Taxonomy and SSU rRNA gene-based phylogeny of two new Euplotes species from China: E. chongmingensis n. sp. and E. paramieti n. sp. (Protista, Ciliophora)

Background The genus Euplotes Ehrenberg, 1830, one of the most complicated and confused taxa, contains about 160 nominal species. It was once proposed to be divided into four genera, two of which were proved to be non-monophyletic. At least 19 new species have been discovered in the past decade, implying that there is a large undiscovered diversity of this genus. Results The morphology of two new freshwater euplotid ciliates, Euplotes chongmingensis n. sp. and E. paramieti n. sp., isolated from Shanghai, China, were investigated using live observations, protargol staining, and Chatton-Lwoff silver staining method. Euplotes chongmingensis is characterized by its small size (40–50 × 25–35 μm), about 24 adoral membranelles, 10 frontoventral cirri, two marginal and two caudal cirri, eight dorsolateral kineties with 11–16 dikinetids in the mid-dorsolateral kinety and a double type of silverline system. Euplotes paramieti n. sp. is 180–220 × 110–155 μm in vivo and strongly resembles E. amieti but having a difference of 57 bp in their SSU rRNA gene sequences. Phylogenetic analyses based on SSU rRNA gene sequence data were used to determine the systematic positions of these new taxa. Conclusions The description of two new freshwater taxa and their SSU rRNA gene sequences improve knowledge of biodiversity and enrich the database of euplotids. Furthermore, it offers a reliable reference for environmental monitoring and resource investigations.

at least four Euplotes species have been sequenced, which may be useful for the identification of new Euplotes species [15][16][17][18].
Borror and Hill [19] split Euplotes into four genera: Euplotes, Euplotopsis, Euplotoides, and Moneuplotes, based on characteristics of cortical structure, endosymbionts, morphometric data, morphogenetic patterns, and ecology. This classification has repeatedly been disclaimed by genetic analyses which show that neither Euplotes nor Euplotopsis is monophyletic [20,21]. Nevertheless, reliable subdivisions have yet to be discovered, therefore most taxonomic studies still recognize Euplotes sensu Ehrenberg, 1830, as we do in the present study.
There is increasing evidence that the biogeographical distribution of ciliates follows the moderate endemicity model [22,23], which indicates there are unknown species in underexplored habitats. This assertion is supported by the discovery in China in the past decade of eight new Euplotes species isolated from previously unsampled habitat or areas [24][25][26][27][28]. Yangtze estuary is an example of a location that has been rarely sampled for its ciliate biodiversity. In the present study, two novel euplotid ciliates were isolated from this estuary. Both were found to be new species of Euplotes. Each is described based on its morphology and SSU rRNA gene sequence following the recommendations in Warren et al. [29]. Their molecular phylogeny was analyzed to determine their evolutionary relationships.

Etymology
The species-group name chongmingensis refers to the area (Chongming Island) where the sample was collected.

Type specimens
The protargol slide with the holotype specimen (No. HK2020102802-1) and a Chatton-Lwoff silver nitrate slide with paratype specimens (No. HK2020102802-2) are deposited in the Laboratory of Protozoology, Shanghai Ocean University.

Etymology
The species-group name paramieti is a composite of the prefix para-(Greek preposition, beside, like) and the species-group name amieti, referring to the similarity of this species to Euplotes amieti.
ZooBank registered details of Euplotes paramieti n. Phylogenetic trees inferred by ML and BI analyses had similar topologies, therefore only the ML is shown with nodal support from both algorithms (Fig. 5

Euplotes chongmingensis n. sp.
Five freshwater species should be compared with E. chongmingensis n. sp. as each has a similar-size body, 10 frontoventral cirri, two marginal cirri, and a double silverline system.  (5 vs. 6), and fewer dorsolateral kineties (7 vs. 8) [30]. The SSU rRNA gene sequence of the type population of E. indica (MN038061) has a 96.83% similarity with E. chongmingensis, and thus is the adelphotaxon of the latter. Euplotes vanleeuwenhoeki was found in Kolleru Lake, India by Serra et al. [31]. It resembles E. chongmingensis in cell size and the pattern of both the ventral and dorsal infraciliature. However, E. vanleeuwenhoeki has three longitudinal furrows on the dorsal surface that reach the posterior region of the cell [31]. Furthermore, these two species are placed in different clades in the SSU rRNA gene tree and they have a sequence dissimilarity of 3.99%.
Euplotes palustris was reported by Hagen [32] from marshy areas in Westfalen, Germany. It resembles E. chongmingensis n. sp. in cell size (45-55 × 35-45 μm in vivo) and dorsal ciliature. However, it can be separated from the new species by having two distinctly weaker frontoventral cirri IV/2 and V/2 (vs. 10 normalsized frontoventral cirri in E. chongmingensis n. sp.) and its transverse cirri are arranged in two groups (vs. in one group) [32]. Molecular information for E palustris has not been reported.

Wing structure
Wings have been reported in populations of Euplotes muscicola Kahl, 1932 Wang, 1930 [11, 30, 49-52]. It has been shown that some freshwater Euplotes can change their morphology, for example by developing wings or a dorsal keel, to defend themselves against the risk of predation [49]. In our non-clonal cultures of E. paramieti n. sp., Daphnia sp., which is considered a potential predator of ciliates [53], was present. The wing in E. paramieti n. sp. may therefore be a predator-induced defense response caused by the presence of Daphnia rather than being a diagnostic character for species circumscription and identification.

Phylogenetic analyses
The phylogenetic trees (ML and BI) are consistent with previous phylogenetic analyses, even with the addition of two new taxa [24,25,30,37,61,62]. The 10 frontoventral cirri and double dorsal silverline pattern are possibly an ancestral character of euplotids considering the shared traits of the basal clade [30,63]. Clades within the SSU rRNA gene tree that are supported by a common morphological trait include: one clade comprising species that lack the V/2 cirrus, i.e., species formerly classified as Euplotides plus E. paramieti n. sp.; and another clade that comprises species with a single dorsal silverline system, i.e., species formerly classified as Monoeuplotes, but excluding E. antarcticus sensu Liu et al., 2020 (MG603602) which was misidentified [13].
Euplotes chongmingensis clusters with E. indica, its most similar freshwater species, with full support (100% ML, 1.00 BI), although they have a SSU rRNA gene sequence dissimilarity of 3.17%. The other two most closely related species, E euryhalinus and E. magnicirratus, are marine, Moreover, their sequences differ from E. chongmingensis by 53 bp and 52 bp, respectively. Therefore, validity of E. chongmingensis as a separate species is supported.
Euplotes paramieti clusters with E. amieti (KJ524911) and four populations of E. eurystomus (FR873716, EF193250, AJ310491, FR873717). The nucleotide difference between E. paramieti and each of these four populations is 41-47 bp. There is another population of E. eurystomus (AF452707) in the sister clade. However, the five populations of E. eurystomus lack reliable morphological descriptions and/or vouchered specimens, therefore their identity cannot be confirmed.

Conclusions
We described two novel ciliates from Shanghai, China: Euplotes chongmingensis n. sp. and E. paramieti n. sp. from River Yangtze estuary, China. The validity of each is supported both by their morphology and their molecular sequences. Some morphological characteristics of the species in the genus Euplotes overlap, so using a multidisciplinary approach could reduce the confusion and ambiguity. Some "well-known" species, e.g., E. eurystomus, should be reinvestigated considering the questionable sequences in GenBank.

Sample collection and identification
Euplotes chongmingensis was collected on 26th Sept. 2019 from a river (31°50′32.6"N, 121°16′02.5"E) on Chongming Island, Shanghai, China, where the water temperature was 17.3 °C and the salinity was 0‰. Euplotes paramieti was collected on 28th Oct. 2020 from the intertidal zone of Hengsha Island (31°18′17.8"N, 121°49′49.7"E), at the estuary of the River Yangtze, Shanghai, China, where the water temperature was 18.8 °C and the salinity was 0.25‰. Non-clonal cultures were established and maintained at room temperature (about 20 °C) in Petri dishes containing mineral water with rice grains added to enrich the growth of bacteria as a food source for the ciliates. Ciliate cells were observed in vivo using bright field and Nomarski differential interference contrast microscopy at magnifications between 100 × and 1000 × [64]. The infraciliature and nuclear apparatus were revealed by protargol staining [65] and the silverline systems were revealed by the Chatton-Lwoff silver nitrate staining method [8]. Counts and measurements were performed at a magnification of 1000 × . Drawings of stained specimens were made with the help of a drawing attachment and photomicrographs. Terminology is mainly according to Curds [6], except for the marginal cirri.

DNA extraction, PCR amplification, and gene sequencing
To avoid contamination, a single cell was picked out using a sterile micropipette and washed five times with distilled water. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, CA) following the manufacturer's instructions but modified such that one quarter of the volume of each reagent was used. PCR amplifications of the SSU rRNA gene were performed with the primers 18S-F (5'-AAC CTG GTT GAT CCT GCC AGT-3') and 18S-R (5'-TG ATC CTT CTG CAG GTT CAC CTAC-3') [66]. Cycling parameters were as follows: initial denaturation at 98 °C for 30 s, 34 cycles of amplification (98 °C, 10 s; 69 °C, 30 s; 72 °C, 1 min), with a final extension of 72 °C for 5 min. PCR product purification and clone sequencing were performed by Sangon Biotech (Shanghai) company.

Phylogenetic analyses
The SSU rRNA sequences of the two new species were aligned with 80 other related ciliate sequences obtained from the National Center for Biotechnology Information (NCBI) database, including all the isolates of Euplotes eurystomus (for GenBank accession numbers, see Fig. 5). Discocephalus ehrenbergi (JX460983) and D. pararotatorius (FJ19639) were chosen as the outgroup taxa. Sequences were aligned using the muscle algorithm [67] in MEGA X [68] with the default parameters. The ends of the resulting alignment were refined by Gblocks (http:// www. phylo geny. fr/ one_ task. cgi? task_ type= gbloc ks), yielding an alignment of 1623 characters.
Maximum likelihood (ML) analysis with 1,000 bootstrap replicates was performed using RAxML-HPC2 on XSEDE 8.2.12 [69] on the CIPRES Science Gateway with the GTRGAMMA model (http:// www. phylo. org). Bayesian inference (BI) analysis was applied on the same platform using MrBayes 3.2.7 on XSEDE [70] on the CIPRES Science Gateway with the best fit model GTR + I + G, which was selected by the Akaike Information Criterion (AIC) in MrModeltest 2.2 [71]. Markov chain Monte Carlo (MCMC) simulations were run for 1,000,000 generations with sampling every 100 generations and a burn-in of 1,000 trees. Tree topologies were manually formatted with