occupying 59.9- 70.4% (64.9% on average) of body length; paroral membrane conspicuous, hook or ‘6’-shaped, proximal end slightly concave (looks like ‘3’); M1 2-rowed, but 3-rowed at distal end (rarely 2-rowed throughout); M2a more or less straight, proximal end curved leftward, 56.1-87.0 μm long, M2a two-rowed at distal and proximal end, one-rowed at middle portion; M1: M2a length ratio about 1: 5; M2b V-shaped, ratio of two arms 1.2-1.8: 1 (right: left), zigzag-pattern of basal bodies in right arm; M3 three-rowed.

18S rRNA gene. The SSU rDNA sequence of Pleuronema marinum is 1,605 base pairs long and has a GC content of 42.3% (OP175946). The genus Pleuronema is monophyletic with poor support (<50 bootstrap value; Fig. 3 ), except for Peniculistoma mytili and Mytilophilus pacificae which are nested inside. Conspecific pairwise similarity of P. marinum is 99.69% and inter-specific sim- ilarities range from 91.11% ( P. setigerum FJ 848874) to 97.38% ( P. sinica EF 486864). The sequence of the unidentified Pleuronema sp. (FJ868876) clusters with and shows higher similarity (99.13%, 99.44%) to both P. marinum populations than to other congeners. The sequence of P. sinica shows a sister relationship with the two sequences of P. marinum and the unidentified Pleuronema sp.

World distribution. Mediterranean Sea, Germany, China, and South Korea. Probably rare species (see below).

Remarks. Dujardin (1841) originally described two Pleuronema species, P. crassa and P. marina (now P. crassum and P. marinum ), and distinguished the two species by the body shape (more elongated in P. marinum ) and habitat (freshwater vs. seawater). Type locality of P. marinum is the Mediterranean Sea. Since the original description, there has been confusion about the validity of P. marinum . The issue seemed to be clarified by Kahl (1931), but it is still in progress ( Carey, 1992; Kadhim and Mahmood, 2014; Alekperov and TahirovА, 2021). Pan et al. (2016) described a Chinese population of P. marinum based on protargol-impregnated specimens and 18S rDNA sequence so that it allows us, as ‘good’ reference, to resolve that issue. In addition, Liu et al. (2022) reported four new species of Pleuronema . These new findings lead us to this monographic treatment of P. marinum as follows.

Perty (1852) synonymized P. crassum with P. chrysalis sensu Ehrenberg (1838) , while synonymizing P. chrysalis Müller, 1786 was not decided as mentioned on page 146. Later, Claparède and Lachmann (1858) synonymized P. crassum and P. marinum with P. chrysalis sensu Ehrenberg (1838) because Ehrenberg described Paramecium chrysalis in 1830 and 1838, which are possibly different species, and the latter was considered as Pleuronema crassa by Dujardin (1841). Considering either P. chrysalis Müller, 1786 or P. chrysalis sensu Ehrenberg (1838) , which were considered as the same species with P. marinum , the synonym is rather complex as reviewed by Small (1964). Diesing (1866) accepted the synonym and additionally included P. chrysalis Müller, 1786 in the list of synonyms, while Fromentel (1876) synonymized P. chrysalis sensu Ehrenberg (1838) and P. marina with P. chrysalis (not P. chrysalis Müller, 1786 ) and considered P. crassa as a valid species. Unfortunately, we did not find that Fromentel considered P. chrysalis Müller, 1786 as a valid species either of Paramecium or Pleuronema . Kent (1881) did not accept the synonyms because P. marinum has larger and longer body size than P. chrysalis and found in marine (vs. freshwater) habitat. Fabre-Domergue (1885) reported a wider form with a subterminal contractile vacuole. Considering the elongated body shape and the contractile vacuole in mid-body of P. marinum , however, the identification/synonyms cannot be accepted. For designating type species of the genus Pleuronema , it is also rather complex because the type is P. crassum (see above for the synonyms). For details, see Small (1964), Foissner et al. (1994), and Aescht (2001).

Kahl (1931) redescribed a German (Sylt) population of P. marinum , which was slightly longer than the type population (120-180 μm vs. 100 μm), and provided addi- tional diagnosis for the species: spherical macronucleus in anterior half of cell; contractile vacuole in right of mid-body; 30-35 somatic kineties on dorsal side. Kahl (1933) briefly described P. marinum collected from Kiel and Sylt. Unfortunately, the key diagnosis (e.g., the position of contractile vacuole) had been abandoned because it is probably that Pleuronema species usually have a terminal/subterminal contractile vacuole.

Noland (1937) described a population called P. marinum with the subterminal contractile vacuole and wider body, while he considered P. marinum sensu Kahl (1931) as a misidentification of Histiobalantium semisetatum Noland, 1937 which has multiple contractile vacuoles. Borror (1963) probably followed Noland and described a Floridan population based on stained specimens. As the Noland’s population, it had the subterminal contractile vacuole and wider body. Carey (1992) probably followed Borror’s classification and an incorrect year 1965 for ‘ Borror (1963) ’ was given in the guide to marine interstitial ciliates. Burkovsky (1970) described a population from the White Sea that differs from Borror’s population and other populations mentioned above in having a slender body and subterminal contractile vacuole as in P. sinica Wang et al., 2009 . However, P. marinum sensu Burkovsky (1970) can be distinguished from P. sinica by the number of somatic kineties (30-34 vs. 41-52) and preoral kineties (2 vs. 1).

Dragesco (1960, 1968) described P. marinum based on silver staining. However, the French populations had the wider body and the terminal contractile vacuole so that it obviously differs from the original and Kahl’s population. Later, Dragesco and Dragesco-Kernéis (1986) considered that P. roscoffensis Dragesco, 1968 might be a synonym of P. marinum . However, the main key to differentiate between P. marinum sensu Dragesco and P. roscoffensis is the presence vs. absence of the curvature at the proximal end of the paroral membrane. The curvature is 3-shaped and is apparent in both the Chinese ( Pan et al., 2016) and the present population of P. marinum .

Agamaliev (1971) described two populations from the Caspian Sea based on stained specimens: the first population is slender with 7-10 macronuclear nodules arranged longitudinally; the second population is characterized by the broad body, the 3 macronuclear nodules in mid-body, and the absence of membranelle 1. Both populations differ from all P. marinum populations mentioned above so that Agamaliev and Suleimanova (2004) established them as new species P. multinucleatum and P. ovatum , respectively. The new species, together with P. ovata Fernandez Leborans and Novillo, 1994 , seem to be not widely known for taxonomists because they are not mentioned in recent/relevant publications ( Wang et al., 2008a; 2008b; 2009; Pan et al., 2016; Liu et al., 2022) and include a homonym (e.g., ovata , ovatum ). Pleuronema ovata sensu Fernandez Leborans and Novillo (1994) differs from P. ovatum sensu Agamaliev and Suleimanova (2004) mainly by the presence (vs. absence) of membranelle 1 and the number of macronuclear nodules (1 vs. 3). For the validity of these species, further study is necessary.

Alekperov and Asadullayeva (1999) reported another population of P. marinum from the Caspian Sea. Further, they considered the Caspian Sea populations described by Agamaliev (1971) as exconjugants because of the nuclear apparatus ( Alekperov, 2005). In terms of the number of somatic kineties (35-38), they considered that it is similar to the White Sea population of P. marinum described by Burkovsky (1970). Even though they did not mention the position of the contractile vacuole, we confirm that the Alekperov and Asadullayeva’s population had the terminal/subterminal contractile vacuole because they did not denote the position when comparing with the populations with terminal/subterminal contractile vacuole. However, Alekperov and Asadullayeva’s population had shorter body length (60-75 μm in vivo). Alekperov (2005) considered that the Agamaliev and Suleimanova’s species (e.g., P. multinucleatum and P. ovatum ) are exconjugants so that the validity of the two new species was doubted. He also provid- ed a drawing and micrograph of P. marinum (wider form), but the latter might be another species of Pleuronema because the proximal end of membranelle 2 is curved to right.

Aladro Lubel (1985) reported a Mexican population with a brief description and illustration. It had rather slen- der body, but with a terminal contractile vacuole. Kadhim and Mahmood (2014) reported an Iraqi population collected from the Tigris River (Baghdad city) and provided a brief description with a single micrograph. However, it had a smaller body length (about 50 μm). Alekperov and TahirovА (2021) provided a micrograph of stained specimen collected from North-East Azerbaijan. They did not provide a description, but based on the stained specimen, we can conclude that it differs from P. marinum mainly by the wider body shape and the hook-shape of membranelle 2a.

The Korean population morphologically and genetically resembles the Chinese population reported by Pan et al. (2016) except for the nuclear apparatus (1 macronucleus with globular or irregular shape vs. usually 4-7 macronuclear nodules gathered slightly above mid-body or sometimes only 1 globular or irregular macronucleus) and the extrusomes (straight or slightly curved vs. only straight). Considering the number of macronuclear nodules, one of the main diagnostic features to identify ciliates, the Chinese population is peculiar and differs from the authoritative redescription by Kahl (1931) ( Wang et al., 2009). The high variation, however, might result from the sexual cycle in P. marinum (e.g., autogamy for the 4-7 nodules, interphasic for the 1 nodule) rather than co-occurrence of another congener ( Lynn, 2008).

Voucher slides. One slide with wet silver nitrate-impregnated specimens was deposited at the National Institute of Biological Resources (NIBRPR0000111138). Six slides (GUC005709 - wet silver nitrate-impregnated; GUC00 5713-5717 - protargol-impregnated) have been deposited in the Jung-lab (J.-H. Jung) in Gangneung-Wonju National University.

Figures

Fig. 2. Pleuronema marinum after wet silver nitrate (A, B, E, G) and protargol impregnation (C, D, F). A. Ventral view of a typical individual showing oral apparatus and silver line system. B. Lateral view showing the excretory pore near mid-body. C-E. Nuclear apparatus fixed in Bouin’s solution (C), concentrated mercury chloride (D), and Champy’s fixative (E). F, G. Oral apparatus. CY, cytopyge; DE, docked extrusome; EP, excretory pore; M1, 3, membranelles 1, 3; M2a, anterior part of membranelle 2; M2b, posterior part of membranelle 2; Ma, macronucleus; Mi, micronuclei; PM, paroral membrane. Scale bars=100μm.

Fig. 3. Maximum likelihood tree of 18S rRNA gene sequences. Bootstrap values are shown on the branches, while the values ≤50% were not shown.The scale bar represents five nucleotide substitution per 100nt.