Overview of the Diversity, Phylogeny and Biogeography of Strombidiid Oligotrich Ciliates (Protista, Ciliophora), With a Brief Revision and a Key to the Known Genera

Strombidiids are common free-living ciliates that have colonized coastal and open oceanic waters across the world. In recent years, numerous new taxa and gene sequences of strombidiids have been reported, revealing a large diversity of both their morphologic and genetic features. Here, we compare the taxonomic characters of all genera in the family Strombidiidae, provide a key to their identification, and investigate their molecular phylogeny. In addition, we analyze their regional distribution based on faunal data accumulated in China and attempt to infer their global distribution based on SSU rRNA gene sequence data. The current work revises the systematics of strombidiids based on morphologic, phylogenetic, and biogeographic evidence and provides a genus-level review of marine strombidiids.


INTRODUCTION
Since the proposal of the "microbial loop" hypothesis (Azam et al., 1983), ciliates have been recognized as one of the major components of marine ecosystems (Azam and Malfatti, 2007;Fenchel, 2008;de Vargas et al., 2015;Bai et al., 2020a,b;Liu Q. et al., 2021). Ciliates, especially species in the subclass Oligotrichia, play essential roles in the microbial loop as grazers of nano-/picoplankton and as food sources for larger zooplankton including copepods and fish larvae Hu et al., 2019;Huang et al., 2021). Consequently, oligotrichs have been a focus in the study of marine science, especially their diversity, phylogeny and biogeography Santoferrara and McManus, 2017;Hu et al., 2019).
Since strombidiids are common members of the microzooplankton community, their distribution and biogeography have received extensive attention from ecologists. Knowledge of this topic, however, remains scant because data on their biogeography are limited by undersampling worldwide (Agatha, 2011;Lu et al., 2020). In recent years, the diversity of strombidiids in Chinese coastal waters has been extensively studied and almost all known genera in the family Strombidiidae have been recorded (Song W. et al., 2013;Liu W. et al., 2017;Song et al., 2018Song et al., , 2019Liu et al., 2019;Hu et al., 2019). Consequently, there is sufficient available data for their distribution in coastal regions of China to be reviewed.
In the last two decades, environmental sequencing-based techniques, e.g., clone library construction, denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), and high-throughput sequencing (HTS), have enabled studies on the distribution of ciliates to be carried out in a culture-independent and taxonomic expertise-free way (López-García et al., 2001;Moon-van der Staay et al., 2001;de Vargas et al., 2015;Xu et al., 2017). The application of environmental sequencing-based techniques has prompted the discovery of new ciliate lineages and previously undiscovered patterns of distribution Santoferrara et al., 2016;Sun et al., 2016Sun et al., , 2019Wang Y.R. et al., 2020). The rapid growth in the number of available SSU rRNA gene sequences allied to detailed observation and documentation of morphological characteristics can thus serve as a reliable database for inferring the taxonomic assignments of environmental sequences. The vast amount of data accumulated in the GenBank database, especially the nearly full-length SSU rRNA gene sequences from clone library studies, also offer the opportunity to infer the biogeographical distribution of strombidiids.
The aims of the present study are to (1) review the systematics of all genera in the family Strombidiidae based on morphological and, where available, molecular data; (2) provide a key to the identification of these genera, and (3) investigate the distribution of marine strombidiids based on taxonomic and SSU rRNA gene sequence data.

Phylogenetic Analyses
The SSU rRNA gene sequences of 65 species of Oligotrichia and Choreotrichia obtained from GenBank were employed to construct phylogenetic trees. Five species (representing five genera) of Halteriida and Hypotrichia were used as outgroup taxa. All 70 sequences were aligned using the MUSCLE algorithm on the GUIDANCE web server with the default parameters (Penn et al., 2010a,b). The ends of the alignment were trimmed using Bioedit (Hall, 1999), yielding a matrix of 1,768 characters. Maximum likelihood (ML) analysis was conducted using RAxML-HPC2 on XSEDE v 8.2.12 (Stamatakis, 2006;Stamatakis et al., 2008) with the optimal model evaluated by the online server CIPRES Science Gateway (Miller et al., 2010). The reliability of internal branches was assessed with a nonparametric bootstrap method featuring 1,000 replicates.
Bayesian inference (BI) analysis was performed with MrBayes 3.2.6 on XSEDE v 3.2.6 (Ronquist and Huelsenbeck, 2003) provided on the CIPRES Science Gateway, with the model GTR+I+ selected by the Akaike information criterion (AIC) in MrModeltest v2 (Nylander, 2004). Markov chain Monte Carlo chains were run for 4 × 10 6 generations with two parallel runs, each with four simultaneous chains, sampling every 100 generations. The first 10,000 generations were discarded as burnin prior to construction of the consensus tree.

Biogeographic Distribution
The regional biogeographic patterns of strombidiids were inferred based on a compilation of faunal data from samples collected from coastal regions of China. Variations in the distribution of strombidiid species from 19 coastal sites (Figure 1) were analyzed. The species richness and the community composition in each site were compared. In addition, among these sites, eight (a-h in Figure 1) were located near Qingdao, Shandong Province, northern China, and 11 (i-s in Figure 1) were located in Guangdong and Hainan Provinces, southern China. The communities were compared between northern and southern China.
To infer the possible worldwide biogeographic distribution of the strombidiids, each annotated SSU rRNA gene sequence was blasted against the GenBank The nucleotide collection database and it's nearest environmental neighbor (NEN), i.e., the environmental sequence with the highest sequence similarity, were retrieved . The locations of the sequences of identified strombidiids and NEN retrieved sequences were marked on a map.

RESULTS AND DISCUSSION
To date, more than 100 species belonging to 12 genera have been assigned to the family Strombidiidae, including 10 species and seven genera that have been described during the past decade (Agatha, 2011;Liu W. et al., 2017;Hu et al., 2019). In addition, molecular data on strombidiids have been extensively collected in the past 15 years and there are now 46 species with available SSU rRNA gene sequences (Gao et al., 2017;Song et al., 2020). Although evolutionary relationships of strombidiids have been discussed in several studies, the molecular phylogenies often reveal unexpected relationships that are not consistent with cladograms based on morphological data, especially at the genus level (McManus and Katz, 2009;Agatha and Strüder-Kypke, 2014). The number of new species and genera in the family Strombidiidae is continually increasing, supporting the assertion that 83%-89% of free-living ciliate species are undescribed (Foissner et al., 2008). The taxonomy and systematics of the family Strombidiidae therefore need to be updated.

Taxonomic Review of Genera in the Family Strombidiidae
The taxonomy of the 12 known genera of Strombidiidae are summarized, including their morphological diagnostic characters, type species, remarks on the history of their establishment, and subsequent revisions (Figures 2, 3).
Type species: Foissneridium constrictum (Meunier, 1910;Agatha, 2011) Extrusome attachment sites arranged along the anterior margin of the girdle kinety are typical in Strombidiidae (Agatha, 2011). In F. constrictum, however, the extrusome attachment sites are pre-equatorial. Moreover, its oral primordium is posterior to the extrusome attachment sites and anterior to the girdle kinety. These are genus-level characters based on which Foissneridium was established.
Although the oral primordium is located anterior to their girdle kinety in both Foissneridium and Opisthostrombidium, the former differs from the latter in that its oral primordium is posterior to the extrusome attachment sites (vs. anterior to extrusome attachment sites). Foissneridium constrictum is the only species in the genus Foissneridium.
Genus Novistrombidium (Song and Bradbury, 1998) Diagnosis: Strombidiidae with incomplete girdle kinety around the equatorial area that is conspicuously open with a large ventral gap through which ventral kinety extends (Song and Bradbury, 1998).
Type species: Novistrombidium testaceum (Anigstein, 1914;Song and Bradbury, 1998) The genus Novistrombidium was established by Song and Bradbury (1998). Agatha and Strüder-Kypke (2014) established two subgenera of Novistrombidium, mainly based on different locations of the oral primordium relative to the girdle kinety and the extrusome attachment sites. In the subgenus Novistrombidium (Novistrombidium) Song and Bradbury (1998), the oral primordium is located between a question mark-shaped field of extrusome attachment sites and the girdle kinety. In the subgenus Novistrombidium (Propecingulum) Agatha and Strüder-Kypke (2014), the oral primordium is located anterior to the stripe of extrusome attachment sites that extends alongside the girdle kinety. Küppers et al. (2019) considered the morphological differences between these two subgenera to be sufficient for them to be elevated to genus rank. However, considering the limited numbers of known species in these two subgenera, we prefer to adopt a conservative approach pending the availability of more morphological and molecular data from more taxa.
Type species: Omegastrombidium elegans (Florentin, 1901;Agatha, 2004) Florentin (1901) Song et al., 2018). (P,Q) Antestrombidium agathae (after Liu et al., 2015b). Numbers at the nodes represent support values in the following order: BI posterior probabilities and ML bootstrap values. Disagreements in topology between the BI and ML trees are indicated by a hyphen. Nodes that were maximally supported (1.00 BI; 100% ML) are represented by filled circles. Scale bar, three substitutions per 100 nucleotide positions. Species discussed in the present study are shown in bold type. and revealed its ciliary pattern for the first time. Based on the girdle kinety performing a " " shape, Agatha (2004) established the genus Omegastrombidium and designated O. elegans the type species.
Type species: Opisthostrombidium montagnesi (Xu et al., 2006;Agatha, 2011) In some Strombidium species, the oral primordium forms anteriorly to the horizontal girdle kinety and extrusome attachment sites, thus differing from the usual arrangement in Strombidium species in which the oral primordium forms posteriorly to the horizontal girdle kinety and extrusome attachment sites. The genus Opisthostrombidium is separated from Strombidium based on this character.
Type species: Parallelostrombidium rhyticollare (Corliss and Snyder, 1986;Agatha, 2004) Corliss and Snyder (1986) described this species under the name Strombidium rhyticollare. Petz et al. (1995) provided a redescription and transferred it to the genus Spirostrombidium based on the spiraled girdle kinety. Later, based on the similar orientation of the ventral and girdle kineties, Agatha (2004) established the genus Parallelostrombidium and designated P. rhyticollare as the type species. Agatha (2004) concluded that the ventral kinety and the posterior portion of the girdle kinety in Parallelostrombidium may or may not be inversely oriented.
Type species: Sinistrostrombidium cupiformum  In strombidiids, the girdle kinety is generally circular or dextrally spiraled. In S. cupiformum, however, the girdle kinety is sinistrally spiraled, based on which the genus Sinistrostrombidium was established .
Type species: Spirostrombidium sauerbreyae (Kahl, 1932) The genus Spirostrombidium was established by Jankowski (1978) and redefined by Agatha (2004). The ciliary pattern of this genus is similar to that of Parallelostrombidium except that the posterior portions of the ventral kinety and girdle kinety have opposite orientations. A key to the identification of species of Spirostrombidium was provided by Wang et al. (2018).
Type species: Strombidium sulcatum (Claparède and Lachmann, 1859) Strombidium is the type and the most speciose genus in the family Strombidiidae; however, some poorly characterized nominal species have been misidentified as Strombidium.
Genus Varistrombidium (Xu et al., 2011) Diagnosis: Strombidiidae with five spirally arranged somatic kineties that run obliquely across the ventral side and parallel to each other, with the longest two extending onto the dorsal side and terminating in the caudal area (Xu et al., 2011).
Type species: Varistrombidium kielum (Maeda and Carey, 1985;Xu et al., 2011) Kahl (1932) described a species collected from marine sand in Kiel Bay, Germany, and identified it as an unknown species of Strombidium. Maeda and Carey (1985)

Key to the Identification of Genera in the Family Strombidiidae
For the identification of each genus, detailed information on the somatic ciliary pattern, and for some genera the position of the oral primordium, is necessary.

Phylogeny of Strombidiid Genera Based on SSU rRNA Gene Sequences
The tree topologies from the BI and ML analyses are similar; therefore, only the ML tree is presented with support values from both methods at the branch nodes (Figure 3). DNA sequences have been reported for 10 of the 12 genera in the family Strombidiidae, the exceptions being Foissneridium and Opisthostrombidium.
Each of the genera Strombidium and Spirostrombidium is polyphyletic, which is consistent with previous studies Song et al., 2020). To date, there is no agreed interpretation of their polyphyly. Species of Strombidium fall into several assemblages, some with morphological support: (1) Strombidium conicum and S. chlorophyllum are basal within the Oligotrichia, and their close relationship corresponds to their morphological similarity in that both species share a special kind of hemitheca; (2) Strombidium basimorphum, S. paracapitatum, and S. biarmatum form a well-supported clade which corresponds with their morphological similarity in that each has two types of extrusome whereas all other strombidiids have only one; (3) Strombidium apolatum, S. rassoulzadegani, S. oculatum, S. purpureum, S. guangdongense, and S. cuneiforme fall into a well-supported clade that also includes Williophrya maedai (present work; Liu et al., 2011Liu et al., , 2013Liu et al., , 2015aLiu et al., , 2016Song W. et al., 2013;Song et al., 2015aSong et al., ,b, 2018Song et al., , 2019Wang et al., 2018). This has been referred to as the "eyespot clade" since all species within it possess a pigment spot, although S. purpureum lacks detailed in vivo information (Gao et al., 2016;Liu et al., 2016). Spirostrombidium is polyphyletic, although the positions of some Spirostrombidium species are not stable and have only low statistical support, which is consistent with previous studies Song et al., 2020).
The genus Parallelostrombidium forms two clusters that correspond to differences in cell shape and somatic ciliary pattern, which is consistent with previous phylogenetic studies Song et al., 2018). One cluster comprises Parallelostrombidium conicum, P. jankowskii, and P. kahli, each of which has an obconical cell shape with a pointed posterior end and their ventral kinety parallel to the girdle kinety except the anteriormost portion. The other cluster comprises five species that share a dorsoventrally flattened cell shape with a rounded posterior end, and only the posterior portion of the ventral kinety is parallel to the girdle kinety.
Species of Novistrombidium are divided into two distantly related assemblages, which corresponds with the separation of this genus into two subgenera and may support their raising to genus rank (Agatha and Strüder-Kypke, 2014;Küppers et al., 2019). The subgenus Novistrombidium (Novistrombidium) is monophyletic whereas the subgenus Novistrombidium (Propecingulum) is not monophyletic as each of its species clusters with other genera.
The three Apostrombidium species and V. kielum form a clade (Figure 3), and previous studies have consistently recovered a close relationship between Apostrombidium and Varistrombidium (Liu et al., , 2016Song W. et al., 2013;Song et al., 2015aSong et al., , 2019Tsai et al., 2015). Morphological data support this finding since both genera have a dorsal split of the girdle kinety and long cilia on the dorsal side (Gao et al., 2016).
In the BI tree (not shown), Antestrombidium agathae clusters with Omegastrombidium elegans and then clusters with Novistrombidium orientale, whereas in the ML tree A. agathae clusters with N. orientale, which together cluster with Omegastrombidium elegans. In several previous studies, A. agathae clusters with O. elegans (Liu et al., , 2016Gao et al., 2016;Song et al., 2019). This finding supports the close evolutionary relationship between Antestrombidium and Omegastrombidium hypothesized in Liu et al. (2015b) and corresponds with the similarity of their morphology. For example, the circular kinety of Antestrombidium appears to be homologous to the -shaped girdle kinety in Omegastrombidium.
Sinistrostrombidium cupiformum clusters with Strombidium tropicum, which is consistent with previous studies Wang et al., 2018;Song et al., 2019). It is noteworthy that the girdle kinety of Strombidium tropicum is slightly spiraled, i.e., the left end of the girdle kinety is positioned higher than the right one, and may represent a ciliary pattern from which Sinistrostrombidium originated (Liu et al., 2015a,b). Nevertheless, the evolutionary relationship between these two species requires further investigation since previous studies have reported that Sinistrostrombidium cupiformum forms an isolated basal branch in some phylogenetic tress (Gao et al., 2016;Liu et al., 2016).

Geographic Distribution of the Strombidiids in China
Eleven of the 12 genera of the family Strombidiidae have been found in Chinese coastal waters (Figures 1, 4), the exception being Foissneridium which has so far only been isolated from the Barents Sea (Meunier, 1910). In terms of species numbers, the genus Strombidium is best represented with 21 species (Figure 4A). This is consistent with findings in other geographic regions such as the northwest and south Atlantic Ocean, the Baltic Sea, and the Mediterranean Sea, where Strombidium is also the strombidiid genus represented by the largest number of species (Dolan and Marrasé, 1995;Santoferrara and Alder, 2009;Agatha, 2011). Ranking second and third are Spirostrombidium (nine species) and Parallelostrombidium (eight species), respectively ( Figure 4A). For Varistrombidium,  Williophrya, Sinistrostrombidium, and Antestrombidium, only one species each has been recorded in Chinese coastal waters. Regarding the distribution of these genera, Strombidium has a most extensive range with occurrences at 15 sites, followed by Parallelostrombidium (10 sites) and Spirostrombidium (nine sites) ( Figure 1B). The comparison of diversity in these sites showed that the highest species richness (14 species) occurred at site k and the lowest was at five sites (h, i, l, n, p) with only one species at each (Figure 1C). At the genus level, sites a, k, and q were the most genus-rich with six genera at each whereas sites d, h, i, l, n, and p each had only one genus ( Figure 1B).
Occurrences of strombidiids in northern and southern China were compared (Figure 4B). Representative of seven genera, i.e., Strombidium, Spirostrombidium, Parallelostrombidium, Novistrombidium, Omegastrombidium, Apostrombidium, and Varistrombidium, were found both in northern and southern China. Opisthostrombidium has only been reported from northern Chinese coastal waters, while Williophrya, Sinistrostrombidium, and Antestrombidium have only been reported from southern Chinese coastal waters. The total number of strombidiids is higher in southern China where 37 species representing 10 genera were collected compared to 30 species and eight genera in northern China. There are two possible reasons for this: (1) the sampling area was wider and the number of sampling sites was higher in southern than in northern China; (2) the environment features of some southern coastal habitats are more suitable for ciliates than those in the north. For example, mangrove wetlands, which are nutrient-rich and could provide a greater quantity and diversity of food resources for ciliates, are only located in southern China.

Global Distribution of the Strombidiids Based on Molecular Information
Most Strombidiidae species for which gene sequence data (mostly SSU rRNA but also ITS and LSU rRNA sequences of some taxa) are available in the GenBank database are from specimens collected from coastal water habitats, including sediments, lagoons, and bays, which are much easier to sample than oceanic habitats. To infer the possible worldwide distribution of the reported species within Strombidiidae, BLAST comparisons against the GenBank Nucleotide collection database were run with SSU rRNA gene sequences of known species. The Sequence Read Archive (SRA) contains only short sequences from high throughput sequencing and thus was not used in the present study.
The nearest environmental neighbor (NEN) for each species, i.e., the environmental sequence with the highest sequence similarity, was obtained as the first BLAST hit. The NENs of strombidiids were from various habitats including mangroves, coastal waters, estuaries, fjords, solar saltern ponds, open ocean waters, and oxygen-depleted marine environments ( Figure 5 and Table 1). Parallelostrombidium conicum was identical to its NEN (100% similarity),  For explanation of locations, see Figure 5.
which was reported from the Bering Sea (KC771186). Five Strombidium species, four Novistrombidium species, three Apostrombidium species, three Spirostrombidium species, Sinistrostrombidium cupiformum, and Varistrombidium kielum each had the same NEN, i.e., EU371386, which was collected from Kongsfjorden, Svalbard (Table 1). Similarly, seven out of eight Parallelostrombidium species, Omegastrombidium elegans, Spirostrombidium schizostomum, and Novistrombidium fistoleramalliei also had the same NEN, i.e., KC771186, which was collected from the Bering Sea. The NENs of Strombidium rassoulzadegani and S. intermedium, and of S. basimorphum and S. paracapitatum, were collected from an oxygen-depleted salt marsh in Massachusetts, United States (AY180033) and the anoxic Framvaren fjord in Norway (EF527106), respectively. For environmental sequencing studies, delineating operational taxonomic units (OTUs) is a key step in the analysis and can significantly affect the results (Nebel et al., 2011). A 5% cutoff (95% sequence similarity) was recommended for microbial eukaryotes by Caron et al. (2009). For ciliates, a 1%-3% cutoff value (i.e., 97-99% sequence similarity) for the SSU rRNA gene is often used (Stoeck et al., 2006;Doherty et al., 2007Doherty et al., , 2010Sun et al., 2017Sun et al., , 2019, although a finer (<1%) cutoff might be needed for some groups (Xu et al., 2013). In the present analysis, the average similarity of the 49 species/populations of strombidiids was 97.9% with their NENs, ranging from 90.3 to 100%. Among these, 17 species/populations had a similarity with their NEN of >99%, 15 had a similarity of 97-99%, 16 had a similarity of 95-97%, and one had a similarity of 90.3%. If a 97% similarity cutoff is used to distinguish different species, 65% of the 49 species/populations are conspecific with their NEN. Even if a much stricter cutoff is used (1%), 35% of the species/populations are conspecific with their NEN. These findings suggest that the species isolated from Chinese coastal waters were likely to be globally distributed based on the 5% cutoff suggested by Caron et al. (2009) with 48 out of 49 species being conspecific with their NEN. Furthermore, it is noteworthy that some species from different genera can have the same NEN, which may indicate that the environmental sampling and sequencing efforts are far from saturation. Increased collection of ciliate environmental sequences from different marine environments, especially those that are difficult to sample, will help improve knowledge and understanding of the biogeographical distribution patterns of marine strombidiids. Also, the enrichment of the full-length or near full-length SSU rRNA gene sequences from various oceanic environments paired with detailed morphological observations will serve to improve the database, thereby contributing to the identification of environmental sequences.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
LL, WeiS, and MS conceived the research. WenS, DX, and XC conducted the analysis and drafted the manuscript. AW critically reviewed the findings and improved the manuscript. All authors contributed to the article and approved the submitted version.