Morphology, morphogenesis and molecular phylogeny of a novel saline soil ciliate, Lamtostyla salina n. sp. (Ciliophora, Hypotricha)

Accepted Manuscript Title: Morphology, morphogenesis and molecular phylogeny of a novel saline soil ciliate, Lamtostyla salina n. sp. (Ciliophora, Hypotricha) Author: Jingyi Dong Xiaoteng Lu Chen Shao Jie Huang Khaled A.S. Al-Rasheid PII: DOI: Reference:

S0932-4739(16)30085-2 http://dx.doi.org/doi:10.1016/j.ejop.2016.09.005 EJOP 25455

To appear in: Received date: Revised date: Accepted date:

27-4-2016 9-9-2016 9-9-2016

Please cite this article as: Dong, J., Lu, X., Shao, C., Huang, J., Al-Rasheid, K.A.S.,Morphology, morphogenesis and molecular phylogeny of a novel saline soil ciliate, Lamtostyla salina n. sp. (Ciliophora, Hypotricha), European Journal of Protistology (2016), http://dx.doi.org/10.1016/j.ejop.2016.09.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

European Journal of Protistology Morphology, morphogenesis and molecular phylogeny of a novel saline soil ciliate, Lamtostyla salina n. sp. (Ciliophora, Hypotricha)

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Jingyi Donga,b,*, Xiaoteng Lua,b,*, Chen Shaob,**, Jie Huangc,**, Khaled A.S. Al-Rasheidd

Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003,

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China b

The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School

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of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China

Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences,

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Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China d

Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia

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*both authors contribute equally **Corresponding author

Chen Shao, e–mail: [email protected]

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Jie Huang, e–mail: [email protected]

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Abstract The morphology and morphogenesis of a new saline soil hypotrich, Lamtostyla salina n. sp., collected from Longfeng Wetland in Daqing, north China, were studied based on live observations and protargol stained specimens. The new species is characterised as follows: body very flexible but not contractile, lanceolate with anterior end broadly rounded, widest at about 1/3 of body length, posterior end narrowly rounded; cortical granules colourless and

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scattered; amphisiellid median cirral row ends ahead of mid–body, composed of 5–13 cirri; 4–20 frontoventral cirri arranged in 2–4 rows; three frontal, one buccal and 2–6 transverse cirri; usually one left and one right marginal row, composed of 17–51 and 20–51 cirri respectively;

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usually two dorsal kineties; two macronuclear nodules and two micronuclei. Morphogenesis is typical of the genus Lamtostyla: parental structures are involved in the formation of

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frontoventral transverse anlagen in the proter. Phylogenetic analyses based on small subunit ribosomal DNA sequence data reveal that the systematic position of Lamtostyla is rather

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unstable with low support values across the tree. However, a well-recognized close relationship between Lamtostyla and Bistichella are shown in all phylogenetic analyses. Keywords: Lamtostyla; morphogenesis; new species; saline soil habitat; SSU rDNA

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phylogeny

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Introduction Recently, hypotrichs have proven to be much more diverse than originally estimated, largely as a result of investigations of new habitats in Asia and South America (Bharti et al. 2015; Bourland 2015; Fan et al. 2015; Foissner 2016; Foissner et al. 2002; Hu et al. 2015; Hu and Kusuoka 2015; Jo et al. 2015; Jung et al. 2015a, b; Kumar and Foissner 2015, 2016; Li et al. 2016; Luo et al. 2015; Lv et al. 2015; Paiva et al. 2015; Pan et al. 2016; Singh and Kamra

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2015; Song et al. 2009, 2011; Zhao et al. 2015). The genus Lamtostyla, established by Buitkamp (1977) with L. lamottei as the type species, is mainly characterised by two or more cirri left of the anterior portion of the amphisiellid

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median row (Berger 2008). Since then 11 other species have been described and reviewed in

detail by Berger (2008). Recently, Luo et al. (2016) described L. ovalis from coast off Qingdao

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in China. Based upon the cirral pattern and cortical granulation, Berger (2008) further separated the Lamtostyla species into three groups, i.e. L. lamottei-group, L. granulifera-group

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and L. longa-group.

In May 2014, saline soil samples from Longfeng Wetland, a district of Daqing in northern China were collected. In March 2015, an unknown hypotrichous ciliate was isolated from

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these soil samples. Observations of its morphology, both in vivo and after protargol staining, demonstrate that it represents a novel species within the genus Lamtostyla. In this paper we describe its morphology and morphogenesis. The SSU rDNA of the new isolate was

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sequenced and analyzed in order to assess its phylogenetic position. Material and Methods

Sampling and cultivation (Fig. 1A–D)

Lamtostyla salina n. sp. was collected from a saline soil sample (soil percolate salinity approximately 20‰, measured by ATAGO MASTER–S/Millα (Cat. No. 2491); pH appoximatelly 10, measured by pH indicator papers) with a strong foul smell (very likely sulphide) in the Longfeng Wetland (46°35'30''N, 125°13'08''E), Daqing, northern China, on May 6th 2014. On March 25th 2015, ciliates were made to excyst by employing the non–flooded Petri dish method (Foissner et al. 2002). We were able to identify the species accurately based on its in vivo morphologic characteristics. Moreover, no other Lamtostyla morphotypes were present in the protargol preparation. The probability is therefore extremely high that our morphological and molecular studies deal with the same species although we were unable to establish clonal cultures. Morphology and morphogenesis Isolated cells were observed in vivo using bright field and differential interference contrast microscopy. The protargol staining method of Wilbert (1975) was used to reveal the nuclear apparatus and the infraciliature. Counts and measurements of stained specimens were 3

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performed at a magnification of 1,000 ×. Drawings of stained cells were made with the aid of a drawing attachment at a magnification of 1,250 ×. In illustrations of morphogenetic processes, old (parental) ciliary structures are depicted by contour whereas new structures are shaded black. Terminology is according to Berger (2008). DNA extraction, PCR amplification, and sequencing Genomic DNA extraction, PCR amplification, and sequencing of the SSU rRNA gene were

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performed according to the method set out in previous studies (Gao et al. 2013). One or more cells was/were isolated from the ciliate cultures and washed three times with filtered water (0.22 µm) to remove potential contamination. These cells were then transferred to a 1.5 ml

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microfuge tube with a minimum volume of water. Genomic DNA was extracted using Dneasy

Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The

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PCR was amplified by PremixTaq (Takara ExTaq version cat. no. DRR003A), with primers

AGG TTC ACC TAC-3’) (Medlin et al. 1988). Phylogenetic analyses

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18S–F (5’-AAC CTG GTT GAT CCT GCC AGT-3’) and 18S–R (5’-TGATCC TTC TGC

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In order to reveal a more reliable and robust phylogeny, we performed preliminary maximum likelihood (ML) analyses with different selections of representative taxa and

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outgroups (e.g. 108, 87, 78, 69 species). Based on these preliminary analyses, a set of 67 SSU rDNA sequences was used in the present study, including sequences of Lamtostyla salina n.

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sp., 48 hypotrichs, 15 closely related environmental samples and three oligotrichs, namely, Favella taraikaensis, Tintinnopsis tocatinensis and Strombidium cuneiforme as the outgroup taxa (see Fig. 7 for accession numbers). Sequences were aligned using MUSCLE with the default parameters on the GUIDANCE server (Gao et al. 2016; Penn et al. 2010). Ambiguous regions and gaps were then removed by Gblocks 0.91b with default parameters (Castresana 2000;

Talavera

and

Castresana

2007)

(http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks). Further modifications were made manually using BioEdit 7.0 (Hall 1999) resulting in a matrix of 1696 characters (available from the authors upon request). ML analyses were conducted with RAxML–HPC2 v.8 on XSEDE (8.2.4) (Stamatakis et al. 2008) on the CIPRES Science Gateway (http://www.phylo.org/sub_sections/portal). The program MrModeltest v.2.0 (Nylander 2004) selected GTR + I + G as the best model with Akaike Information Criterion (AIC), which was then used for Bayesian inference (BI) analysis. BI analysis was performed with MrBayes on XSEDE 3.2.6 (Ronquist and Huelsenbeck 2003), with a run of 4,000,000 generations at a sampling frequency of 100 and a burn–in of 10,000 trees. All remaining trees were used to calculate posterior probabilities using a majority rule consensus. Node support came from 4

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1,000 bootstrap replicates. TREEVIEW v1.6.6 (Page 1996) and MEGA 4.0 (Tamura et al. 2007) were used to visualize tree topologies. Results Lamtostyla salina n. sp. Diagnosis. Size in vivo 100–160 × 45–50 μm. Body very flexible, lanceolate with anterior

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end broadly rounded and posterior end narrowly rounded. Cortical granules colourless and scattered, about 0.5 µm across. 18–29 adoral membranelles. Amphisiellid median cirral row ends ahead of mid–body, composed of 5–13 cirri. 4–20 frontoventral cirri arranged in 2–4

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rows left of amphisiellid median cirral row. One buccal cirrus. 2–6 transverse cirri. Usually

one left and one right marginal row, composed of 17–51 and 20–51 cirri respectively. Usually

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two bipolar dorsal kineties. Two macronuclear nodules. Usually two micronuclei. Saline soil habitat.

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Type locality. Longfeng Wetland (46°35'30''N, 125°13'08''E), Daqing, Heilongjiang Province, northern China.

Deposition of type slides. The holotype slide (registry no. Leo2015032501A) with the

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holotype specimen (Figs 2D, E, 5F) marked and one paratype slide (no. Leo2015032501B) have been deposited in the collection of the Laboratory of Protozoology, Ocean University of China, China. Another paratype slide (no. NHMUK 2016.8.1.1) has been deposited in the

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Natural History of Museum, London, UK.

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Etymology. The species–group name salina refers to the saline habitat where the type specimen was discovered.

Morphology (Figs 2A–E, 5A–N; Table 1). Size 100–160 × 45–50 μm in vivo, but 90–160 × 28–67 μm after protargol staining. Body very flexible, but not contractile, lanceolate with anterior end broadly rounded and posterior end narrowly rounded, widest at about one third of body length, slightly twisted about main axis (Figs 2A, B, 5A, B). Dorsoventrally flattened ca. 1.5:1. Usually with conspicuous grooves along marginal rows. Two ellipsoidal macronuclear nodules, about 26 × 14 µm in size (after protargol staining), located along cell midline or slightly left of it, one each in anterior and posterior half of cell, respectively. Usually, two micronuclei (ca. 2 µm across). Contractile vacuole about 10 µm across when fully extended, positioned at about 33%–40% of body length near left margin (Figs 2A, B, 5B, C). Cortical granules inconspicuous and colourless, about 0.5 µm across, scattered throughout cell surface (Figs 2C, 5E). Cytoplasm colourless to greyish, containing numerous lipid droplets (ca. 1–6 µm across) that render cell opaque and dark at low magnification (Fig. 5A, B). Locomotion mainly by slowly crawling on substrate and debris. In polyxenic cultures, cells usually aggregate around rice grains. 5

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Adoral zone 20–32 μm long, shaped like a question mark, occupying 16–28% (average about 20%) of body length in protargol preparations. 18–29 (on average 20, n = 24) adoral membranelles, cilia up to 15 μm long. Paroral and endoral about 12 μm and 16 μm long respectively, optically intersecting at anterior regions of both. Three enlarged frontal cirri, cilia about 15 µm long. Single buccal cirrus, cilia about 10 µm long, right of anterior end of paroral. Amphisiellid median cirral row (ACR) composed of 5–13 cirri, commences at about level of

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distal end of adoral zone, terminates at 13–39% of body length. 4–20 cirri arranged in 2–4 frontoventral rows (FVR) left of ACR. Of 23 investigated specimens, 19 cells possessed three FVRs, two possessed two FVRs, and the other two possessed four FVRs. FVR1–4 composed of

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1–4, 2–6, 2–10, and 7–9 cirri. 2–6 transverse cirri, pretransverse ventral cirri absent. Usually one right and one left marginal row with cirri about 10 μm long. Right and left marginal rows

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(when each of them is single) commence more or less at same level of mid–region of adoral zone but not confluent at posterior end, composed of 20–51 and 17–51 cirri respectively (Figs

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2D, 5F). Sometimes, a second or a third marginal row presents on either/both sides, e.g. three out of 24 investigated specimens possess two right marginal rows; four out of 24 cells possess two left marginal rows and only one has three left marginal rows (Fig. 5J–L). Interestingly, in

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most non–divisional individuals, the marginal cirri not arranged regularly, i.e. several marginal cirri not outlined very well and seem not fully differentiated, as well as being aggregated close to each other to form several relatively larger patches of basal bodies (Figs 2D, 5I). Usually,

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two bipolar dorsal kineties (Fig. 2E), but rarely three (one out of 24 cells; Fig. 5N). Morphogenesis (Figs 3A–I, 4A–D)

Stomatogenesis. In the opisthe, stomatogenesis commences with the formation of an oral primordium (Fig. 3A). Basal bodies of this oral primordium increase in number and then adoral membranelles begin to organize at the anterior end (Fig. 3C, E). As the formation of adoral membranelles proceeds posteriad, the anlage for the undulating membranes (UM–anlage = anlage I) is formed to the right of the oral primordium (Fig. 3F). Later, the UM–anlage produces the left frontal cirrus at its anterior end. Subsequently, the UM–anlage splits longitudinally into two streaks from which the paroral and endoral are formed (Figs 3H, 4A, C).

In the proter, the parental adoral membranelles are retained intact during the morphogenetic process, so changes to the oral structure are confined to the paroral and endoral. The UM–anlage is formed from the dedifferentiation of the parental undulating membranes (Fig. 3C, E). In subsequent stages, the basic development of the UM–anlage follows a similar pattern to that in the opisthe (Figs 3F, H, 4A, C). Development of the frontoventral transverse cirri. From the earliest stages found, frontoventral transverse cirri anlagen (FVT–anlagen) appear as a small group of basal bodies 6

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which connect with the anterior end of the oral primordium (Fig. 3A, arrow). Apparently, the parental ACR and the FVRs are disaggregated and involved in the FVT–anlagen formation. Later, 5–7 (based upon the following stages) thread–like FVT–anlagen are formed to the right of the UM–anlage in the proter as primary primordia (Fig. 3C, E). Then, the FVT–anlagen split into two sets in the middle stage, so that a set of 5–7 anlagen (not including the UM–anlage) each for the proter and opisthe is formed (Fig. 3F, H). Subsequently, when the

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segregation of cirri from the FVT–anlagen is complete, anlage n–1 produces the posterior portion of ACR; and anlage n forms the anterior portion of ACR (Fig. 4A, C).

Development of marginal rows and dorsal kineties. As is usual for most hypotrichs,

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marginal row anlagen and dorsal kinety anlagen are formed at two levels within the parental rows and kineties. Subsequently, the new marginal cirri/kineties develop and replace the old

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ones. Unfortunately, the development of multiple marginal rows is not observed. Dorsomarginal rows, caudal cirri and dorsal kinety fragmentation are lacking (Figs 3G, I, 4B,

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D).

Division of nuclear apparatus. The nuclear apparatus divides in the usual way for hypotrichs. Briefly, the two macronuclear nodules fuse to form a single mass during the divide mitotically (Figs 3B, D, G, I, 4B, D).

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mid–divisional stage and then divide twice prior to cytokinesis. Micronuclei were observed to

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SSU rDNA sequence and phylogenetic analyses (Fig. 7)

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The SSU rDNA sequence of Lamtostyla salina n. sp. was deposited in the GenBank database with the accession number KX641150. The length and GC content of the SSU rDNA sequence are 1677 bp and 45.80%, respectively. Phylogenetic trees inferred from the SSU rDNA sequences using two different methods (ML and BI) show similar topologies; therefore, we present only the ML tree with bootstraps and posterior probabilities from both algorithms (Fig. 7).

The new species Lamtostyla salina n. sp. nests within a poorly supported clade (node values below 50) containing Bistichella variabilis, Orthoamphisiella breviseries, Uroleptoides magnigranulosus, Parabistichella variabilis, one unidentified species of Gonostomum and nine sequences from environmental samples. This assemblage then forms a polytomy with a weakly supported cluster (node values below 50) composed of Lamtostyla ovalis and Bistichella cystiformans. The present phylogeny is still far from being robust, noting the low support values across the tree, despite the fact that several preliminary phylogenetic analyses were performed using different taxon sampling and outgroup species. Discussion Comparison with closely related species (Fig. 6A–F; Table 2) 7

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In terms of the number of cirri in the ACR (more than four) and two macronuclear nodules, Lamtostyla salina n. sp. should be compared with L. lamottei Buitkamp, 1977, L. australis (Blatterer and Foissner, 1988) Petz and Foissner, 1996, L. islandica Berger and Foissner, 1988, L. ovalis Luo et al., 2016, L. perisincirra (Hemberger, 1985) Berger and Foissner, 1987 and L. procera (Foissner et al., 2002) Berger, 2008. Lamtostyla lamottei, the type species of the genus, is only described after protargol

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preparations. Thus, some important features of the living cells are unavailable. L. salina n. sp. can be easily separated from L. lamottei, however, by: (i) cell outline (lanceolate vs. parallel

body margins with both ends broadly rounded); (ii) cirral arrangement left of anterior portion

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of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) rows vs. three cirri in which the anterior one shifted leftwards and the posterior two aligned) (iii) number of dorsal

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kineties (two or three vs. four); (iv) number of transverse cirri (2–6 vs. two) (Fig. 6A; Berger 2008; Buitkamp 1977).

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Lamtostyla salina n. sp. differs from L. australis mainly in: (i) cell outline (lanceolate vs. elongate elliptical); (ii) cortical granules (present vs. absent); (iii) rear end of ACR (on average 13–39% vs. 38–46% of body length in protargol preparations); (iv) cirral arrangement left of

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anterior portion of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) vs. four cirri more or less aligned); (v) endoral and paroral (intersecting vs. parallel); (vi) number of 1988).

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dorsal kineties (usually two vs. constantly three) (Fig. 6B; Berger 2008; Blatterer and Foissner

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Compared with Lamtostyla islandica, L. salina n. sp. differs in (i) cell size (100–160 × 45–50 μm vs. 60–80 × 20–25 μm) in vivo; (ii) cell outline (lanceolate vs. body margins parallel, both ends broadly rounded); (iii) cirral arrangement left of anterior portion of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) vs. three cirri more or less aligned); (iv) number of right (20–51 vs. 13–16) and left (17–51 vs. 13–15) marginal cirri; (v) cortical granules (present vs. absent); (vi) number of dorsal kineties (usually two vs. constantly three); and (vii) arrangement of endoral and paroral (about same level vs. overlapping only by about half of their length) (Fig. 6C; Berger 2008; Berger and Foissner 1988). Lamtostyla salina n. sp. differs from L. ovalis in: (i) cell outline (lanceolate vs. oviform); (ii) cortical granules (present vs. absent); (iii) symbiotic algae (absent vs. present); (iv) ACR (continuous vs. segmented into two unequal parts); (v) cirral arrangement left of anterior portion of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) vs. three or four cirri more or less aligned); (vi) number of dorsal kineties (usually two vs. constantly three); (vii) pretransverse ventral cirri (absent vs. present); (viii) biotope (terrestrial vs. brackish water) (Fig. 6D; Luo et al. 2016). Lamtostyla salina n. sp. can be separated from L. perisincirra by: (i) body size (100–160 × 45–50 μm vs. 50–80 × 20–30 μm) in vivo; (ii) cell outline (lanceolate vs. both ends broadly 8

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rounded); (iii) cortical granules (present vs. absent); (iv) cirral arrangement left of anterior portion of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) vs. three cirri in which the anterior one shifted leftwards and the posterior two aligned); (v) number of dorsal kineties (usually two vs. constantly three); (vi) number of micronuclei (usually two vs. constantly three) (Fig. 6E; Berger 2008; Berger et al. 1984; Hemberger 1985). Lamtostyla salina n. sp. can be distinguished from L. procera by: (i) body size (100–160 ×

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45–50 μm vs. 130–210× 15–25 µm) in vivo; (ii) body length: width ratio (2.7: 1 vs. 6.1: 1) in protargol preparations; (iii) cell outline (lanceolate vs. vermiform); (iv) cortical granules (present vs. absent); (v) adoral zone length: body length (16–28% vs. 11–18%); (vi) cirral

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arrangement left of anterior portion of ACR (4–20 cirri (on average 10) arranged in 2–4 (on average 3) vs. three or four cirri more or less aligned); (vii) number of cirri forming ACR

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(5–13, on average eight vs. 11–23, on average 15) cirri; (viii) type of ACR (continuous vs. segmented into two unequal parts and overlapping at the level of buccal vertex); (ix) number

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of right marginal cirri (20–51 vs. 48–87); (x) number of left marginal cirri (17–51 vs. 43–80) (Fig. 6F; Berger 2008; Foissner et al. 2002).

There are three closely related species which also need to be compared.

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Lamtostyla salina n. sp. differs from Lamtostylides halophilus in: (i) cell outline (lanceolate vs. elliptical); (ii) body length (100–160 vs. 55–100); (iii) arrangement of cortical granules (scattered vs. loosely arranged); (iv) position of contractile vacuole (33%–40% of

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body length vs. midbody); (v) cirral arrangement left of anterior portion of ACR (4–20 cirri

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(on average 10) arranged in 2–4 (on average 3) rows vs. 1 or 2 cirri arranged in 1 row); (vi) number of adoral membranelles (18–29 vs. 15–19) and cirri in ACR (5–13 vs. 3–5) (Foissner et al. 2002).

For the comparison with Bistichella, see the section of “phylogenetic analyses”. Parabistichella variabilis can be separated from Lamtostyla salina by midventral pairs (present vs. absent) (Jiang et al. 2013). Morphogenetic comparison

Hitherto, the ontogenesis of five Lamtostyla species, namely, L. australis, L. islandica, L. perisincirra, L. decorata and L. longa has been reported and their morphogenetic pattern can be summarized as follows: posterior and anterior portions of amphisiellid median cirral row form from anlage n–1 and anlage n respectively. Except for anlage n, each FVT–anlage originates from parental structures. Anlage n–1 originates within the ACR whereas anlage n forms right of ACR. Based on the early stage (Fig. 3A) and morphogenesis of congeners, it can be concluded that in Fig. 3C, anlage I–V each derive from undulating membranes, buccal cirrus, FVR1, FVR2 and ACR respectively, while anlage VI forms right of ACR (Berger 2008). Phylogenetic analyses 9

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The new sequence obtained in this study, and sequences from unidentified environmental samples, provide the opportunity to further infer the phylogenetic relationships of Lamtostyla. Since the present topology is rather unstable with low support values across the tree, however, the

phylogenetic

position

of

Lamtostyla

remains

undetermined.

Nevertheless,

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well–recognized close relationship between Lamtostyla and Bistichella Berger, 2008 has been shown in a previous study (Luo et al. 2016) and this is recovered, although with low support,

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in all the phylogenetic analyses we performed. Morphologically however, Lamtostyla differs significantly from Bistichella in: (i) number of buccal cirri (one vs. more than one); (ii) transverse cirri (present vs. absent). Besides, Bistichella lacks the typical ACR composed of at

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least two parts (Berger 2008). Given the low support values, the present taxon sampling is not

sufficient to unravel a robust phylogeny for this complex group and, therefore, phylogenetic

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analyses based on additional gene data with more taxa and the sequence of the type species (L. lamottei) are needed to provide further insights into the phylogeny and reclassification of

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amphisiellid ciliates. Acknowledgements

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This work was supported by the Natural Science Foundation of China (Project numbers: 31372148, 31430077; 15–12–1–1–jch), the Fundamental Research Funds for the Central Universities (201564022) and King Saud University Deanship for Scientific Research, Prolific

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Research Group (PRG–1436–01). Many thanks are also given to Dr. Zhao Lv for her kind help

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with molecular sequencing and Prof. Weibo Song, OUC, anonymous reviewers and associate editor for their constructive suggestions. References

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Hu, X., Kusuoka, Y., 2015. Two oxytrichids from the ancient Lake Biwa, Japan, with notes on morphogenesis of Notohymena australis (Ciliophora, Sporadotrichida). Acta Protozool. 54, 107–122.

Hu, X., Fan, Y., Warren, A., 2015. New record of Apoholosticha sinica (Ciliophora, Urostylida) from the UK: morphology, 18S rRNA gene phylogeny and notes on morphogenesis. Int. J. Syst. Evol. Microbiol. 65, 2549–2561. Jiang, J., Huang, J., Li, L., Shao, C., Al-Rasheid, K.A.S., Al-Farraj, S.A., Chen, Z. Morphology, ontogeny, and molecular phylogeny of two novel bakuellid–like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera. J. Eukaryot. Microbiol. 40: 78–92. Jo, E., Jung, J.H., Min, G.S., 2015. Morphology and molecular phylogeny of two new brackish water ciliates of Bakuella (Ciliophora: Urostylida: Bakuellidae) from South Korea. J. 11

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Eukaryot. Microbiol. 44, 799–809. Jung, J.H., Park, K.M., Min, G.S., 2015a. Morphology and molecular phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora, Sporadotrichida), with a brief discussion of the Cyrtohymena undulating membranes pattern. J. Eukaryot. Microbiol. 62, 280–297. Jung, J.H., Park, K.M., Min, G.S., Berger, H., Kim, S., 2015b. Morphology and molecular

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phylogeny of an Antarctic population of Paraholosticha muscicola (Kahl, 1932) Wenzel, 1953 (Ciliophora, Hypotricha). Polar Sci. 9, 374–381.

Kumar, S., Foissner, W., 2015. Biogeographic specializations of two large hypotrich ciliates:

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Australocirrus shii and A. australis and proposed synonymy of Australocirrus and Cyrtohymenides. Eur. J. Protistol. 51, 210–228.

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Kumar, S., Foissner, W., 2016. High cryptic soil ciliate (Ciliophora, Hypotrichida) diversity in Australia. Eur. J. Protistol. 53: 61–95.

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Li, J., Chen, X., Xu, K., 2016. Morphology and small subunit rDNA phylogeny of two new marine urostylid ciliates, Caudiholosticha marina sp. nov. and Nothoholosticha flava sp. nov. (Ciliophora, Hypotrichia). J. Eukaryot. Microbiol. 63, 460–470

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Luo, X., Gao, F., Al-Rasheid, K.A., Warren, A., Hu, X., Song, W., 2015. Redefinition of the hypotrichous ciliate Uncinata, with descriptions of the morphology and phylogeny of three urostylids (Protista, Ciliophora). Syst. Biodivers. 13, 455–471.

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Luo, X., Yi, Z., Gao, F., Pan, Y., Al-Farraj, S.A., Warren, A. 2016. Taxonomy and molecular

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phylogeny of two new brackish hypotrichous ciliates, with the establishment of a new genus (Protozoa, Ciliophora). Zool. J. Linn. Soc. doi: 10.1111/zoj.12451.

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Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D., Pupko, T., 2010. GUIDANCE: a web server for assessing alignment confidence scores. Nucleic. Acids. Res. 38, W23–W28. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Singh, J., Kamra, K., 2015. Molecular phylogeny of Urosomoida agilis, and new

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combinations: Hemiurosomoida longa gen. nov., comb. nov., and Heterourosomoida lanceolata gen. nov., comb. nov. (Ciliophora, Hypotricha). Eur. J. Protistol. 51, 55–65.

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Science Press, Beijing.

Song, W., Wilbert, N., Li, L., Zhang, Q., 2011. Re-evaluation on the diversity of the

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polyphyletic genus Metaurostylopsis (Ciliophora, Hypotricha): ontogenetic, morphologic, and molecular data suggest the establishment of a new genus Apourostylopsis n. g. J.

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analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.

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Fig. 1A–D Sampling sites. (A) Portion of Google map, showing the locations of Longfeng Wetland (46°35'30''N, 125°13'08''E), Daqing, northern China. (B, C) Surroundings of the sampling site, arrows indicate where the soil samples were collected. (D) Photograph showing the non–flooded Petri dish. Fig. 2A–K Morphology of Lamtostyla salina n. sp. from life (A–C) and after protargol

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staining (D–K). (A) Ventral view of a representative individual, arrow depicts the contractile vacuole. (B) Ventral view showing a different body shape. (C) Cortical granules. (D, E) Ventral and dorsal view of infraciliature of holotype specimen, arrow marks buccal cirrus,

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arrowheads indicate marginal cirri aggregated closely to each other to form several relatively larger patches of basal bodies. (F–H) Frontoventral ciliatures of different individuals. (I)

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Individual with two right marginal rows. (J, K) Individuals with two (J) and three (K) left marginal rows. ACR, amphisiellid median cirral row; AZM, adoral zone of membranelles; E,

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endoral membrane; FC, frontal cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; P, paroral membrane; RMR, right marginal row; TC, transverse cirri; 1−4 (D, F−H), frontoventral rows 1−4; 1, 2 (E), dorsal kineties 1, 2. Scale bars = 65 μm (A); 60 μm

M

(B); 30 μm (D, E); 6 μm (C).

Fig. 3A–I Early and middle stages of morphogenesis in Lamtostyla salina n. sp. after protargol

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staining. (A, B) Ventral view and nuclear apparatus of an early divider, arrow indicates the

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differentiation of frontoventral transverse cirri anlagen. (C−E) Ventral view of a slightly later divider (C), note the nuclear apparatus (D) and the magnified view of the oral primordium and frontoventral transverse cirri anlagen (E). (F, G) Ventral and dorsal view of a middle divider with seven frontoventral transverse cirri anlagen. (H, I) Ventral and dorsal view of a later divider with seven frontoventral transverse cirri anlagen. LMA, left marginal row anlagen; Ma, macronuclear nodules; Mi, micronuclei; OP, oral primordium; RMA, right marginal row anlagen; 1, 2, dorsal kineties anlagen 1, 2; I−VII, frontoventral cirri anlagen I−VII. Scale bars = 30 μm.

Fig. 4A–D Late stages of morphogenesis in Lamtostyla salina n. sp. after protargol staining. (A, B) Ventral and dorsal view of a late divider with seven frontoventral transverse cirri anlagen. (C, D) Ventral and dorsal view of a late divider with eight frontoventral transverse cirri anlagen. Unfortunately, the frontoventral transverse cirri in the opisthe are not seen (arrow). DKA, dorsal kineties anlagen; LMA, left marginal row anlagen; Ma, macronuclear nodules; Mi, micronuclei; RMA, right marginal row anlagen; 1, 2, dorsal kineties anlagen 1, 2; I−VIII, frontoventral cirri anlagen I−VIII. Scale bars = 30 μm. 14

Page 14 of 25

Fig. 5A–N Photomicrographs of Lamtostyla salina n. sp. from life (A–E) and after protargol staining (F–N). (A, B) Ventral views of representative individuals, note the contractile vacuole (arrow). (C, D) Ventral views of a slightly squeezed specimen, arrow in (C) points to the contractile vacuole; arrows in (D) mark grooves along marginal rows. (E) Ventral view to show the scattered cortical granules (arrows). (F) Ventral view of holotype specimen. (G, H) Frontoventral views of two different specimens. (I) Ventral views to show several left

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marginal cirri aggregated closely to each other to form several relatively larger patches of basal bodies (arrows). (J–L) Ventral views to show multiple marginal rows (arrows). (M)

Transverse cirri (arrow) and a patch (not pretransverse cirrus, arrowhead). (N) Dorsal view to

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show dorsal kineties (arrows). Scale bars = 65 μm (A); 60 μm (B–D); 30 μm (F, G, H, J).

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Fig. 6A−H Comparative illustrations of several Lamtostyla species after protargol staining. (A) L. lamottei (from Buitkamp 1977). (B) L. australis (from Blatterer and Foissner 1988). (C) L.

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islandica (from Berger and Foissner 1988). (D) L. ovalis (from Luo et al. 2016). (E) L. perisincirra (from Berger et al. 1984). (F) L. procera (from Foissner et al. 2002). Scale bars =

M

25 µm (A, B); 20 µm (C); 15 µm (E); 30 µm (D); 80 µm (F).

Fig. 7 Maximum likelihood (ML) tree inferred from the SSU rDNA sequences showing the systematic position of Lamtostyla salina n. sp. (in bold). Bootstrap values above 50 for the

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Maximum likelihood and/or Bayesian inference are given at the individual nodes. All branches

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are drawn to the scale bar, which corresponds to one substitution per 100 nucleotide positions.

15

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Table 1. Morphometric characterisation of Lamtostyla salina n. sp. Charactera

Min Max Mean M SD

CV

n

Body length

90 160 134.8 140 19.3 14.29 24

Body width

28

Body, length: width ratio

67

50.9 51.5 9.2 18.09 24

1.87 3.93 2.7 2.65 0.5 17.11 24 20

Adoral zone of membranelles, length

32

27.2 27.5 2.9 10.78 24

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Adoral zone of membranelles, length: body length ratio 0.16 0.28 0.2 0.20 0.0 13.97 24 7

15

11.9

Endoral, length

10

20

16.5 17 2.3 14.07 24

Anterior body end to begin of paroral, distance

6

12

8.8

8

1.7 19.22 23

Anterior body end to begin of endoral, distance

4

15

8.4

8

2.3 27.79 24

Anterior body end to begin of buccal cirrus, distance

8

18

12.0

11 2.6 22.05 23

Frontoventral rows, number

2

4

3.0

3

19

10.8 10 3.4 31.26 23

6

31

17.2 16 6.1 35.79 23

8

55

31.5 34 12.3 39.16 21

48

57

52.5 52.5 6.4 12.12 2

17

42

32.4 35 7.7 23.71 21

row 1, distance

Anterior body end to posterior end of frontoventral

M

row 2, distance

Anterior body end to posterior end of frontoventral row 3, distance

d

Anterior body end to posterior end of frontoventral row 4, distance

us

6

an

Anterior body end to posterior end of frontoventral

Anterior body end to posterior end of ACR Anterior body end to posterior end of frontoventral row 1, distance: body length ratio

Anterior body end to posterior end of frontoventral row 2, distance: body length ratio

Anterior body end to posterior end of frontoventral row 3, distance: body length ratio

Anterior body end to posterior end of frontoventral row 4, distance: body length ratio

Anterior body end to posterior end of amphisiellid median row, distance: body length ratio

12 2.0 16.96 23

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Paroral, length

Ac ce pt e

1

0.4 14.21 23

0.04 0.14 0.08 0.08 0.0 34.11 23 0.05 0.24 0.13 0.11 0.0 36.89 23 0.07 0.53 0.24 0.23 0.1 42.23 21 0.36 0.36 0.36 0.36 0.0

0.14

2

0.13 0.39 0.25 0.26 0.1 29.15 21

Adoral membranes, number

18

29

20.2 20 2.0 10.00 24

Buccal cirri, number

1

1

1.0

1

0.0

0.00 23

Frontal cirri, number

2

3

3.0

3

0.2

6.90 24

Transverse cirri, number

2

6

3.7

4

1.0 25.74 21

1

Page 16 of 25

2

4

3.0

3

0.4 14.21 23

Frontoventral row 1, cirri number

1

4

2.1

2

0.9 43.13 23

Frontoventral row 2, cirri number

2

6

3.1

3

1.0 32.38 23

Frontoventral row 3, cirri number

2

10

5.3

5

2.3 44.43 21

Frontoventral row 4, cirri number

7

9

8.0

8

1.4 17.68 2

Amphisiellid median row, cirri number

5

13

8.0

8

2.0 24.99 23

4

20

10.7 10 4.4 40.51 23

Cirri left of anterior portion of amphisiellid median cirral row, total number

1

2

1.1

Right marginal row 1, number

b

20

51

38.3 38 6.8 17.79 24

Right marginal row 2, number

b

31

39

33.7 31 4.6 13.72 3

1

3

cr

Right marginal rows, number

ip t

Frontoventral row, number

1.2

0.3 30.03 24

0.5 42.12 24

17

51

35.5 34.5 8.0 22.67 24

Left marginal row 2, number

b

9

31

16.8 13.5 9.9 59.38 4

Left marginal row 3, number

b

an

Left marginal row 1, number b

1

us

Left marginal rows, number

1

20

20

20.0 20

2

3

2.0

2

0.2 10.20 23

2

2

2.0

2

0.0

1

3

1.6

15

44

25.7 26 6.2 24.28 23

Anterior macronuclear nodule, width

8

21

13.9 12 4.0 28.82 23

Micronuclei, length

1

3

2.4

2

0.6 25.77 23

1

3

2.2

2

0.6 25.16 23

Dorsal kineties, number Micronuclei, number

Ac ce pt e

d

Anterior macronuclear nodule, length

M

Macronuclear nodules, number

Micronuclei, width

-

1

0.00 23

1.5 0.7 43.31 18

1

a

2

Measurements in μm. “-” data not applicable. Abbreviations: ACR, Amphisiellid median row;

3

CV, coefficient of variation in %; M, median; Max, maximum; Mean, arithmetic mean; Min,

4

minimum; n, number of cells measured; SD, standard deviation.

5 6 7

All data are based on protargol-stained specimens.

b

-

numbered from inner to outer.

2

Page 17 of 25

1

Table 2. Morphological and morphometric comparison of Lamtostyla salina n. sp. with some

2

congeners. Lamtostyla

Lamtostyla

Lamtostyla

Lamtostyla

Lamtostyla

Lamtostyla

Lamtostyla

salina n. sp.

lamottei

australis

procera

islandica

perisincirra

ovalis

90–130 ×

130–210 ×

60–80 ×

50–80 ×

70–110 ×

30–40

15–25

20–25

20–30

40– 60

100–160 ×

vivo

45–50

granules BL/BW, ratioa

present

-

absent

absent

absent

absent

absent

2.7: 1

-

3.5:1

6.1:1

3.4:1

2.8:1

1.7:1

ca. 23%

24%–30%

11%–18%

ca. 27%

ca. 35%

13–39%

ca. 26%

38–46%

ca. 30%

5–13

ca. 10

8–17

11–23

3

4

Adoral zone

16–28%, on

length/BL,

average

a

ratio

20%

ACR length/BL, a

Cirri of ACR,

number

average 10

2–6

2

3 or 4

2–4

Ac ce pt e

TC, number

4–20, on

d

ACR, total

M

number Cirri left of

DK, number

2

4

3

0–6 2

Blatterer

Data source

ca. 27%

present

Buitkamp

and

Foissner et

work

(1977)

Foissner

al. (2002)

(1988)

ca. 30%

ca. 30%

ca. 30%

5 or 6

6–8

8–16

3

3

3–7

2–5

4–6

3

3

Berger et al.

Luo et al.

(1984)

(2016)

an

ratio

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Cortical

ca. 100

cr

Body size in

us

Charactera

3 or 4 3 Berger and Foissner (1988)

3

a

4

Abbreviations: ACR, amphisiellid median cirral row; BL, body length; BW, body width; DK,

5

dorsal kineties; TC, transverse cirri.

6 7

Data are based on protargol-stained specimens. Measurements in µm. “-” data not available.

8 9

1

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