Rediscovery of Remarkably Rare Anaerobic Tentaculiferous Ciliate Genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea)

Simple Summary

Rare organisms represent a challenge for researchers in all fields of study in biology. In the realm of ciliatology, the genera Dactylochlamys and Legendrea are considered to be such cases. Very little information has accrued in over a century since their first descriptions; only a few published reports, online images or videos of rarely encountered individual specimens exist. Dactylochlamys and Legendrea are also morphologically remarkable for their likely independently evolved tentacle-like structures. Recently, the first molecular data were published for the species L. loyezae. In our study, we present more robust phylogenetic analysis based on three molecular markers of Dactylochlamys pisciformis and all of the three known Legendrea species, showing that they likely represent a new anaerobic lineage of ciliates. We first provide a detailed morphological characterization of both genera using modern microscopy and staining methods. We identify and discuss the bacterial (Syntrophaceae) and archaeal endosymbionts harbored by both genera based on 16S rRNA gene sequences. We also discovered that Legendrea preys on gastrotrichs, which is supported by molecular data and a unique video material documenting the feeding behavior of one Legendrea species. This study brings essential information needed to better understand the phylogeny, life strategies, and rarity of these organisms and emphasizes the importance of citizen science.

Abstract

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

1. Introduction

The phylum Ciliophora comprises a bewildering diversity of ciliated unicellular eukaryotes inhabiting virtually every biotope on Earth as both free-living organisms or symbionts. Their enormous morphologic variety is matched by the many indispensable ecologic roles they play in terrestrial, freshwater, and marine microbial food webs. Eukaryovorous ciliates, an important guild of microorganisms, use various morphologic features for prey capture, and tentacles represent one of the most intriguing structures of this kind. Tentacles, as a feature of the ciliate cortex, have evolved several times in very distantly related lineages; e.g., the family Mesodiniidae and classes Phyllopharyngea and Litostomatea [1]. Ciliates of the class Litostomatea are known mainly as free-living aerobic eukaryovorous predators (subclasses Rhynchostomatia and Haptoria) or anaerobic endosymbionts inhabiting the guts of invertebrate and vertebrate hosts (subclass Trichostomatia) [2,3]. In addition to these varied lifestyles, litostomateans also exhibit some spectacularly diverse morphologic features, e.g., the skeletal plates of the endosymbiotic entodiniomorphids, the triple proboscises of Teuthophrys Chatton and Beauchamp, 1923, and the tentacles and papillae of Actinobolina Strand, 1928, Dactylochlamys Lauterborn, 1901, and Legendrea Fauré-Fremiet, 1908 [1,4,5].

The rarely encountered tentaculiferous ciliate genera Legendrea and Dactylochlamys have been assigned to the litostomatean family Spathidiidae based solely on their morphology [1,6]. The most distinctive features of Legendrea are tassel-like, ciliated, and extrusome-bearing tentacles which, in two species (L. bellerophon Penard, 1914 and L. loyezae Fauré-Fremiet, 1908), are highly extensible and have been assumed, but not proven, to be involved in prey capture [7]. On the other hand, the tentacles of Dactylochlamys pisciformis Lauterborn, 1901, the type and only species of the genus, are morphologically quite different from those of Legendrea and are more similar in appearance to those of Actinobolina and Belonophrya Andre, 1914, yet their functions remain unknown [4,5]. Legendrea and Dactylochlamys are vanishingly rare genera that may elude even the most widely traveled ciliatologists. To date, there have been only a few published reports, website images, and videos of Legendrea species and Dactylochlamys [5,8]. Thus, little information has accrued in over a century since their first descriptions.

In the genus Legendrea, three currently accepted species were originally described based on shared morphological features: L. loyezae, the type species, L. bellerophon, and L. pespelicani Penard, 1922. Four other species previously included in Legendrea by Penard (original combinations L. crassa, L. interrupta, L. porculus, and L. simplex) have been transferred to other genera in the order Spathidiida [9,10,11].

We present a phylogenetic analysis based on the ribosomal RNA cistron of D. pisciformis and all three Legendrea species, and also provide more detailed morphological characterization of Legendrea and Dactylochlamys, using protargol-impregnated specimens and scanning electron microscopy. We also present unique video material, including documentation of the feeding behavior of one Legendrea species, and discuss the putative bacterial (Syntrophaceae) and archaeal (Methanobacterium sp., Methanocorpusculum sp., and Methanosaeta sp.) endosymbionts harbored by both genera based on 16S rRNA gene sequences. Our results provide unique data essential to a better understanding of the phylogeny, life strategies, and rarity of these remarkable organisms.

2. Materials and Methods

2.1. Sampling

Sulfidic sediment samples from four freshwater localities in Belgium, the Czech Republic, Germany, and the USA were collected in 50 ml Falcon tubes. All populations in the original raw samples were kept in closed tubes for two to three weeks. Attempts to establish enriched and clonal cultures were unsuccessful.

The Dactylochlamys pisciformis population VB2A was found in the Venusbassin in Tiergarten Park in Berlin, Germany (52°30′52.5″ N 13°22′14.2″ E), and the MRATIN population in a eutrophic pond, Mratín, Czech Republic (50°11′58.7″ N 14°33′17.2″ E). The Czech populations of Dactylochlamys sp. (MOKOTP1Q), Legendrea loyezae (MOKOTL), and L. ornata (MOKOTP1) were found in a small permanent pond adjacent to the Vltava river in Prague (49°59′24.9″ N 14°24′04.1″ E). The US population of L. ornata (SAGEGLEN) came from a small permanent eutrophic pond near Boise, Idaho (43°40′57.20″ N 116°15′15.44″ W), the Belgian population of L. ornata came from a freshwater garden pond in Tontelange, Province de Luxembourg (49°43′36.7″ N 5°48′32.1″ E), and the GTUB population from a garden mesocosm (see [12]). The Czech population of L. pespelicani (VLKOV) was found in a small permanent concrete-lined freshwater reservoir in the village of Vlkov (49°09′04.0″ N 14°43′22.4″ E).

2.2. Morphological Characterization

Living cells were hand-picked with glass micropipettes and studied at magnifications of 100–1000× with brightfield and differential interference contrast illumination, using an Olympus BX51 microscope equipped with an Olympus DP70 camera (Olympus Corp., Tokyo, Japan). Cells of VB2A were fixed with 4% (v/v) formalin and impregnated with protargol (Polysciences, Warrington, PA, USA) according to a modified Dieckmann’s protocol [13,14,15]. In vivo measurements were made from photomicrographs of uncompressed, freely swimming cells, using calibrated software. Counts and measurements from protargol preparations were made at 1000× magnification. For scanning electron microscopy (SEM), cells were fixed for at least 30 min in 2.5% glutaraldehyde or in a 1:1 (v/v) solution of 5% glutaraldehyde and 2% osmium tetroxide, processed according to [14], and examined JSM-IT200 scanning electron microscope (JEOL LV, Peabody, MA, USA). Except as noted, morphological terminology and size classification follows [1,2,9,14]. For clarity, we refer to the cortical pellicular projections of Dactylochlamys pisciformis and the three species of Legendrea as “tentacles” and the trichocyst-bearing oral bulge structures of L. pespelicani as “papillae”.

2.3. DNA Extraction, Amplification, and Sequencing

In total, 3 cells from the VB2A population and single cells from the VLKOV, MOKOTL, MOKOTP1, and MOKOTP1Q populations were hand-picked with glass microcapillary pipettes, washed 5 times with distilled water, and added into 30 µL of DNA/RNA shield (Zymo Research, Irvine, CA, USA). The total DNA was isolated using the MasterPure^TM Complete DNA and Purification Kit following the manufacturer’s instructions. Three individuals of SAGEGLEN were picked for DNA extraction with a modified Chelex method [16].

The 18S rRNA gene (complete or almost complete) of SAGEGLEN and VLKOV was amplified using the primers EukA 5′-AACCTGGTTGATCCTGCCAGT-3′ and EukB 5′-TGATCCATCTGCAGGTTCACCT-3′ [17], VB2A and MOKOTL were amplified with primers 82F 5′-GAAACTGCGAATGGCTC-3′ [18] and EukB, and the annealing temperature was 55 °C. MOKOTP1 was amplified with the primers 82F and the newly designed LEGR3 5′-TTCTCCTTCCTCTAGGTGATAAGG-3′, and the annealing temperature was 55 °C. An approximately 1200 bp long fragment of the ITS region and 5′ end of the 28S rRNA gene was amplified using the primers ITS-F 5′-GTAGGTGAACCTGCGGAAGGATCATTA-3′ [19] and LO-R 5′-GCTATCCTGAGRGAAACTTCG-3′ [20], and the annealing temperature was 54 °C. The 16S rRNA gene fragment (approx. 460 bp) of methanogenic Archaea was amplified using the primers Arc915F 5′-AGGAATTGGCGGGGGAGCAC-3′ and ArcR1326 5′-TGTGTGCAAGGAGCAGGGAC-3′ [21,22], and the annealing temperature was 55 °C. The PCR products were purified using EXOSAP (Applied Biosystems, Waltham, MA, USA). Amplicons were directly sequenced on an ABI PRISM 3100 sequencer (Applied Biosystems). For taxonomic assignment of archaeal 16S rRNA gene fragments, the BLASTn algorithm was used (https://blast.ncbi.nlm.nih.gov, accessed on 29 March 2023).

The V4 variable region fragment of 16S rRNA gene (approx. 300 bp) from VLKOV and VB2A populations was amplified from extracted DNA (see above) for Illumina sequencing, using barcoded primers 515F 5′-GTGYCAGCMGCCGCGGTAA-3′ and 806R 5′-GGACTACNVGGGTWTCTAAT-3′ [23,24]. Paired-end (2 × 250 bp) Illumina amplicon sequencing (NovaSeq6000) and demultiplexing was performed at SEQme company (Dobříš, Czech Republic). Microbiome bioinformatic analyses were performed using the QIIME2 2022.8 platform with DADA2 plugin for denoising and chimera removal [25,26]. Amplicon sequence variants (ASVs) were taxonomically classified using the classify-sklearn naïve Bayes taxonomy classifier (via q2-feature-classifier plugin) [27] against the Silva 138 99% OTUs reference sequences of the 515F/806R region [28]. Dominant archaeal ASVs were identified as putative symbionts based on their relative frequency.

2.4. Phylogenetic Analyses

An alignment of 18S rRNA gene sequences included 167 taxa, comprising 135 litostomateans and 32 outgroup taxa representing Armophorea and CONThreeP [29]. Five litostomatean sequences are newly reported in this study and all other sequences were obtained from GenBank. The dataset of concatenated 18S-ITS-28S rRNA region sequences consisted of 168 taxa, including 6 newly determined sequences and 162 sequences (135 litostomateans and 32 outgroup taxa) obtained from GenBank. The sequences were aligned using the G-INS-i algorithm [30] on the MAFFT server (https://mafft.cbrc.jp/alignment/software/, accessed on 15 March 2023). The alignment was manually edited using AliView [31] to trim primer sequences. The length of the final 18S rRNA gene data set was 1825 positions and the concatenated data set comprised 3163 positions. Phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. The best fit substitution model (GTR + I + Γ) was selected using Modeltest-NG [32,33]. ML analysis was performed in RAxML-NG [34] on the CIPRES portal (http://www.phylo.org/sub_sections/portal/, accessed on 15 March 2023) under the GTR + I + Γ substitution model. Statistical support was assessed using 1000 bootstrap pseudoreplicates and convergence assessed using weighted Robinson–Foulds distance. Bayesian analysis was performed using MrBayes 3.2.7 [35] on the CIPRES portal using the GTR + I + Γ substitution model. For the 18S rRNA gene alignment, Markov chain Monte Carlo (MCMC) analyses of four chains (three hot, one cold, temperature 0.2) and for the concatenated alignment six chains (five hot, one cold, temperature 0.1) were each run for 10 million generations, with a sampling frequency of 1000 generations. The first 25% of sampled trees were removed as burn-in. Convergence was assessed using RWTY [36]. Uncorrected p-distances were calculated using MEGA v10.2.6.

3. Results

3.1. Morphological Descriptions and Diagnoses

3.1.1. Dactylochlamys Pisciformis Lauterborn, 1901

Amended diagnosis based on [10,37,38] and current study. Moderately small-to-medium-sized spathidiid. Shape fusiform to clavate. Cortex rugose. One equatorial, globular to ellipsoidal macronucleus with one adjacent ellipsoidal micronucleus. Contractile vacuole subterminal. Somatic kineties follow left-hand spiral course. Long fine somatic cilia interspersed with very slender, rigid, tubular, capitate, retractile tentacles. Long filiform extrusome occupies lumen of each tentacle, and extends into cytoplasm when tentacles retract. Oral bulge truncate conical; base surrounded by short oblique pectinelles at anterior ends of somatic kineties.

Description based on VB2A (Figure 1 and Figure 2; Table S1; Video S1). Size 85–110 × 25–35 μm in vivo (n = 2), about 60 × 18 μm after protargol impregnation, length:width ratio from 2.5–3.7 in vivo (n = 2) and about 3.6 in protargol preparations. Shape fusiform to clavate. Colorless. Single equatorial globular to ellipsoidal macronucleus about 8 × 6 μm with many small, scattered nucleoli in protargol preparations; single adjacent globular micronucleus about 2–3 μm across (Figure 2C). Contractile vacuole subterminal; excretory pores not observed (Figure 1A,B,F,G). Long, fine filiform extrusomes occupy lumen of tentacles; proximal ends extend into cytoplasm. Cortical granules not observed. Cytoplasm filled with refractive globules and large food vacuoles.

About 10–12 ordinarily spaced somatic ciliary rows on slightly wavy left-hand spiraling ridges, short inclined ciliated pectinelle between anterior end of each somatic kinety and base of oral bulge, each pectinelle composed of three or four basal bodies. Kineties composed of monokinetids bearing long fine cilia (12–20 μm long), interspersed with tentacles. Tentacles very slender, tubular, retractile, about 15 µm long when extended, inconspicuously capitate when retracted, and each encloses an argyrophilic extrusome. Dorsal brush not observed.

Oral bulge truncate conical, inconspicuous in vivo, about 2 µm high, 4 μm in diameter after protargol impregnation. Oral bulge extrusomes and cytopharyngeal basket not observed (Figure 2).

Based on our own observations, we accept Kahl’s synonymization of D. hystrix Wetzel, 1928 with D. pisciformis [10]).

3.1.2. Legendrea Fauré-Fremiet, 1908

Amended diagnosis: Moderately small to large spathidiid. Tentacles, each with circumtentacular kinety and extrusome bundle, sometimes highly extensible, located on dorsal and ventral margins or restricted to posterior one-half of cell. Sometimes with papillae in oral bulge. Inhabits anoxic or microoxic freshwater biotopes.

• Legendrea loyezae Fauré-Fremiet, 1908

Amended diagnosis (based on [7,8,9,10,39] and current study): With characters of genus. Size 70–132 × 62–85 μm in vivo. Shape broadly elliptical to almost spherical. Eccentric posterior contractile vacuole may appear as two vacuoles due to median cleft at posterior end when tentacles retracted. Up to 25 tentacles clustered posteriorly, never anterior to cell equator, trail limply behind swimming cells. Tentacles more or less tubular when retracted, distal ends mushroom-shaped when extended, often with an eccentric distal luminal vacuole, tentacular extrusome bundles 4.5–14 µm long, layer of smaller rod-shaped extrusomes beneath terminal cap of tentacles. Extended tentacles reach up to 6.5 times retracted length.

Description based on in vivo observations of MOKOTL (n = 3; Figure 3; Video S2): Size 85–102 × 55–102 in vivo, contractile. Shape broadly obovate to nearly globular, anterior end obliquely truncate; up to 25 flexible, trailing tentacles restricted to posterior one half of cell. Horseshoe-shaped macronucleus, micronuclei not observed. Contractile vacuole eccentrically located at posterior end, sometimes indented by median pellicular cleft when tentacles retracted, occupies entire posterior end in diastole (Figure 3C,D). Extrusomes in oral bulge, morphology not determined, tentacles with two types of extrusomes; one type curved, filiform, closely packed in central bundles at distal ends of tentacles (about 4.5 µm long); second type comprises layer beneath distal ends of tentacles, small (about 1.5 µm long), rod-shaped (Figure 3F,H,I). Ejected extrusomes not observed. Tentacles extensible, covered with granular layer near junction with cell body, distinct cortical ruffles (collar) not observed. Cytoplasm colorless but cells appear dark under low magnification. Cells packed with refractive globules, food vacuoles with ingested prey. Cells swim steadily at moderate pace and rotate rightward about long axis (Video S2).

Ciliature holotrichous, somatic cilia about 6 µm long in closely spaced longitudinal kineties; number of kineties not determined. Each tentacle with single circumferential subterminal kinety. Dorsal brush inconspicuous with approximately 3 µm long clavate cilia; number and morphology of brush rows not determined.

Oral bulge inconspicuous, less than 3 µm high, occupies two-thirds of anterior end, eccentric (i.e., displaced slightly ventrally). Oral basket inconspicuous.

Remark: We were unsuccessful in obtaining protargol preparations of L. loyezae.

• Legendrea pespelicani Penard, 1922

Amended diagnosis based on [9] and current study: With characters of genus. Size 180–210 × 60–90 μm in vivo. Broadly spatulate in lateral view, laterally compressed. Oral bulge oblique, invariably contains one trichocyst-bearing papilla at either end, usually one or two papillae in between; thus, oral bulge outline moniliform or dumbbell-shaped when viewed anteriorly. Macronucleus filiform, very long, tortuous strand. About six stout, mushroom-shaped, ciliated, extrusome-bearing tentacles commence at or anterior to equator, distributed at irregular intervals on dorsal and ventral margins of cell, always one posterior polar tentacle. Tentacular extrusomes longer (about 25 µm) than those in tentacles of L. ornata or L. loyezae. Distinctly spathidiid somatic ciliary pattern. Circumtentacular kineties composed of many more dikinetids than in L. ornata. Ciliary rows interrupted by tentacles while deviating around tentacles in L. ornata. Tentacle extension characteristic for L. loyezae and L. ornata not observed.

Description based on VLKOV (Figure 4, Figure 5 and Figure 6; Table S2; Videos S3–S5): Size about 180 × 90 μm in vivo, about 165 × 120 after protargol impregnation, length:width ratio 2 in vivo and 1.4 in protargol preparations. Shape broadly spatulate in lateral view, laterally compressed in vivo, less so in protargol preparations. Cells appear dark in vivo under low magnification. Single very long (up to 331 µm), tortuous, filiform macronucleus; nucleoli prominent in vivo (Figure 4I and Figure 5). Five to nine scattered micronuclei (2.5–4 µm across). Extrusomes of one type, restricted to closely packed bundles in distal ends of tentacles and in oral bulge papillae, curved, filiform (about 25 × 1 µm), do not impregnate with protargol. Contractile vacuole large, terminal, several subterminal excretory pores on right side (Figure 4F and Figure 5C). Cortex flexible (Figure 4C). Cortical granules not observed. Cytoplasm filled with refractive globules (about 2–4 µm across) and large food vacuoles. Swims lazily (Videos S3–S5).

Somatic ciliation holotrichous, longitudinal ciliary rows interrupted by tentacles. Somatic cilia about 8 µm long, arranged in about 70 rows. Ciliary pattern distinctly spathidiid (i.e., right-side kineties curve dorsally to meet circumoral kinety, left kineties curve ventrally). Each tentacle with ciliated subcapital circumtentacular kinety composed of dikinetids (about 35 on average). Stout (about 8 µm high × 10 µm across) mushroom-shaped tentacles (six on average) at irregular intervals on dorsal, ventral margins, invariably one tentacle at posterior pole (Figure 6A). Active tentacle extension not observed. Total of 3 dorsal brush rows composed of dikinetids bearing 3.5 µm long clavate cilia, rows commence to left of dorsal oral bulge papilla; B1 and B2 extend posteriorly about 20% of cell length; B3 highly unusual, i.e., proximal part similar in morphology and length to B1 and B2 but posterior part composed of patchily distributed monokinetids and groups of dikinetids extending about 50% of cell length (Figure 6C,D,F).

Oral bulge oblique (about 40° to long axis of cell), convex anteriorly, long, narrow (about 80 × 5 µm). Circumoral kinety about 80 µm long, 5 µm wide between papillae, invariably encloses one extrusome-bearing papilla at dorsal and ventral end, usually one or two additional papillae between (two of nine individuals had only two oral bulge papillae: one dorsal, one ventral). Oral bulge papillae about 3.5 µm high × 10 µm across. Outline of circumoral kinety moniliform or dumbbell-shaped when viewed anteriorly due to deviation around large papillae (Figure 5C,D,G). Large broadly conical oral basket extending nearly entire length of cell (Figure 5H).

Stomatogenesis holotelokinetal; proliferation of basal bodies first occurs in opisthe kineties in line with proter dorsal brush rows. Developing anterior ends of opisthe kineties curve ventrally (Figure 5K).

• Legendrea ornata (Stokes, 1887) Penard, 1914
• Original combination, Holophrya ornata Stokes, 1887

Amended diagnosis based on [7,10,40] and current study: With characters of genus. Size 70–180 × 60–90 µm. Shape oblong ellipsoidal when swimming, broadly ovoidal when at rest with tentacles extended. Rows of tentacles at alternating angles on dorsal and ventral margins, commence in anterior one-fourth of cell, continuous around posterior end. Extrusome bundles about 5–7 × 5 µm in vivo. Tentacles highly extensible, up to >25 times retracted length. Ciliary pattern distinctly enchelyodontid, unlike spathidiid pattern of L. pespelicani, i.e., anterior ends of somatic kineties perpendicular to circumoral kinety. Ciliary rows not interrupted by tentacles as seen in L. pespelicani. Fewer (ten on average) circumtentacular kinety dikinetids than seen in L. pespelicani (35 on average). Three dorsal brush rows; B1 and B2 approximately equal in length, longer than B3.

Description based on BELG, GTUB, MOKOTP1, and SAGEGLEN (Figure 7, Figure 8, Figure 9 and Figure 10; Table S3; Videos S6 and S7):

With characters of genus. Size 88–141 × 30–80 μm in vivo. Shape narrowly to broadly oblong when swimming, broadly ovoidal to almost globular when at rest with tentacles extended. Anterior end obliquely truncate; posterior end broadly rounded. Macronucleus horseshoe-shaped or tortuous strand (49–80 × 3.5–5.5 µm), numerous nucleoli approximately 1 µm in diameter in protargol preparations, inconspicuous in vivo, multiple (2–7) globular micronuclei (about 2.5 µm across) in protargol preparations, not discernible in vivo (Figure 7, Figure 8C and Figure 9B). Contractile vacuole large, terminal, multiple excretory pores at right posterior side of cell. Two types of extrusomes: ordinary rod-shaped subcortical mucocysts (about 1 × 0.5 µm); curved, filiform tentacular extrusomes (about 6 × 1 µm), impregnate densely with protargol. Retracted mushroom-shaped tentacles in continuous row at alternating angles on dorsal, ventral, and posterior margin, commence in anterior one-fourth of cell, retracted tentacles with telescoping ruffled cortical collar (Figure 7A,J and Figure 10A,C); tentacles highly extensile (up to >25 times retracted length), used to immobilize prey (Video S7). Cortex flexible. Cytoplasm colorless, filled with refractive globules, prokaryotic symbionts (Figure 9F,G). Rod-shaped prokaryotic ectosymbionts in SEM preparations of some populations (Figure 10D). Swims at a moderate pace, rotating on the long axis (Video S6).

Somatic cilia about 12 µm long in about 30 longitudinal rows, pattern of infraciliature distinctly enchelyodontid, i.e., anterior ends of somatic kineties perpendicular to circumoral kinety. Ciliary rows not interrupted by tentacles. Dorsal brush inconspicuous; three rows of dikinetids with 3 µm long clavate cilia, brush rows commence to left of circumoral kinety; B1 and B2 of equal length (about 15 µm), B3 slightly shorter (Figure 8A,F).

Oral bulge inconspicuous, slightly oblique. Circumoral kinety narrow elliptical, composed of dikinetids. Oral bulge extrusomes not observed. Oral basket inconspicuous in vivo, obconical, extends about one-half length of cell in protargol preparations (Figure 8D, Figure 9A,D).

Stomatogenesis holotelokinetal, basal body proliferation commences first in somatic kineties in line with dorsal brush rows (Figure 9H).

3.2. Molecular Data and Phylogenetic Analysis

Analyses of the concatenated data set revealed that the genera Legendrea and Dactylochlamys form a supported clade (bootstrap value 80, Bayesian posterior probability 1) together with Arcuospathidium sp. and Apertospathula oktemae; Legendrea sequences formed a fully supported clade (Figure 11 and Figure S1).

As the 18S rRNA gene sequences of L. loyezae and L. pespelicani are identical and differ from that of L. ornata by only one nucleotide, the analysis based on the 18S rRNA gene did not resolve the relationships between Legendrea species (Figure S2), and neither did the analysis of the concatenated dataset resolve the relationships among the three Legendrea morphospecies. L. ornata MOKOTP1 and SAGEGLEN sequences cluster together. Sequences of L. loyezae OP352778 and L. pespelicani VLKOV are more closely related to each other than to L. loyezae MOKOTL (Figure 11 and Figure S1). However, the published sequence of L. loyezae OP352778 represents only the V4 region of the 18S rRNA gene (1007 bp), while L. pespelicani VLKOV and L. loyezae MOKOTL are concatenated sequences (18S-ITS-28S). Genetic distances (uncorrected p-distances) between ITS–28S fragments (1183 bp) of L. ornata MOKOTP1, L. loyezae MOKOTL, and L. pespelicani VLKOV range between 0.008 and 0.009. Only amplification of the ITS-28S rRNA gene region of Dactylochlamys sp. MOKOTP1Q isolate was successful. Newly determined sequences are available in GenBank (accession numbers: OP985785–OP985794).

We also obtained two partial 18S rRNA gene sequences of prey gastrotrichs (family Chaetonotidae) from a cell of L. loyezae (MOKOTL; Figure 3B) and from the only cell of L. ornata from MOKOTP1 (GenBank accession numbers: OQ848030–OQ848031).

Using Sanger sequencing of 16S rRNA gene fragments, we identified two methanogenic archaeal endosymbionts as Methanobacterium sp. (L. pespelicani VLKOV) and Methanosaeta sp. (L. loyezae MOKOTL) (GenBank accession numbers: OQ843028–OQ843029). Using Illumina sequencing of the V4 region of 16S rRNA gene, we identified Methanocorpusculum sp. as the dominant archaeal methanogenic symbiont of D. pisciformis VB2A, representing around 95% of all archaeal ASVs. Methanobacterium sp. was the only archaeal ASV present in L. pespelicani VLKOV. In L. pespelicani VLKOV, we also identified a bacterial symbiont belonging to the Syntrophaceae (Table S4).

4. Discussion

4.1. Morphological Comparison of Legendrea Species and Similar Species

Legendrea pespelicani can be easily distinguished from L. ornata and L. loyezae by size (180–210 vs. <180 μm), cell shape (broadly spatulate vs. oblong, resp. broadly obovate), and by possession of oral papillae (present vs. absent). L pespelicani also differs from L. ornata by number of tentacles (4–8 vs. 10–43), number of circumoral kineties (30 vs. 10 in average), number of somatic kineties (66–70 vs. 28–36), and the disposition of the ciliary rows (interrupted by tentacles vs. deviating around the tentacles). L. loyazae can be distinguished from L. ornata by the position of tentacles (bundle at the posterior vs. margin of the cell on ¾ of the cell length), and from both other species by the eccentric posterior contractile vacuole which appears as two vacuoles due to median cleft at posterior. All Legendrea species are different to any other spathidiid in terms of tentacles (present vs. absent), except Dactylochamys pisciformis, which are not similar to those of Legendrea (very slender, not ciliated vs. thick with circumtentacular kineties). L. pespelicani differs from Spathidium papilliferum in morphology of the oral papillae (circumtentacular kineties present vs. absent).

4.2. Remarks on the Rarity and Ecology of Dactylochlamys and Legendrea

Both Dactylochlamys and Legendrea spp. have been very rarely reported, although Lauterborn and Penard suggested that the populations could be quite abundant [9,37]. Our experience was similar: the VB2A sample was unusually rich in Dactylochlamys (approx. 4 cells/mL of water and sediment), but the abundance quickly declined in several days, and we were unable to maintain the ciliate in long-term culture. In other cases (MRATIN, MOKOTP1Q), only a few cells were found in the whole volume of a 50 mL sample. In the case of Dactylochlamys, Penard cautioned about possible misidentification, because some swarmers of suctorian ciliates (Phyllopharyngea: Suctoria) (e.g., Enchelyomorpha vermicularis Smith, 1899) appear similar in vivo (compare Figure 1F,I) [9]. Thus, mentions in the literature not accompanied by illustrations are questionable, e.g., [41,42]. In the case of Legendrea, we found two relatively rich localities in the Czech Republic and one in the USA; other known localities are, for example, in France, Germany, Belgium, and Poland. This might suggest that the rarity of Dactylochlamys and Legendrea is rather a matter of specific habitat conditions than endemicity.

Interestingly, gastrotrich rRNA gene partial sequences (family Chaetonotidae) were recovered from cells of L. loyezae and L. ornata, indicating ingestion of gastrotrichs as prey. The process of L. ornata from sediments of a German freshwater pond catching and digesting a gastrotrich (family Chaetonotidae) is the first documentation of the role played by the tentacles in prey capture (Video S7). Chaetonotidae is a species-rich and widely distributed gastrotrich family that commonly inhabits hypoxic lacustrine sediments [43]. Although chaetonotid gastrotrich 18S rRNA gene sequences were the only eukaryotic contaminants from Legendrea, the importance of gastrotrichs in the diet of Legendrea species remains unknown.

4.3. Phylogenetic Analysis and the Relationship of Tentaculiferous Ciliates

A recent report (8) included a 994 bp V4-V9 rRNA region sequence from L. loyezae. Phylogenetic analysis could not resolve a position of Legendrea within Haptorida. The problems with attempting to resolve relationships within Litostomatea based on analysis of the rRNA cistron have been emphasized previously [44,45]. Consistent with the studies mentioned above, we did not succeed in resolving the internal relationships among Legendrea species in spite of addition of two more markers, i.e., using three times more positions in comparison to [8].

Despite pronounced morphological differences, all three identified Legendrea species have identical (L. loyezae and L. pespelicani) or almost identical (L. ornata) 18S rRNA gene sequences. However, the existence of the three Legendrea morphospecies is further supported by the analysis based on additional molecular markers; namely, part of the ITS region and part of the 28S rRNA gene. Clustering of L. loyezae OP352778 with L. pespelicani was interpreted as an artifact due to the short length of the former (1007 nucleotides) and ambiguous sites in both of the sequences of L. loyezae. This view is also supported by the p-distances of the ITS–28S fragments of each of the three Legendrea species, which range from 0.008 to 0.009. On the other hand, Jankowski even erected a monotypic genus for each of the three Legendrea species solely on morphology, but we consider with respect to the morphological and molecular analysis presented above [46].

We also obtained a partial ITS-28S rRNA gene sequence of a second Dactylochlamys population (MOKOTP1Q), but the material was insufficient for morphometric analysis. Molecular data and in vivo observation suggest that it is possibly a new species (Video S9).

Our analysis showed that Dactylochlamys and Legendrea are not closely related to Actinobolina, contradicting the traditional notion of the family Actinobolinidae, comprising the genera Actinobolina, Belonophrya, Legendrea, and Dactylochlamys, based on the presence of tentacle-like structures which have evolved independently [1,10,47]. As noted previously, homoplasies are rife throughout the Spathidiidae and, together with many plesiomorphies, have so far obscured the evolutionary relationships in the group [44,48,49,50]. Interestingly, Dactylochlamys and Legendrea are members of the same well-supported clade despite their obvious morphological differences. The other members of the clade (i.e., Apertospathula oktemae and an undescribed Arcuospathidium sp.) belong to spathidiid genera which do not bear any tentacle-like structures [2]. Spathidium papilliferum Kahl, 1930, another only distantly related spathidiid, has oral bulge papillae superficially similar to those of L. pespelicani but lacking circumtentacular kineties [10,48]. Analysis also showed that a supported clade including S. papilliferum (DQ411857, DQ411858; morphologically uncharacterized population) and an Epispathidium species (KT246081, KT246094; undescribed) are not closely related to the other S. papilliferum (KY556645, KY556652), suggesting possible misidentification (Figure 11). Thus, the diversity of tentacle- or papillae-bearing spathidiids could be even higher than currently indicated. The genus Belonophrya is highly similar to Actinobolina, but molecular data and ultrastructural details of its tentacles are still unavailable [51]. Another tentacle-bearing ciliate, Holophrya ornata is now synonymized with Legendrea bellerophon based on its morphological characteristics (Figure 12) [40].

Given the complex relationships between tentaculiferous lineages in Spathidiidae or Haptoria, it is not surprising that tentacles of other litostomateans are also likely not homologous. Tentacles in the family Mesodiniidae, possibly a sister group to Litostomatea, are placed anteriorly, are reinforced by a cylindrical structure of 14 microtubules, and bear extrusomes [1,52,53]. Another tentacle-bearing species with unclear phylogenetic position is Enchelyomorpha vermicularis, once included in Actinobolinidae, tentacles of which are also reinforced by microtubules and do not bear extrusomes, which is now considered to be the swarmer of a globular suctorian on morphological grounds but has not yet been sequenced [1,54]. Tentacles of the members of the class Phyllopharyngea have a completely different morphology compared to both Legendrea and Dactylochlamys. Typical suctorian feeding tentacles are formed by two concentric cylinders of microtubules, the inner one being reinforced by microtubule fibrils, and the outer with various taxon-specific microtubular structures [1,55]. Although, as indicated by their name, suctorians have been assumed to “suck” cell contents from their prey, this mechanism has been cast into doubt by Rudzinska [56]. In the parasitic subclass Rhynchodia, only a single sucking tentacle-like structure, probably a transformed cytostome, is present [1].

4.4. Putative Prokaryotic Endosymbionts

Using Sanger sequencing of the partial 16S rRNA gene, we identified putative methanogenic endosymbionts of L. pespelicani as Methanobacterium sp. and Methanosaeta sp. in L. loyezae. Using Illumina sequencing, we corroborated the identity of the archaeal symbiont of L. pespelicani as Methanobacterium sp. and identified the archaeal symbiont of Dactylochlamys pisciformis as Methanocorpusculum sp. Autofluorescence typical of archaeal methanogens was also observed in Dactylochlamys (Video S8) [57]. Interestingly, it seems that there is some symbiont diversity among the studied species, where genera belonging to the same clade and even each of the Legendrea species harbor different methanogenic symbionts. Methanogenic Archaea commonly form syntrophic symbioses with anaerobic ciliates using the hydrogen and acetate produced by the host mitochondria for their metabolism, e.g., [21,58,59,60]. This suggests that both Dactylochlamys and Legendrea are most likely facultative or obligate anaerobes, potentially having hydrogen-producing mitochondria. The role of these prokaryotes as endosymbionts requires confirmation by fluorescence in situ hybridization and/or transmission electron microscopy. Nevertheless, our results indicate that both Dactylochlamys and Legendrea represent a novel anaerobic lineage of ciliates. Interestingly, in addition to archaeal symbionts, we also identified a putative bacterial symbiont related to the family Syntrophaceae. The Syntrophaceae are strictly anaerobic and grow only in the presence of hydrogen-utilizing partners, such as archaeal methanogens [61]. Further investigations of symbiotic interactions between the host, archaeal, and bacterial endosymbionts in Legendrea are needed.

4.5. The Role of Citizen Science in Ciliatology

Taxonomic and biogeographic studies of a wide variety of groups, including protists, have always been plagued by the problem of undersampling. This fact, in part, underlies the uncertainty and contentiousness associated with such topics as protist endemicity [62,63,64]. Since the number of academic professionals dedicated to and funded for mainly taxonomic work has rapidly declined, the importance of the much larger population of “expert amateurs” (interested individuals without formal academic credentials in the particular specialty) in species identification, specimen collection, ecology, and biogeographical data collection has come into clearer focus [65,66,67,68,69,70]. The role of the expert amateur was central to science world-wide prior to the professionalization of science in the early 20th century, after which contributions by non-academics were increasingly ignored or criticized as inferior. Alfred Kahl, one of the most notable ciliatologists of the last century, a high school teacher by training, was probably an unfortunate object of this developing attitude [38,71]. Although a formal definition of “citizen-science” infers that it occurs mainly as an activity in collaboration with, or under the direction of, academic professionals usually as part of structured projects [72], we consider the data generated by expert amateurs working independently to be of considerable value, especially with regard to rare taxa such as Legendrea. Most of the information that has accrued in the century since the first description of the genus Legendrea has been gathered almost exclusively by expert amateurs and made publicly available in online forums and social media. The study of Weiss et al. and this study are perfect examples of the fruitfulness of such cooperation [8].

5. Conclusions

Although both genera, Legendrea and Dactylochlamys, presented in this study are considered to be remarkably rare, we were able to collect enough data to not only corroborate their phylogenetic position close to or within Spathidiidae but also revealed that both genera are closely related to each other despite apparent differences in their tentacle-like structures. We studied their morphology with modern methods and found out that these two ciliate genera are in fact anaerobes harboring prokaryotic endosymbionts. Our work demonstrates that for studying rare organisms, both modern single-cell methods and the contribution of citizen science are essential. Thus, we encourage the increasing collaboration of academic professionals with their expert amateur counterparts, inclusion of their data in peer-reviewed research publications, and acknowledgement of their contributions.