Description of a New Freshwater Bloom-Forming Dinoflagellate with a Diatom Endosymbiont, Peridiniopsis Minima Sp

Algological Studies 145/146 (2014), p. 119–133 Article Published online May 2014

Description of a new freshwater bloom-forming dinoflagellate with a diatom endosymbiont, Peridiniopsis minima sp. nov. (Peridiniales, Dinophyceae) from China

Qi Zhang, Guoxiang Liu* & Zhengyu Hu

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydro- biology, Chinese Academy of Sciences, Wuhan 430072, P.R. China

With 4 figures and 2 tables

Abstract: A new freshwater dinoflagellate, Peridiniopsis minima Zhang, Liu et Hu sp. nov. (Peridiniales, Dinophyceae), from the Jiulongjiang River, Fujian Province, China, is described. The dinoflagellate can form serious brown freshwater blooms in the river. The cell is very small and the plate formula is: Po, x, 3', 1a, 6'', 5c, 4s (?), 5''', 2''''. The dinoflagellate is characterized by the presence of a eukaryotic endosymbiotic alga. The dinoflagellate cell possesses two different types of nuclei: a dinokaryon and a eukaryotic nucleus from the diatom endosymbiont. Our molecular analyses based on SSU, LSU and ITS rDNA sequences revealed that these freshwater diatom-harbouring Peridiniopsis species forms a strongly supported subclade, very dis- tant from Peridiniopsis borgei, which is the type species of Peridiniopsis, and distinctively separated from marine diatom-harbouring dinoflagellates. The phylogenetic analyses based on endosymbiont SSU rDNA sequences indicate that the diatom endosymbionts of Peridiniopsis species are closely related to centric diatoms, such as Discostella and Cyclotella.

Keywords: Peridiniopsis, Peridiniopsis minima, Dinoflagellate, Phylogeny, Diatom endosymbiont

Introduction

The cosmopolitan genus Peridiniop sis was established by Lemmermann (1904, p. 134), wi th P. borgei Lemmermann as the type species. Peridiniopsis is the second largest genus of thecate, freshwater dinoflagellate (ca. 20 species) (Popovský & Pfie- ster 1990). According to the revision by Bourrelly (1968 ), this genus differs from Peridinium in the number of the anterior intercalary plates (0a or 1a in Peridiniopsis,

*Corresponding author: [email protected]

© E. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany www.schweizerbart.de DOI: 10.1127/1864-1318/2014/0159 1864-1318/0159 $ 3.75 120 Qi Zhang, Guoxiang Liu & Zhengyu Hu whereas 2a or 3a in Peridinium). Species of Peridiniopsis have the plate tabulation 3-5', 0-1a, 6-8'', 5''', 2'''' (Bourrelly 1968). However, this taxonomic criterion seems to be an oversimplification. Based on morphological and molecular evidence, some Peridiniopsis species have been transferred to new genera in recent years (e.g. Bol- tovskoy 1999, Calado et al. 2009). Several dinoflagellates are known to possess two different types of nuclei: a dinokaryon and an ordinary eukaryotic nucleus from an endosymbiont (reviewed by Schnepf & Elbrächter 1999). Endosymbionts have been observed in Durinskia baltica (Levander) Carty et Cox [= Peridinium balticum (Levander) Lemmermann] by Tomas & Cox (1973), in Durinskia capensis Pienaar, Sakai et Horiguchi by Pienaar et al. (2007), in Kryptoperidinium foliaceum (Stein) Lindeman [= Peridinium foliaceum (Stein) Biecheler] by Dodge (1983), Jeffrey & Vesk (1976) and Kempton et al. (2002), in Peridinium quinquecorne Abé by Horiguchi & Pienaar (1991), in D inothrix paradoxa Pascher by Horiguchi & Chihara (1993), for Gymnodinium quadrilobatum Horiguchi et Pienaar by Horiguchi & Pienaar (1994) and in Galeidinium rugatum Tamura et Horiguchi by Tamura et al. (2005). Some Peridiniopsis species also possess an endosymbiotic diatom or diatom-like alga. Takano et al. (2008) revealed that Peridiniopsis kevei Grigorszky et Vasas and P. penardii (Lemmermann) Bourrelly are two diatom-ha rboring dinoflagellates. Zhang et al. (2011) r ecently described a new variety of a freshwater bloom-forming dinoflagellate, Peridiniopsis penardii (Lem- mermann) Bourrelly var. robusta Q. Zhang, G.X. Liu et Z.Y. Hu, from China with a similar type of endosymbiont. In the summer of 2011, an extensive algal bloom was observed in Jonglongjiang River, Fujian Province, China. We investigated the causative organism and found that the dense bloom caused by a small unidentified peridinioid dinoflagellate. After examining its morphological characteristics and thecal plate arrangement, we considered it to represent a new species. Based on the morphological and molecular evidence, we demonstrated the phylogenetic affinities of this new dinoflagellate with a similar type of endosymbiont.

Material and methods

Collection and preservation Plankton samples were collected from Jiulongjiang River, Fujian Province, China (117°15 '14''E, 25° 12'35'' N) by Sen Li n, on 16 Augus t 201 1 at the time of bloom formation. Samples were preserved in 10 % formalin and 90 % ethanol. Ethanol-fixed samples were frozen at –20°C until analysis.

Morphospecies identification For observation of the thecal plates, formalin-fixed cells were sterilized with 0.1 % Fluorescent Brightener 28 (Sigma, UK) (Fritz & Triemer 1985) and observed using Description of a new dinoflagellate with a diatom endosymbiont 121 epifluorescence microscopy (EFM) (Leica DM5000B, Germany). Ethidium bromide (EB)-stained cells were prepared by a common method for observation of nuclei (Liu et al. 2008). We also observed cells for differential interference contrast (DIC) and phase contrast (PC) microscopy using a Leica DM5000B microscope. Micrographs were taken with a Leica DFC 320 digital camera.

Cells isolation and DNA extraction About 20 cells were isolated from ethanol-fixed samples under an inverted microscope for each PCR reaction (Olympus CKX41, Japan). Individual dinoflagellate cells were then placed in a 200 μL PCR tube. The DNA extraction wa s performed by treating the cells with Proteinase K (pK) which is commonly used in molecular biology to digest protein and remove contaminations from preparations of nucleic acids. Proteinase K (1 μL 200 μg/ml) was then added, and the tubes were maintained at 55°C for 50 minutes. Samples were then incubated at 95°C for a further 10 minutes to inac- tivate the proteinase K and facilitate DNA denaturation. The tubes were then cooled at 4°C in preparation for PCR amplification (Ki et al. 2005). Finally, the elution volume of DNA used in the PCR was about 5 μL.

Single-cell PCR and sequencing The sequences of nuclear-encoded rDNA (SSU, LSU and ITS[ITS1/5.8S/IT S2]) and plastid-encoded 23S rDNA from the dinoflagellate and SSU rDNA from the endosymbiont nucleus were determined using the sing-cell PCR method. The details of this method are described by Zhang et al. (2011). Polymerase chain reaction condi- tions for partial 23S rDNA amplifications using primers have been described previously (Zhang et al. 2000). The methods of PCR amplifications of nuclear-encoded rDNA and endosymbiontic SSU rDNA were the same as described by Zhang et al. (2011) . All amplicons were sequenced from both s ides using PCR primers. The PCR products were analyzed on an ABI 3700 sequencer (Applied Biosystems, USA). The sequences were deposited in GenBank under the Accession numbers: JQ639752, JQ639767, JQ639770, JX027617, and JX141779.

Phylogenetic analyses The SSU, LSU, ITS and endosymbiont SSU sequences were downloaded from Gen- Bank. Perkins us marinus was used as an outgroup in the SSU, LSU and ITS phylogenies. Bolidomonas mediterranea was used as an outgroup in the endosymbiont SSU phylogeny. The SSU, LSU, ITS and endosymbiont SSU consisted of 49 sequences with 1599 characters, 51 sequences with 529 characters, and 38 sequences with 506 characters, 40 sequences with 1304 characters, respectively. After the elimination of identical and apparently erroneous sequences, we created four sets of alignments using Clustal X (v1.8) (Thompson et al. 1997) and Bioedit (v7.0.9.1) (Hall 1999). We 122 Qi Zhang, Guoxiang Liu & Zhengyu Hu analyzed conversion/transversion and genetic distances using MEGA (v4.0.0.4103) (Tamura et al. 2007). The phylogenies were estimated using Maximum Likelihood(ML) and Bayesian Inference (BI) as implemented in Paup 4.0* (v4.0b10) (Swofford 2002) and MrBayes (v3.1.2) (Huelsenbeck & Ronquist 2001). The program Modeltest (v3.07) (Posada & Crandall 1998) was used to explore the model of sequence evolution that best fits the data set by the hierarchical likelihood ratio test (hLRT) (Huelsenbeck & Cran- dall 1997). In ML analyses, a heuristic search option with r andom addition of sequences (100 replicates) and the nearest neighbor interchange branch-swapping algo- rithm (NNI) were used for tree searching. All Bayesian Markov Chain Monte Carlo (MCMC) analyses were run with seven Markov chains (six heated chains, one cold) for 1,000,000 generations. Trees were sampled every 100 generations. We obtained posterior probability (PP) values for the branching patterns in BI trees as well as bootstrap (BP) values in ML trees. The evolutionary model used in ML and BI analyses for the SSU, ITS, endosymbiont SSU phylogenies was TrN+I+G. The GTR+G model was selected for LSU phylogeny. Bootstrap values and posterior probabilities for some clades obtained across the phylogenies were presented on the nodes.

Results

Taxonomic descriptions Peridiniopsis minima Zhang, Liu et Hu sp. nov. Figs. 1–3 Diagnosis: Unicellular, freshwater thecate dinoflagellate. Cells measure 8–15 μm in length , 6–12 μm in width. The plate tabulat ion is Po, x, 3', 1a, 6'', 5c, 4s (?), 5''', 2''''. Cells are compressed dorsoventrally, oval or spherical in ventral view and subcircular in apical view. Cells contain numerous discoid brownish chloroplasts and an eyespot within the sulcus. The m edian cingulum is well excavated and descending. The sulcus is excavated and extends to the antapex. Dinoflagellate with a diatom endosymbiont, possessing both a dinokaryotic and a eukaryotic nucleus. Habitat: Freshwater rivers. Type Locality: Jiulongjiang River, Fujian Provin ce, China. Holotype: FJ-2011-01 (HBI) , from Jiulongjiang River, collected by Shen Lin on Au- gust 28, 2011. It is kept in 10 % formalin at the Freshwater Algal Herbarium (HBI), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China. Etymology: The variety epithet minima describe the size of the cell. Distribution: Known from Jiulongjiang River, Fujian Province, China.

The cell is almost pentagonal to rhombic in ventral view and dorsoventrally flattened. Dimensions: 8–15 μm in length and 6–12 μm in width (Fig. 1A-C). The thecal plates are thin and smooth. Epitheca and hypotheca are almost equal in size. The hypotheca is slightly more rounded than the epitheca. The cingulum is well excavated and descending, displaced about half of its own width, measuring 1.5–2 μm. The sulcus is Description of a new dinoflagellate with a diatom endosymbiont 123

Fig. 1. A–F. P. minima. A, B. Single cell showing the cell shape. C. Single cell showing nucleus. D. Single cell showing the eyespot. E. Numerous discoid chloroplasts. F. Stained cell showing the dinoflagellate nucleus (DI) and endosymbiotic nucleus (EN). Scale bars = 1 μm. Symbols used: Po, apical pore; x, canal plate; X', apical plates; X'', precingular plates; X''', postcingular plates; X'''', antapical plates; Xc, cingular plates; Xa, intercalary plates; Pc, pedun- cle cover plate; Sa, anterior sulcal plate; Sd, right sulcal plate; Ss, left sulcal plate; Sm, median sulcal plate; Sp, posterior sulcal plate; X is serial number. N, nucleus; E, eyespot; DI, dinokaryotic nucleus; EN, endosymbiotic nucleus; DIC, differential interference contrast; EFM, epifluorescence microscopy.

excavated and extends into the antapex. Numerous discoid chloroplasts are brownish in color and are situated in the periphery of the cell. An elongated red eyespot is located ventrally along the proximal part of the sulcus, immediately below the ventral ridge. The cell contains two different types of nuclei. One is ordinary dinokaryotic and located in the middle of the cell, while the other is smaller and situated on the upper side of the dinokaryon (Fig. 1D–F). The apical pore plate (Po) is small and circular in shape. The plate X is elongate. The arrangement of the epitheca is basically symmetrical. Apical plate 1' is rhombic, rather large, and wide. The lower edge of this plate is short. Plates 2' and 3' are irregularly six-sided. Precingular plates 1'', 2'', 5'', and 6'' are four-sided whereas Plates 3'' and 4'' are five-sided. One of the most distinctive features is the shape and location of the small 1a plate. This anterior intercalary plate is rhombic and appears to be inserted between plates 2', 3', 3'', and 4'' (Fig. 2A–E and Fig. 3B–C). 124 Qi Zhang, Guoxiang Liu & Zhengyu Hu

Fig. 2. A–I. The plate pattern of P. minima. A. The epithecal plate pattern seen in a ventral view. B. Dorsal view of the plate pattern. C, D. Epithecal plate pattern, right view. E. Epithecal plate pattern, dorsal view. F. The hypothecal plate pattern seen in a dorsal view. G. Antapical view of the plate pattern. H. Cingular plate pattern. I. Plate pattern of the sulcal series. Scale bars = 1 μm.

The sulcus consists of four or five plates. No dissection of the theca was attempted to specify the number of sulcal plates. There is an anterior sulcal plate (= Sa), a posterior sulcal plate (= Sp), a right sulcal plate (= Sd), and a left sulcal plate (= Ss). It is not certain whether a median sulcal plate (= Sm) is present. The sulcus does not extend to the epitheca. The Sp plate widens along the hypotheca where it reaches the antapex (Fig. 2I and Fig. 3A). The cingulum is composed of five plates (1c–5c). Plate 1c is rather short and it contacts plate 1' on the epitheca and plate 1''' on the hypotheca. Plate 4c is relatively longer than the other cingular plates (Fig. 2H and Fig. 3C–D). Description of a new dinoflagellate with a diatom endosymbiont 125

Fig. 3. A–D. Diagrammatic plate pattern of P. minima. A. Ventral view. B. Dorsal view. C. Plate pattern of epitheca. D. Plate pattern of hypotheca.

The hypotheca is composed of five postcingular plates (5''') and two antapical plates with a similar size (2''''). Plates 1''', 2''', 4''', and 5''' are irregularly four-sided whereas plate 3''' is irregularly five-sided (Fig. 2F–G and Fig. 3D).

Sequence divergence The SSU region p-values between species in the genus Peridiniopsis (p = 0.003–0.012) were lower than any ITS region p-values observed within the genus (p = 0.044–0.130). The SSU sequence divergence values between Peridiniopsis borgei and diatom-harboring Peridiniopsis (p = 0.046–0.052) were much higher than the values within diatom- harboring Peridiniopsis. The sequence divergence values between diatom-harboring Peridiniopsis and other diatom-harboring dinoflagellates (p = 0.043–0.086 in the SSU sequences and p = 0.307–0.362 in the ITS sequences) were also consistently higher than the values within diatom-harboring Peridiniopsis (Table 1). 126 Qi Zhang, Guoxiang Liu & Zhengyu Hu G. rugatum quinquecorne P. nieiP. kevei P. borgei P. D. baltica K. foliaceum Pe.

var. robusta var. -distances) based on ITS sequences (upper right) and SSU rDNA sequences (lower left) in this study. p -distances) based on ITS sequences (upper right) and SSU rDNA 00.004 0– 0.130 0.1260.004 – 0.0440.012 0.0030.046 0.126 0.012 0.088 – 0 – 0.0470.043 – –0.077 – 0.044 – – 0.0790.060 0.080 0 – 0.318 – 0.0600.066 0.010 – 0.308 0.048 0 – 0.345 0.065 – – 0.052 0.358 0.044 0 – – – 0.079 0.050 – 0.307 0.086 0.053 – 0.060 – – 0.077 0.307 0 0.066 0.362 0.081 0.064 – 0.067 0.345 0.072 – – 0.072 – – 0.069 0 0.406 – 0.072 0.088 – – – – 0.063 – 0 – 0.084 – – – – – 0 P. minimaP. penardii P. penardii P. P. P. minima Peridiniopsis penardii Peridiniopsis var. penardii robusta Peridiniopsis niei Peridiniopsis kevei Peridiniopsis borgei Durinskia baltica Kryptoperidinium foliaceum Peridinium quinquecorne Galeidinium rugatum Table 1. Distance values (pairwise uncorrected Table Description of a new dinoflagellate with a diatom endosymbiont 127

Phylogenetic analyses SSU, LSU and ITS rDNA (analysis of hosts): Most phylogenetic hypotheses based on the SSU, LSU, and ITS obtained from host nuclei had weakly defined backbone topologies but several well-supported internal clades (Fig. 4A–C). Only the ML tree is shown with the bootstrap and posterior probability values from the different analytical methods. In general, the PP values were higher than the BP values because the BP is normally more conservative than the PP. In the SSU rDNA analysis, the phylogenetic results showed that the species currently classified in Peridiniopsis were not a monophyletic group. The type species P. borgei was not affiliated with any other Peridiniopsis species. All of the dinoflagellates with a diatom endosymbiont formed a clade, although the support values were not high (BP/PP = –/0.66 in the SSU phylogeny and –/0.83 in the LSU phylogeny). In all analyses, the sequences from diatom-harboring Peridiniopsis species formed a relatively highly supported subclade that was distinct from other diatom-harboring dinoflagellates (e.g., Durinskia baltica, Kryptoperidinium foliaceum, Galeidinium rugatum, and Peridinium quinquecorne) (BP/PP = 96/1.00 in the SSU phylogeny, 100/1.00 in the LSU phylogeny, and 80/1.00 in ITS phylogeny). SSU analysis of endosymbionts and other diatom taxa: The two analytical methods yielded more or less the same topology, so only the ML tree is shown (Fig. 4D). Nuclear-encoded SSU rDNA from the endosymbiont nuclei showed that P. minima, P. kevei, P. penardii, and P. penardii var. robusta were closely related to the centric diatom Thalassiosirales species. The endosymbiont of P. minima was closely related to some Cyclotella species. The other freshwater diatom-harboring Endodiadinium species were included in a centric diatom Discostella subclade. However, the endosymbiont of marine Peridinium quinquecorne was affiliated to a member of the bipo- lar centric genus Chaetoceros. Furthermore, the endosymbionts of D. baltica and K. foliaceum were included with a pennate diatom in the Nitzschia clade.

Discussion

Identity of P. minima P. salina Trigueros, together with P. lindemannii (Lefèvre) Bourrelly, were the small- est recorded species of the genus Peridini opsis (Trigueros 2000). The cells of P. minima were s imilar in size to those of P. salina and P. lindemannii. P. lindemannii was pentangular furnis hed with several hypothecal spines (Bourrel ly 1968) where as P. minima and P. salina were oval furnished without any spi nes. In addition, P. lindemannii differed by the pr esence of seven precingular plates. P. minima was very similar to P. salina with respect to cell shape, but we found it to be different from this species with respect to habitat and plate tabulation. P. salina usually occurred in brackish waters, and in contrast, P. minima was found in freshwater. The plate pattern of P. minima was basically symmetrical whereas the plate pattern of P. salina was 128 Qi Zhang, Guoxiang Liu & Zhengyu Hu

Fig. 4. Maximum-likelihood phylogenies constructed from dinoflagellate SSU rDNA sequences (A), LSU rDNA sequences (B), ITS rDNA sequences (C), and endosymbiont SSU rDNA sequences (D). The numbers on the nodes indicate the posterior probabilities/bootstrap support values based on Maximum Likelihood and Bayesian Inference. Only values > 0.50 are shown. The Endodiadinium species are shown in a gray square. Description of a new dinoflagellate with a diatom endosymbiont 129 Po, x, 3', 1a, 6'', 5c, 4s(?), 5''', 2'''' yes freshwater rivers yes P. minima P. d 2 rare 3 or 4 no Po, x, 4', 0a, 6'', 5c, 5s, 5''', 2'''' yes freshwater rivers and lakes yes P. niei P. c Po, x, 3', 1a, 6'', 5c, ?s, 5''', 2'''' yes freshwater rivers and lakes rhombicyes pentagonal oval P. kevei P. species. b Peridiniopsis Po, x, 4', 0a, 6'', 5c, 5s, 5''', 2'''' yes freshwater rivers and lakes rhombic yes var. var. penardii P. robusta a Po, x, 4', 0a, 6'', 5c, 44343 00101 55555 2 rare 0 or 4 numerous robust5s, 5''', 2'''' 1, robust yellowish brown yellow brown yellow brown yellow-green brownish freshwater rivers, lakes and ponds yes P. penardii P. Diatom endosymbiont yes Anterior intercalary plate Cingular plate Plate tabulation Apical plate Hypothecal spine Colour Habitat Cell size (μm)Cell shapeEyespot 25–43 × 20–38 oval 30–43 × 27–38 26–42 × 23–45 26–48 × 15–35 pentagonal to 8–15 × 6–12 Zhang et al. (2011) Liu et al. (2008) Lemmermann (1910) and Popovský & Pfiester (1990) Grigorszky et al. (2001) Table 2. Morphology comparison of diatom-harbouring Table a b c d 130 Qi Zhang, Guoxiang Liu & Zhengyu Hu asymmetrical. P. minima differe d f urther from P. salina by having 3 rather than 4 apical plates and 5 rather than 6 cingular plates. Although their 1a plates were very similar with respect to plate shape and size, the 1a plate of P. minima was bordered by 2', 3', 3''and 4'', whereas the one of P. salina was bordered by 2', 3', 2''and 3'' (Trigueros 2000 ). The plate pattern of P. kulczynskii (Wołoszyńska) Bourrelly was identical to P. minima, but it was much larger than P. minima, measuring 28–48 μm in length and 23–46 μm in width (Wołoszyńska 1916, Thompson 1951). In conclusion, P . minima was considered a new species, which differed from the aforementioned species by cell shape, size, habitat and plate tabul ation.

Comparison between diatom-harbouring Peridiniopsis species Morphological comparisons with diatom-harboring Peridiniopsis species are shown in Table 2. The plate pattern of P. niei was identical with the one of P. penardii and P. penardii var. robusta. The cells of P. penardii were usually furnished with two (sometimes zero or four) small antapic al spines and a short/ absent spine, wh ereas the cells of P. penardii var. robusta were u sually furnished with numerous robust antapical spines and a conspicuous apical spine (Zhang et al. 2011). P. kevei and P. minima had similar plate patterns, but they had different cell shapes and sizes. The rhombic P. kevei was clearly larger than the oval P. minima. As previously reported by Zhang et al. (2011), P. niei did not possess an endosymbiotic diatom. However, we found that P. niei also possessed endosymbiont SSU rDNA in this study.

Phylogeny of diatom-harbouring Peridiniopsis species Regardless of their diversity in morphology and habitat, the presented results indicated that the dinoflagellates with an endosymbiont of diatom origin are monophyletic. The same results had been obtained by Horiguchi & Takano (2006), Pienaar et al. (2007) and Takano et al. (2008). Furthermore, the freshwater diatom-harboring Pe- ridiniopsis species clustered into a highly supported subclade that was distinct from marine diatom-harboring dinoflagellates. D. baltica has been reported from marine and freshwater habitats (Tomas & Cox 1973, Carty & Cox1986).

Origin of endosymbionts The phylogenetic trees based on endosymbiont SSU rDNA showed that the diatom- harboring dinoflagellates are not monophyletic. The endosymbionts of D. baltica and K. foliaceum were closely related to Nizschia-like diatoms, while P. quinquecorne clustered with Chaetoceros-like diatoms. However, the endosymbionts of freshwater dinoflagellates clustered with other diatoms. Takano et al. (2008) recently described two freshwater dinoflagellates, P. kevei and P. penardii, from Japan, which also had endosymbionts related to Thalassiosira/Skeletonema-like diatoms. In our phylogenic analyses, three diatom-harboring freshwater dinoflagellates had endosymbion ts re- Description of a new dinoflagellate with a diatom endosymbiont 131 lated to Discostella-like diat oms, i.e., P. kevei, P. penardii, and P. penardii var. robusta. This agrees with the hypothesis proposed by Zhang et al. (2011). However, the endosymbiont of P. minima was found to be included in a clade consisting of Cyclotella species. Due to their characteristic valve morphology an d ultrastructure (e.g., marginal fultoportulae and rimoportulae positioned between the costae), all stelligeroid taxa of Cyclotella (Kützing) Brebisson have been transferred into the genus Discostella Houk et Klee (Thalassiosirales) (Houk & Klee 2004). Takano et al. (2008) indicated that there was a serial replacement of endosymbionts from an original pennate Nitzschia-like diatom to a centric diatom, such as Thalassiosira, or from an original centric diatom to a pennate diatom. Furthermore, these freshwater dinoflagellates have taken up a marine diatom (Thalassiosira/Skele- tonema-like diatom) as an endosymbiont. More freshwater diatoms have now been sequenced, however, and the endosymbionts of marine dinoflagellates have been shown to originate from marine diatoms whereas the endosymbionts of diatom-harboring Peridiniopsis species was found to have originated in freshwater diatoms in our study. One reasonable explanation for this is that after colonizing the freshwater environ- ment, the common ancestor of diatom-harboring Peridiniopsis species obtained the endosymbiont from a freshwater Cyclotella/Discostella-like ancestor, w hich then di- versified to give rise to several different but closely related species.

Acknowledgements

This work was financial supported by the National Natural Science Foundation of China (Grant no. 30970501 and 31270252). We thank the staff at the Research group of Algae Taxonomy and Resources Utilization, Institute of Hydrobiology, the Chinese Academy of Sciences (IHB, CAS) for helpful suggestions. We also thank Sen Lin and Lin-Lin Zheng at IHB, CAS for sampling collection.

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Manuscript received June, 29, 2013, accepted March 19, 2014