Plagiopyla ramani, Nitla & Serra & Fokin & Modeo & Verni & Sandeep & Kalavati & Petroni, 2019

CONSIDERATIONS ON PLAGIOPYLA RAMANI SP. NOV. AND PLAGIOPYLA NARASIMHAMURTII SP. NOV.

The two newly described species clearly belong to the genus Plagiopyla , owing to the presence of typical morphological traits and due to the molecular results. Indeed, the 18S rRNA based phylogeny showed that the two new Plagiopyla species fall within the riboclass Plagiopylea in the clade of the family Plagiopylidae , clustering with the Plagiopyla spp. already present in the database (i.e. P. frontata and P. nasuta ).

Identification at species level was somewhat more challenging. Indeed, available morphological data regarding the species included in the genus are scarce. Descriptions of other species, especially in former literature, generally lack some morphological information that would have been important for species discrimination (i.e. presence and number of vacuole pore/s, presence and morphology of extrusive organelles, cytoproct-related ciliary rows, etc.). Also, morphometric data are generally fragmentary or missing. In the present paper, we report an overview of past species descriptions, which are summarized in Table 7, where the scarcity of available details is evident. Nevertheless, we succeeded in discriminating P. ramani and P. narasimhamurtii , as true novel freshwater species, describing them according to the most recent criteria of integrative taxonomy.

If compared with other Plagiopyla species from the freshwater environment, i.e. P. nasuta , P. simplex and P. megastoma , the two newly described species have reduced body sizes and a lower number of somatic kineties.

In detail, P. ramani is the only species of the genus so far described with multiple vesicular micronuclei. Additionally, it shows 2–5 pores of contractile vacuoles and a curved type of extrusomes: this combination of features has never been reported before for any other freshwater Plagiopyla species (for further comparison see Table 7).

As for Plagiopyla narasimhamurtii , it has some traits in common with other congeners, such as the overall body shape and the nuclear composition, with one ovoid macronucleus and one compact-type micronucleus (except for P. ramani and P. stenostoma , which have several micronuclei). However, P. narasimhamurtii has a peculiar trait that allowed us to propose this species as novel, e.g. the presence of two types of extrusomes, differing in appearance and structure; this feature has never been reported for any other Plagiopyla species, except for P. nasuta according to de Puytorac et al. (1985) (see discussion of P. nasuta and for further comparison see Table 7). Straight extrusomes of P. narasimhamurtii closely resemble ‘extrusome type I’ of Sonderia vorax at ultrastructural level; interestingly, the latter plagiopylid carries two types of such structures as well ( Modeo et al., 2013).

CONSIDERATIONS ON PLAGIOPYLA NASUTA STEIN, 1860

Plagiopyla nasuta View in CoL was described for the first time by Stein (1860) who provided a brief report. It was later recorded by other authors without providing any comprehensive account ( Kent, 1880 –82; Engelmann, 1862). The first proper description was presented by Levander (1894). Later, reports of P. nasuta View in CoL were frequent: this species has been described many times by different authors who made remarkable efforts to depict its features ( Roux, 1899; Penard, 1922; Wetzel, 1928: Kahl, 1931; Jankowski, 1964; Mahajan & Nair, 1971; Bick, 1972; Dragesco, 1972; Agamaliev, 1978; Dragesco & Dragesco-Kerneis, 1986; Sola et al., 1988; Foissner et al., 1995; Şenler & Yildiz, 2004).

Plagiopyla nasuta View in CoL described by Wetzel (1928) was indicated by Kahl (1931) as P. nasuta var. wetzeli View in CoL due to the narrower body shape and the smaller cell size. Jankowski (2007) elevated this variety to the rank of species, Plagiopyla wetzeli . In the present study, we consider the organism described by Wetzel as P. nasuta View in CoL , because we could not detect significant differences with the original description of this species.

Similarly, Plagiopyla varians , described by Maskell (1886) from Wellington ( New Zeland ) is hereby considered as P. nasuta View in CoL , because its morphological description and the provided pictures do not highlight significant differences to P. nasuta View in CoL , except for the presence of two contractile vacuoles. Maskell describes a freshwater organism, 125 µm in length, with a single macronucleus and two contractile vacuoles, ‘…resembling P. nasuta, Stein View in CoL , but differs in the position of the nucleus, the two contractile vesicles, and the variation of the oral fossa’. A similar organism has been more recently described by Vuxanovici (1963) who described it as P. nasuta var. bivacuolata .

Despite all these contributions, P. nasuta has never been redescribed up to now according to requirements of integrative taxonomy (morphology, ultrastructure and molecular characterization of a marker gene).

Concerning molecular analysis, only a single 18S rRNA sequence of P. nasuta was available in on-line databases (accession number Z29442 View Materials ) before our study; this sequence was provided by Embley and colleagues ( Embley et al., 1995), allowing in the mid-1990s the first phylogenies of plagiopylids together with P. frontata and Trimyema sequences. Due to the sequencing technology available at the time, it is possible that their sequence could have included nucleotide errors; indeed, four nucleotides were not properly resolved. Remaining differences between that and our sequence are represented by five mismatches, four of which are present in highly conserved columns when aligned with other sequences from Plagiopylea. Therefore, it is feasible that only the remaining mismatch located in a variable site represents a real difference between the two populations, which are then extremely similar.

In the morphological analysis, we focused our attention on the same traits proposed by previous authors, as well as on new, in our view significant, morphological details. Thus, we produced a detailed formal redescription based on a more complete set of morphological and morphometric data, also enriched by the results of the first SEM investigation of this species.

Our morphometric analyses agree with some of the previous studies while deviating from other

MIN: minimum value; MAX: maximum value; X: arithmetic mean; SD: standard deviation; CV: coefficient of variation (%); N: number; n: number of specimens analyzed; SI: silver impregnation; SEM: scanning electron microscopy; FE: Feulgen staining.

descriptions ( Table 8). Indeed, in past reports the size of P. nasuta varied conspicuously in length, ranging from 50–70 µm ( Wetzel, 1928) to 100–240 µm ( Kent, 1880 –82). The original description by Stein (1860) did not report any precise measurements for the species, but he mentioned that its size was almost identical to that of Pleuronema chrysalis (i.e. about 70–120 µm in length); Kahl (1931) described a P. nasuta about 80–180 µm long, presumably measured in live conditions. Plagiopyla nasuta redescribed in the present work does not vary so greatly either in live or in stained specimens ( Table 6), and is much more similar in size (i.e. 86–100 µm in length) to those reported by Levander (1894) and Sola et al. (1988) ( Table 8).

COMPARISON WITH PREVIOUS DESCRIPTIONS OF PLAGIOPYLA NASUTA

In our study, the use of silver staining and SEM analysis allowed the detection of some features never reported before, which should be considered, in our opinion, for diagnostic purposes. In detail: (1) in our P. nasuta from Kolleru, the striated band terminates beneath the equatorial line of the body, in proximity to the anterior region of the cytoproct, at a distance of four parallel somatic kineties from the latter. Our observation agrees with the schematic drawings by de Puytorac et al. (1985), but disagrees with the study of Sola et al. (1988), in which the striated band terminates in contact with the cytoproct. Foissner et al. (1995) focused on the presence of the striated band, which they considered a sensory structure, although not mentioning where it terminates. In our study, this structure gains importance as an additional diagnostic trait, since we have also introduced its length as an additional feature in morphometric analysis. (2) Several authors described the composition of oral and somatic ciliature ( Jankowski, 1964; Bick, 1972; de Puytorac et al., 1985; Dragesco & Dragesco-Kerneis, 1986; Sola et al., 1988; Foissner et al., 1995), although none of them gave an exhaustive description of their junction. Indeed, all interpreted the somatic cilia in continuity with the oral kineties. The present morphological analysis clarifies this issue by revealing the presence of a small gap between the somatic cilia and the oral lips. (3) The micronucleus had always been recorded by past authors, although none of them gave a precise definition regarding its composition. After our comparative analysis among different Plagiopyla species we have decided to define the micronucleus of P. nasuta as ‘compact-type micronucleus’, because it is formed by a compact mass of chromatin. This definition was used by Fokin ( Fokin, 1997; 2010 /2011) for the description of Paramecium species, but also seems appropriate for the genus Plagiopyla . (4) As for previous Plagiopyla descriptions, we also detected the presence of a very dense ciliature in P. nasuta , aligned along the left side of cytoproct. Two cytoproct dense rows were depicted by de Puytorac et al. (1985), but they did not highlight this feature, nor even indicate the position of the cytoproct. In our opinion, the composition of this dense ciliature should be considered another useful trait for species identification. (5) Our analysis detected the presence of a single type of extrusomes (straight) as already reported by Foissner et al. (1995), while the study by de Puytorac et al. (1985) proposed two types of extrusomes, i.e. curved-long and curved-short. As TEM pictures published by de Puytorac and colleagues (1985) are unfortunately scarcely explanatory, and a detailed description of extrusomes has not been provided, in our view the presence of a single type of straight extrusomes has to be considered a diagnostic characteristic for P. nasuta . Dragesco and Dragesco-Kernéis raised the importance of extrusomes, although they doubted they would be considered a diagnostic feature ( Dragesco & Dragesco-Kernéis, 1986). In our opinion, these structures are useful for species identification, because they constitute a stable characteristic among organisms of the same species. Therefore, they should be taken into account as additional information in cell description, together with other morphological features; in keeping with this view, we also reported the presence of cortical granules in our P. nasuta .

Plagiopyla nasuta View in CoL type populations were retrieved in Germany and Czech Republic from freshwater habitats by Stein (1860), although, later on, several authors described this species from marine and brackish environments. Gourret & Roeser (1886), for instance, reported P. nasuta var. marina View in CoL from the Gulf of Marseille, while Lynch (1930) from Monterey Bay in California. Subsequently, Kahl (1931) observed some similarities between the latter two descriptions and decided to reassign those two marine P. nasuta View in CoL to Plagiopyla marina View in CoL sp. nov.

Since then, many other Plagiopyla View in CoL specimens from marine environments have been recorded and identified as P.nasuta View in CoL by different authors, while P.marina View in CoL has been completely neglected during the specimen identification process. Thus, previous authors attributed significantly different features to the species P. nasuta View in CoL , as is well evident in Table 8. Consequently, great prudence should be used when considering all organisms listed as authentically ascribable to the species P. nasuta View in CoL . Moreover, P. nasuta View in CoL has apparently been found by different authors both in freshwater and marine environments, even if a salt resistance test has never been performed to the best of our best knowledge. In our opinion, marine populations need additional investigation, including with molecular markers, to evaluate possible similarity/synonymy of these populations with P. marina View in CoL .

NEOTYPIFICATION OF PLAGIOPYLA NASUTA STEIN, 1860

No type or voucher slides are available from the P. nasuta populations described by Stein (1860) and other authors (see above). Moreover, past descriptions of this species present some details that are not in agreement with each other: body size, presence/absence and shape of extrusomes, type of habitats, etc. Thus, it seems wise to define P. nasuta objectively by the designation of a neotype (ICZN, 1999; Foissner, 2002), associated with an 18S rRNA sequence deposited in online database. According to Article 75.3 of the ICZN (1999), our designation is accompanied by the publication of the following statements:

1. The taxonomic status of the present species is somewhat unclear because the original description does not report morphometrics and subsequent redescriptions do not agree in some important features, such as body size, extrusomes, habitats, etc. (see above for details).

2. For a differentiation of P. nasuta from related taxa, see above and further in the text.

3. The neotype specimen ( Figs 12–16; Table 6), from Kolleru Lake is described in detail (see above); thus, recognition of the neotype designated is ensured and associated with an 18S rRNA sequence deposited in GenBank ( KY563719 View Materials ) .

4. It is generally known that no type material is available from species described by Stein (1860). Further, there is no indication that other authors made permanent preparations of the present species.

5. There is strong evidence that the neotype is consistent with P. nasuta as originally described by Stein (1860) and subsequentely by other authors. For a detailed comparison, see above and below. Moreover, the 18S DNA sequencing confirms the identity of the species at a molecular level (see above).

6. Unfortunately, the neotype population does not come from very near the original type locality (freshwater bodies in Czech Republic and Germany vs. freshwater Kolleru Lake, in India; distance about 6000 km). However, both sites are freshwater habitats and P. nasuta is a cosmopolitan ciliate (see further), so this point should not be over-interpreted as suggested by Foissner (2002): ‘ neotypes of protists, especially ciliates, should be freed from the type locality regulation of Article 75.3.6 of the Code, provided that neotypification is based on a through redescription of the organism and usable neotype material has been deposited in an acknowledged repository’. Other authors have already performed ciliate neotypification despite the same issue: as examples, see Foissner et al. (2002), Berger (2004), Li et al. (2007, 2010) and Modeo et al. (2013). A detailed description of the neotype locality is provided in the ‘Material and Methods’ and ‘Results’ sections.

7. The slide containing the neotype specimen is deposited in the collection of the ‘ Museo di Storia Naturale dell’Università di Pisa’ (Calci, Pisa, Italy), with the code ‘2017-3 ’.

CONSIDERATIONS ON THE PHYLOGENY OF THE FAMILY PLAGIOPYLIDAE

The ingroup topology obtained in our phylogenetic analysis agrees with previous topologies ( Modeo et al., 2013; Xu et al., 2013; Liu et al., 2015), showing four distinctive clades corresponding to the four families of the class Plagiopylea.

In particular, the family Plagiopylidae consists of two sister clades including sequences of characterized and uncharacterized Plagiopyla species, plus Lechriopyla mystax . Unfortunately, sequences of Paraplagiopyla kiboko and Pseudoplagiopyla sinistra are still not available in on-line databases, so it was not possible to obtain an exhaustive phylogeny of the family, following the current systematic definition of the group ( Lynn, 2008).

The monophyly of Plagiopylidae is supported by the present analysis, although the clade of Plagiopyla also includes the sequence belonging to L. mystax . The latter clusters with some uncharacterized plagiopylids and our two novel species, which can undoubtedly be assigned to the genus Plagiopyla based on their morphology (see discussion above).

The phylogenetic tree made by Lynn & Strüder-Kypke (2002) did not show this discrepancy because only the sequences of P. nasuta and P. frontata were available at the time. The availability of more sequences of Plagiopyla and related environmental sequences would have helped to understand that L. mystax is not the sister genus of Plagiopyla but, more likely, a congener.

Lechriopyla mystax View in CoL is an endosymbiont of sea urchin guts; it shares some morphological traits with the genus Plagiopyla View in CoL such as the oval-reniform, holotrichously ciliated body, the slit-like oral aperture, the striated band on the dorsal surface, the single macronucleus and the contractile vacuole on the posterior end of the body ( Lynch, 1930; Berger & Lynn, 1984, Lynn & Strüder-Kypke, 2002). The morphology of the inner buccal cavity is the main feature used to differentiate Lechriopyla View in CoL from the genus Plagiopyla View in CoL ( Fig. 17): L. mystax View in CoL has a buccal cavity bent towards the posterior part of the cell with respect to the longitudinal body axis and the presence of the so called furcula ( Fig. 17B) (a large fibrous structure that appears to support the oral cavity according to Lynn & Strüder-Kypke, 2002) lacking in all other plagiopylids ( Small & Lynn, 1985; Lynn & Small, 2000).

According to the morphological affinity between these two genera and our phylogenetic results, we suggest that the ciliate described by Lynch as L. mystax ( Lynch, 1930) View in CoL , studied for its fine structure by Berger & Lynn (1984) and molecularly characterized on newly collected specimens by Lynn & Strüder-Kypke (2002), is indeed a Plagiopyla View in CoL , with some peculiar features on its buccal region, such as the tubular inner part bent backward, together with the

MIN: minimum value; MAX: maximum value; X: arithmetic mean; SD: standard deviation; CV: coefficient of variation (%); N: number; n: number of specimens analyzed; SI: silver impregnation; SEM: scanning electron microscopy; FE: Feulgen staining.

furcula. Thus, we propose that the genus Lechriopyla represents a junior synonym of Plagiopyla and that L. mystax should be renamed as Plagiopyla mystax comb. nov.

As an alternative, it could be hypothesized that when the authors collected L. mystax specimens from sea urchin gut for their study ( Lynn & Strüder-Kypke, 2002), a few Plagiopyla specimens had been erroneously selected (e.g. both Plagiopyla minuta and P. nyctotherus occur in the gut of sea urchins according to: Beers, 1954; Poljansky & Golikova, 1959; Berger & Lynn, 1984) and their DNA preferentially amplified with respect to L. mystax ’s DNA. In this case, the sequence published as L. mystax ( AF527757 View Materials ) could be attributed to an uncharacterized Plagiopyla , and Lechriopyla would remain a valid genus needing a molecular characterization. Considering that there is no evidence of the occurrence of such a technical error, and that these authors are extremely skilled in comparative morphology and species identification, we consider the first hypothesis to be more reliable, and consequently propose to synonymize the two genera.

Our conservative interpretation leads us to maintain the monophyly of the genus Plagiopyla . Indeed, we chose a ‘lumper’ approach ( Corliss, 1976) given the high morphological and molecular affinities between the genera Lechriopyla and Plagiopyla

CRITICAL REVISION OF THE FAMILY PLAGIOPYLIDAE

The present critical revision is based on several, selected publications concerning members of the family, i.e. what we consider to be milestone works regarding morphological description, molecular characterization, ecology, and distribution. As some other papers have not been included, the present section is meant as a non-comprehensive overview of plagiopylid literature.

To date, family Plagiopylidae comprises four genera: Plagiopyla , Lechriopyla , Paraplagiopyla and Pseudoplagiopyla . Some of them share common features, such as the holotrichously ciliated body, the peculiar oral apparatus and the striated band. However, it is not the case for Paraplagiopyla , whose morphology raises some doubts on its systematic account.

Paraplagiopyla kiboko , surveyed by Thurston & Grain (1971) in the stomach contents of Hippopotamus amphibius from Queen Elisabeth Park ( Uganda), has been described as 48–110 µm long and 25–62 µm wide, after staining. The surface of its body is not uniformly ciliated: only a peripheral ciliated furrow departs from the oral groove, although protargol staining reveals lines of non-ciliated kinetosomes on the remaining cell surface. The striated band is lacking. One elongated macronucleus, a single micronucleus and two contractile vacuoles are present; no extrusomes detected.

The attribution of Paraplagiopyla to the family Plagiopylidae has already been questioned by Lynn (2008) who remarked that, ‘ Paraplagiopyla , if truly a plagiopylean, is an exception as its somatic kineties are restricted to a narrow furrow that extends around the

For Plagiopyla spp. described in the present work (in bold) we reported measurements from fixed specimens (silver and Feulgen stainings); N: number; ND: no data; (l): measurements from live specimens; (f): measurements from fixed specimens; (ns): not specified if measurements were from live or fixed specimens; AV: average value, calculated on minimum and maximum measurements reported in cited literature; * trait observed or measured from the representative illustration; °/°° indicates the source of the information within the column if more than one reference is indicated. For Plagiopyla nasuta described in the present work (in bold) we reported measurements from fixed specimens (silver and Feulgen stainings); MA: macronucleus; MI: micronucleus; SB: striated band; CVP: contractile vacuole pore; ND: no data; (l): measurements from live specimens; (f): measurements from fixed specimens; (ns): not specified if measurements were from live or fixed specimens; Ellips.: Ellipsoidal; Irreg.: Irregular; Spher.: Spheroidal; Renif.: Reniform; * trait observed or measured from the representative illustration; # Misinterpreted by authors as trichocysts.

edges of the flattened cell’. Moreover, it lacks the striated band which, in our opinion, is an apomorphic feature of the family. For all these reasons, we propose the exclusion of this genus from the family Plagiopylidae .

As for Pseudoplagiopyla sinistra , its description is poor. Authors ( Small & Lynn, 1985) did not mention either the place/habitat of retrievement or some important morphological features (i.e. body size, striated band, contractile vacuole, extrusomes, etc.). Given the limited available data, any change in its systematic position is not advisable, and we do recommend a redescription of the species, possibly using a multidisciplinary integrated approach also comprising the analysis of molecular markers.

Regarding Lechriopyla , as previously discussed, the only species belonging to that genus has been moved and included as a new combination in the genus Plagiopyla . Moreover, two novel species of Plagiopyla have been described. Therefore, the genus Plagiopyla now comprises three additional species: P. ramani sp. nov., P. narasimhamurtii sp. nov. and P. mystax comb. nov., reaching a total of 14 included species ( Fig. 18).

We analysed all the considered descriptions of Plagiopyla spp. in order to pinpoint possible synonyms and uncertain/wrong species attributions.

The majority of described Plagiopyla spp. ( Table 7) have distinctive features, except for P. cucullio , the description of which is poor. Actually, this marine species could be easily misidentified either as P. frontata , P. ovata or P. marina . Indeed, Wallengren (1918) himself proposed to synonymize Plagiopyla nasuta var. marina (former description of P. marina ) found by Gourret & Roeser (1886) with P. cucullio . Since Kahl (1931) and Jankowski (2007) kept P. cucullio separate from P. marina , we still prefer to treat P. cucullio as a distinct species, although we do notice a marked resemblance with P. frontata .

The type of habitat, in most cases, could help discriminate Plagiopyla species: P. simplex , P. megastoma , P. ramani and P. narasimhamurtii are typically found in freshwater; Plagiopyla cucullio , P. frontata , P. ovata and P. marina in marine environment; while P. mystax , P. minuta and P. nyctotherus are endocommensals of sea urchins. The only exception is P. nasuta that, apparently, was described both from freshwater and marine environments (see discussion above). Further details on species discrimination are treated in the following compendium of the family Plagiopylidae , providing a diagnosis and a key for species identification. Nevertheless, due to the scarcity of morphological details, it was not possible to insert P. cucullio in the present taxonomic key. Further investigation and, perhaps, a redescription of P. cucullio are necessary to give a detailed account of the species features.