Morphology and molecular phylogeny of three heterotrichid species (Ciliophora, Heterotrichea), including a new species of Anigsteinia

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Morphology and molecular phylogeny of three heterotrichid species (Ciliophora, Heterotrichea), including a new species of Anigsteinia Xiangrui Chena,1 , Ji Hye Kimb,1 , Shahed Uddin Ahmed Shazibb , Choon Bong Kwonb , Mann Kyoon Shinb,∗ a b

School of Marine Sciences, Ningbo University, Ningbo, 315211, China Department of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan, 44610, South Korea

Received 8 February 2017; received in revised form 14 June 2017; accepted 17 June 2017

Abstract Heterotrichs are generally larger than ciliates of other groups with a seemingly cosmopolitan distribution, and recent studies have demonstrated that they exhibit great biodiversity. In the present work, we investigated the morphology and small subunit rRNA (SSU rRNA) gene sequences of three heterotrichous species, including that of a new one, Anigsteinia paraclarissima spec. nov. The new organism is morphologically very similar to A. clarissima, however, it can be distinguished from the latter by the larger dimensions and more somatic kineties (25–32 vs. 18–26), and its sequence similarity of SSU rRNA gene is 97.14% which indicate that it is a distinct species. Detailed morphological and molecular data for Blepharisma bimicronucleatum are supplied together for the first time in this study. In addition, the morphology of a poorly known species, Spirostomum yagiui is redescribed and an improved species diagnosis is provided. Finally, based on phylogenetic analyses of SSU rRNA gene sequences data, the Spirostomum group contains two main clades based on the type of the macronucleus. Spirostomum yagiui was newly sequenced here and clustered with the other S. yagiui populations and positioned within the Spirostomum assemblage. The Anigsteinia clade, including A. paraclarissima and A. clarissima, clustered within the family Spirostomidae. © 2017 Elsevier GmbH. All rights reserved. Keywords: Anigsteinia; Blepharisma; Molecular phylogeny; Morphology; Spirostomum

Introduction Heterotrichous ciliates are generally large and free-living with a cosmopolitan distribution. Hence, many of them are known since more than 200 years (Foissner et al. 1992; Lynn 2008; Müller 1773, 1786; Song et al. 2003). Species identification in this group is, however, challenging. This is because many forms share very similar morphological features, even

∗ Corresponding

author. Fax: +82 52 259 1694. E-mail address: [email protected] (M.K. Shin). 1 Both authors contributed equally to this study.

infraciliature, and relatively few characters can be used reliably for species separation (Song et al. 2009). Another reason is that most of the known species have not been investigated in sufficient detail and many characters overlap. Furthermore, although molecular information plays a more important role in taxonomic and systematic studies of ciliates, such data are available for only few species of heterotrichs (Boscaro et al. 2014; Fernandes et al. 2013, 2014, 2015, 2016; Shazib et al. 2014, 2016; Yan et al. 2015, 2016). In recent years, it has been proposed that both morphological and molecular data should be used for ciliated protozoa species-level descriptions (Boscaro et al. 2014; Bourland

http://dx.doi.org/10.1016/j.ejop.2017.06.005 0932-4739/© 2017 Elsevier GmbH. All rights reserved.

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2015; Gao et al. 2016; Warren et al. 2017). Careful comparisons with forms reported in the literature must be carried out, especially when erecting a new taxon or providing an improved diagnosis (Song et al. 2003). During a faunistic investigation of the free-living ciliates in South Korea, nine heterotrichous species were found and redescribed (Jang et al. 2012; Kim et al. 2012; Kim and Shin 2015; Lee and Shin 2009). As a new contribution, the present work describes three heterotrichous ciliates, namely the new species Anigsteinia paraclarissima spec. nov., and Blepharisma bimicronucleatum Villeneuve-Brachon, 1940 and Spirostomum yagiui Shigenaka, 1959. Moreover, a phylogeny based on small-subunit rRNA (SSU rRNA) gene sequences is provided for these three species and other heterotrichous ciliates.

Material and Methods Sample collection, observation, and identification Anigsteinia paraclarissima spec. nov. was collected from estuarine brackish water (salinity 10‰, water temperature about 14 ◦ C) of the Taehwagang River (N35◦ 33 10 , E129◦ 16 52 ), Ulsan, South Korea on 2nd April, 2009. Samples were taken from the surface of sediment using a spoon, and then diluted with untreated water from the collection site. Blepharisma bimicronucleatum was collected from terrestrial humic soil in Hwangsung Park (N35◦ 51 35 , E129◦ 12 53 ), Gyeongju, South Korea on 2nd June, 2014. The sample comprised 500 g of soil with decomposed leaves from the top 5 cm layer. Ciliates were stimulated to excyst and emerge from the soil sample by employing the non-flooded Petri dish method described by Foissner et al. (2002). Spirostomum yagiui population-1 was collected from estuarine brackish water (salinity 6‰) of the Woldaecheon River (N33◦ 29 32 , E126◦ 26 07 ), Jeju Island, South Korea on 11th September, 2010; population-2 was collected from estuarine brackish water (salinity 1‰) near Okgye beach in Gangneung (N37◦ 37 36 , E129◦ 03 03 ), Gangwon-do, South Korea on 7th June, 2014. Samples were transferred to the laboratory with green algae (Ulva lactuca) and some pieces of aquatic plant stems and leaves. Raw cultures were maintained for 1–2 weeks at room temperature. Because for none of these three species a clone could be established, living cells were randomly selected from the original sample or raw cultures and observed using bright field and differential interference contrast microscopy (Axio Imager A1; Carl Zeiss). The protargol silver staining method according to Wilbert (1975) was used to reveal the infraciliature. The protargol reagent was synthesized following the protocol of Pan et al. (2013). Counts, measurements, and drawings of stained specimens were carried out with the help of a camera lucida. Terminology and systematics mainly follow Lynn (2008).

DNA extraction, sequencing, and comparison Three to five living cells from each population and species were isolated from the original sample or raw cultures and washed several times to remove contaminants, and subsequently transferred to 1.7ml microtubes. Total genomic DNA extraction was performed using the RED Extract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA), as described by Shazib et al. (2014). The universal eukaryotic primers 18S-like forward (5 -AAC CTG GTT GAT CCT GCC AG3 ) and 18S-like reverse (5 -CAC TTG GAC GTC TTC CTA GT-3 ) designed by Medlin et al. (1988) were used to amplify the SSU rRNA gene by polymerase chain reaction (PCR). The polymerase reaction was done using the TaKaRa high fidelity Ex Taq DNA polymerase kit (TaKaRa Bio-medicals, Otsu, Japan) following the manufacturer’s instructions. PCR cycling parameters followed the protocol in Shin et al. (2000). PCR products were checked visually on 1.2% agarose gels, purified, and bi-directionally sequenced with PCR primers and the internal primer 18S 528F (5 CCG CGG TAA TTC CAG CTC-3 ) (Alves-de-Souza et al. 2011) using an ABI 3730XL automatic sequencer operated by Macrogen Inc. (Seoul, Korea). Sequence fragments from each species were checked, trimmed, and assembled into contigs using Geneious pro software ver. 8.0.5 (Biomatters, http://www.geneious.com/).

Phylogenetic analyses Phylogenetic analyses of SSU rRNA gene sequences were performed using an alignment comprising 159 homologous sequences of Heterotrichea and Karyorelictea (as an outgroup) (Table 1). Sequences were aligned in MAfft ver. 7.0 using the Q-INS-I strategy (Katoh and Toh 2008). The alignment was checked and trimmed at both end using Geneious. Hypervariable sites were masked and removed using Gblocks ver. 0.91b with default setting (Castresana 2000; Talavera and Castresana 2007), which resulted in a matrix of 1464 unambiguously aligned nucleotide characters. GTR + I (=0.4690) + G (=0.4430) was the best-fit model selected by jModeltest ver. 2.0.1 under the Akaike Information Criterion (Guindon and Gascuel 2003; Posada 2008). Bayesian inference (BI) analyses was performed using MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck 2003) using the best-fit model (GTR + I + G). Two parallel runs were performed over 1 million generations with every 100th tree sampled, and the first 25% sampled trees were discarded as burn-in. Maximum likelihood (ML) analysis was carried out using PhyML ver. 3.0 (Guindon et al. 2010) under the best selected evolutionary model (GTR + I + G). Support for internal branches on the ML tree was assessed using nonparametric bootstrap analysis (1000 replicates). Pairwise uncorrected p-distances and numbers of nucleotide differences in the SSU rRNA gene sequences were calculated in MEGA ver. 6.06 (Tamura et al. 2013).

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Table 1. GenBank accession numbers of all taxa analyzed in Fig. 6 except the members of Blepharismidae, Spirostomidae and outgroup. Species

GB number

Species

GB number

Family Climacostomidae Climacostomum virens Climacostomum virens Climacostomum virens Climacostomum virens

KP970234 X65152 KJ651814 EU583990

Stentor amethystinus Stentor amethystinus Stentor amethystinus Stentor amethystinus Stentor amethystinus

AM713191 FN659807 FN659808 AY775566 KP970242

Family Condylostomatidae Condylostentor auriculatus Condylostentor auriculatus Condylostoma arenarium Condylostoma cf. arenarium Condylostoma curva Condylostoma curva Condylostoma minutum Condylostoma minutum Condylostoma spatiosum Condylostoma spatiosum Condylostoma tropicum Condylostoma sp. Condylostoma sp. Condylostoma sp. Condylostoma sp. Chattonidium setense Condylostomides sp. Condylostomides sp.

KP970235 DQ445605 JQ282895 FJ998021 KJ651827 EU379939 KJ651815 DQ822482 HM140390 DQ822483 FJ868178 FJ998022 FJ868179 AM295496 KJ651816 AM295495 KP970236 AM713188

Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor coeruleus Stentor elegans Stentor igneus

KP970244 KJ651825 AF357145 DQ136037 FN659814 FN659815 FN659816 KJ651823 FN659813 DQ132978 KP970243 AM713189 FN659809 FN659810 FN659811 FN659812 JQ282899 FN659817 KP970245

Family Fabreidae Fabrea salina Fabrea salina Fabrea salina Fabrea salina

EU583991 DQ168805 DQ168806 KJ651817

Stentor cf. katashimai Stentor muelleri Stentor muelleri Stentor muelleri Stentor multiformis

FN659818 KJ651824 FN659819 FN659820 FN659822

Family Folliculinidae Eufolliculina uhligi Folliculina sp.

U47620 EU583992

Stentor multiformis Stentor polymorphus Stentor polymorphus

FN659821 KP970246 AM713190

Family Gruberiidae Gruberia lanceolata Gruberia sp.

KJ651818 L31517

Stentor polymorphus Stentor polymorphus Stentor polymorphus

JQ282898 FN659823 AF357144

Family Maristentoridae Maristentor dinoferus

AY630405

Stentor roeselii Stentor roeselii

KP970247 AF357913

Family Peritromidae Peritromus faurei Peritromus kahli Peritromus kahli Peritromus sp.

EU583993 KP970237 AJ537427 KJ651830

Stentor roeselii Stentor roeselii Stentor roeselii Stentor roeselii Stentor sp.

FN659824 FN659825 KP970248 KJ651826 JQ282900

Family Stentoridae Stentor amethystinus

EF492142

Stentor sp. Stentor sp.

KJ651828 KF639913

Results and Discussion

Anigsteinia paraclarissima spec. nov. (Figs 1A–D, 2A–G; Tables 2 and 3)

Family Spirostomidae Stein, 1867 Genus Anigsteinia Isquith, 1968

Diagnosis. Extended living cells about 300–600 × 80–125 ␮m in size, oral length about half of body length; 47–89 adoral membranelles; 25–32 somatic kineties; moniliform macronucleus with 17–38 nodules;

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Fig. 1. A–R. Morphology and infraciliature of Anigsteinia paraclarissima spec. nov. (A–D), Blepharisma bimicronucleatum (E–H), and five previously reported Anigsteinia species (I–R). A: Right-lateral view of a typical individual of A. paraclarissima. B, C: Ciliature of right (B) and left (C) side of holotype specimen, arrow shows paroral membrane. D: Arrangement of cortical granules. E: Paroral membrane. F: Right-lateral view of a typical individual of B. bimicronucleatum. G, H: General infraciliature of right (G) and left (H) side of same specimen, arrows in (H) mark the micronucleus. I–K: A. clarissima (from Yan et al. 2016). L–N: A. salinara (L, M, from Anigstein 1912; N, from Kahl 1928). O: A. longissima (from Kahl 1932). P: A. oligonucleata (from Dragesco 1966). Q, R: A. candida (Q from Yagiu and Shigenaka 1956, R from Al-Rasheid 2000). AZM – adoral zone of membranelles, CV – contractile vacuole, Ma – macronuclear nodules, PM – paroral membrane, SK – somatic kineties. Scale bars 100 ␮m (A–C), 50 ␮m (F–H), 10 ␮m (D, E).

cortical granules colourless, spherical, about 1 ␮m across; brackish water habitat. Type locality. Estuarine brackish water of the Taehwagang River (N35◦ 33 10 , E129◦ 16 52 ), Ulsan, South Korea. Type deposition. The protargol-impregnated slide containing the holotype specimen (Fig. 2G) marked with an ink circle has been deposited in the National Institute of Biological Resources (NIBR), Incheon, South Korea, with registration number (NIBRPR0000107883). Etymology. The species-group name paraclarissima is a composite of the Greek prefix para+ (beside, at, along, during) and the species-group name clarissima, and refers to the

similar morphology of A. clarissima and A. paraclarissima. Gene sequence. The SSU rRNA gene sequence derived from one cell isolated from the same sample as the holotype with the length 1721 bp, GC content 46.5%, which is deposited in GenBank (accession number HM140405). Description. Cell size of extended specimens about 300–600 × 80–125 ␮m in vivo. In general, body slender and elongated, only slightly contractile with tapered, narrowly rounded posterior end; anterior portion bluntly pointed with conspicuous coracoid structure like Blepharisma (Figs 1A, 2A–C). Large oral region starting slightly below anterior end and extending to about 50% of cell length. Bilaterally

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Fig. 2. A–G. Photomicrographs of Anigsteinia paraclarissima spec. nov. from life (A–E) and after protargol staining (F, G). A: General right-lateral view of a typical individual, arrow shows lacunar contractile vacuole system. B: Right view of stumpy individual filled with some green food vacuoles. C: Anterior region of cell showing the inclusions, including shining droplets and food vacuoles. D: Posterior portion of buccal field, arrow indicates paroral membrane. E: The arrangement of colorless cortical granules. F: Oral region in right-lateral view, arrow shows paroral membrane. G: Left lateral view of a typical individual, showing the general macronuclear nodules. Scale bars 100 ␮m (A, B, G), 50 ␮m (C, D, F), 25 ␮m (E).

flattened with width to thickness ratio about 3:1. Pellicle flexible and thin, with colorless spherical cortical granules (0.8–1.0 ␮m across) sparsely and irregularly arranged between kineties (Figs 1D, 2E). Cytoplasm somewhat grayish and opaque, packed with fusiform granules (about 4 × 2 ␮m in size) and abundant food vacuoles (10–20 ␮m across) (Fig. 2C). Macronucleus moniliform, composed of 17 to 38 nodules of similar size and shape (Figs 1A, C, 2B, G). Micronucleus difficult to recognize in vivo and in protargol preparations. Contractile vacuole terminally positioned and with long anteriad collecting canal on dorsal side (Figs 1A, 2A). Locomotion by gliding over sand grains or organic debris. Ciliary pattern as shown in Figs 1B, C, 2F, G. Twenty-five to 32 somatic kineties, including 5–7 postoral shortened rows. Cilia about 10–12 ␮m long in vivo. Adoral zone composed of 47–89 membranelles, with cilia about 20 ␮m in length. Paroral membrane difficult to recognize in vivo, but very conspicuous after protargol-impregnation, proximal portion composed of densely packed pairs of basal bodies, while the distal part comprises loosely lined units (Figs 1B, 2D, F).

Comparison of Anigsteinia paraclarissima spec. nov. with congeners Anigsteinia paraclarissima spec. nov. was collected from the Taehwagang River, South Korea in 2009 and it’s phy-

logenetic position was discussed by Shazib et al. (2014), who, however, misidentified it as A. clarissima. Here we describe its morphology and molecular characteristics in detail. Prior to this study, five nominal species of Anigsteinia were recognized: A. clarissima (Anigstein, 1912) Isquith, 1968 (type species of Anigsteinia Isquith, 1968); A. salinara (Florentin, 1899) Isquith, 1968; A. longissima (Kahl, 1932) Isquith and Repak, 1974; A. candida (Yagiu and Shigenaka, 1956) Isquith, 1968; and A. oligonucleata Isquith and Repak, 1974 (= Blepharisma clarissimum sensu Dragesco (1966)). Morphological characteristics are shown in Fig. 1I–R and compiled in Table 3. Anigsteinia salinara was originally found in a high salinity (about 65‰) pond near Lorraine, France, by Florentin (1899). About 30 years later, Kahl (1928) collected it in a similar habitat near Hamburg, Germany. The original and subsequent descriptions provide important characters for separation (macronucleus composed of 50–100 spherical fragments irregularly distributed in the cytoplasm vs. macronucleus moniliform with 17–38 interconnected nodules arranged in a line; fewer somatic kineties, 16–18 vs. 25–32) (Figs 1L–N; Table 3; Florentin 1899; Kahl 1928). Anigsteinia longissima, discovered by Kahl (1932) in brackish water (salinity about 3–9‰) near Cuxhaven (Germany) is much slender and longer than A. paraclarissima (500–1000 ␮m long, length/width ratio about 8–12:1 vs. 300–600 ␮m long, length/width ratio about 3.5–5.0:1). It

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Table 2. Morphometric data on Anigsteinia paraclarissima spec. nov. (A. par), Blepharisma bimicronucleatum (B. bim) and Spirostomum yagiui (S. yag-J, Jeju population; S. yag-G, Gangwon-do population). Character

Species

Min

Max

Mean

M

SD

CV

n

Body, length (␮m)

A. par B. bim S. yag-J S. yag-G

289 115 137 163

569 211 267 253

391.7 166.7 194.7 217.6

366 165 192 224

81.5 23.5 33.4 22.5

20.8 14.1 17.2 10.3

14 35 19 25

Body, width (␮m)

A. par B. bim S. yag-J S. yag-G

58 45 46 91

134 75 96 149

95.3 60.0 66.6 117.2

130 60 64 115

26.0 7.3 12.6 16.1

27.3 12.2 19.0 13.7

15 31 19 25

Oral area, length (␮m)

A. par B. bim S. yag-J S. yag-G

112 66 86 84

238 99 200 119

153.8 83.7 165.1 102.4

145 83 170 110

40.8 7.7 26.6 8.8

26.1 9.2 16.1 8.6

10 32 20 24

Somatic kineties, number

A. par B. bim S. yag-Ja S. yag-Ga

25 15 15 19

32 36 21 24

28.3 20.0 18.7 21.8

27.5 17 19 22

2.3 6.3 1.7 1.3

8.1 31.5 9.3 6.0

12 20 19 24

Adoral membranelles, number

A. par B. bim S. yag-J S. yag-G

47 28 85 86

89 38 124 106

73.0 34.6 107.3 97.5

71 35 106 96

13.2 2.6 10.1 4.6

18.1 7.5 9.4 4.7

10 26 19 24

Ma nodules, number

A. par B. bim S. yag-J S. yag-G

17 1 1 1

38 1 1 1

23.2 1.0 1.0 1.0

23 1 1 1

5.4 0.0 0.0 0.0

23.3 0.0 0.0 0.0

14 31 19 25

Ma, length

A. parb B. bim S. yag-J S. yag-G

10 37 79 108

51 66 141 168

21.4 48.9 108.7 144.1

20 47 110 146

11.6 7.5 17.7 22.6

54.2 15.3 16.3 15.7

14 31 18 8

Ma, width

A. parb B. bim S. yag-J S. yag-G

2 25 12 25

28 45 17 39

14.9 34.0 14.1 33.0

13 34 13 34

5.9 4.5 1.9 3.3

39.6 13.2 13.1 10.0

14 31 19 25

Mi, number

B. bim S. yag-J

2 1

2 4

2.0 1.8

2 2

0.0 1.0

0.0 54.1

8 16

All data based on protargol-impregnated specimens. Measurements in ␮m. Abbreviations: CV, coefficient of variation in %; M, Median; Ma, macronucleus; Max, maximum; Mean, arithmetic mean; Mi, micronucleus; Min, minimum; n, number of specimens investigated; SD, standard deviation. a Shortened and fragmented somatic kineties were excluded. b Macronuclear nodules were selected randomly in each individual.

has a terminally positioned, Spirostomum-like contractile vacuole, namely huge and rectangular (vs. spindle-shaped) (Fig. 1O; Table 3; Kahl 1932). Anigsteinia candida was first discovered in Hiroshima Bay, Japan (Fig. 1Q; Yagiu and Shigenaka 1956). Al-Rasheid (2000) reinvestigated this species using protargol preparation based on an Arabian Gulf population (Fig. 1R). It can be easily distinguished from A. paraclarissima by the macronucleus character (181–287 macronucleus beads distributed in the axial part of the cell vs. macronucleus moniliform with

17–38 interconnected nodules arranged in a line generally near the dorsal side) (Table 3; Al-Rasheid 2000; Yagiu and Shigenaka 1956). Only one freshwater species has been reported in this genus, Anigsteinia oligonucleata Isquith and Repak, 1974. The species was first collected from Lake Leman near Excenevex, France, and described as Blepharisma clarissima by Dragesco (1966) only based on live cells. Anigsteinia oligonucleata deviates from A. paraclarissima by the body shape (anterior half evenly narrowing from cytopharynx

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Table 3. Comparison of Anigsteinia paraclarissima n. sp. (A. par.) with five congeners, A. clarissima (A. cla.), A. longissima (A. lon.), A. oligonucleata (A. oli.), A. salinara (A. sal.), and A. candida (A. can.). Species

BL in vivo (␮m)

BL/BW ratio

No. SK

No. Ma

Habitat (S, ‰)

Ref.

A. par. A. cla.

300–600 160–380 230–300 500–1000 320–400 160–360 160–250 330–520 365–475a

3.5–5 ca. 10 5.5–7b 12–20 ca. 7b 3.5–5b ca. 4.5b 6–9 ca. 7.3

25–32 22–25 18–26 NA 25–30b 16–18 NA 22–27 24–33

17–38 32–47 14–26 ca. 30b 8–12 50–75 70–100 181–287 200–260

Estuarine, (1) Marine, intertidal beach Marine, intertidal beach (31) Brackish, (3–9) Freshwater High-saline, (65) High-saline or brackish Brackish Brackish, (15–28)

[1] [2] [3] [4] [5] [6] [7] [8] [9]

A. lon. A. oli. A. sal. A. can.

Abbreviations: AM, adoral membranelles; BL, body length; BW, body width; Ma, macronuclear nodules; NA, not available; SK, somatic kineties. References: [1] present work; [2] Anigstein (1912); [3] Yan et al. (2016); [4] Kahl (1932); [5] Dragesco (1966); [6] Florentin (1899); [7] Kahl (1928); [8] Yagiu and Shigenaka (1956); [9] Al-Rasheid (2000). a Data from protargol-impregnated specimens. b Data from drawing.

forming obvious neck area vs. dorsal and ventral sides slightly convex and without neck area), fewer macronuclear nodules (8–12 vs. 17–38), and habitat (freshwater vs. brackish) (Fig. 1P; Table 3; Dragesco 1966; Isquith and Repak 1974). Anigsteinia clarissima was originally described by Anigstein (1912) from marine samples collected from Kiel, Germany, and has been reported or described subsequently by nearly 30 authors all over the world (Agamaliev 1968; Anigstein 1912; Bock 1952; Czapik and Jordan 1976; Dragesco 1960, 2002; Dragesco and Dragesco-Kernéis 1986; Fauré-Fremiet 1950; Fenchel 1969; Hartwig 1973; Hirschfield et al. 1973; Kahl 1928, 1932; Raikov 1960; Ricci et al. 1982; Yagiu 1943). Unfortunately, almost all of these authors did not provide details of the infraciliature and it is difficult to know whether their identification was justified. Based on exhaustive morphological and molecular characterization of Anigsteinia clarissima collected from Qingdao, China, Yan et al. (2016) clarified the historic confusion and improved the diagnosis for this type species. Anigsteinia paraclarissima spec. nov. is morphologically very similar to A. clarissima, however, the new species can be distinguished from A. clarissima based on several minor differences (taxonomic data for A. clarissima is based on the original and Qingdao populations): (1) body length (300–600 ␮m vs. 160–380 ␮m; 230–300 ␮m); (2) the ratio of body length to width (3.5–5.0:1 vs. ca.10:1; 5.5–7.0:1); (3) number of somatic kineties (25–32 vs. 22–25; 18–26); (4) undulating membrane in vivo (inconspicuous, almost invisible vs. conspicuous; conspicuous, especially near the oral cavity) (Figs 1I–K; Table 3; Anigstein 1912; Yan et al. 2016). These two species show 47 nucleotide differences with a genetic divergence of 2.86% in their SSU rRNA gene sequences, supporting them as two valid morpho-species (Nebel et al. 2011). Family Blepharismidae Jankowski in Small and Lynn, 1985 Genus Blepharisma Perty, 1849

Blepharisma bimicronucleatum Villeneuve-Brachon, 1940 (Figs 1E–H, 3A–J; Tables 2 and 4) 1940 Blepharisma bimicronucleata n. sp. – VilleneuveBrachon, Arch. Zool. Exp. Gén., 82: 49, Fig. XVI (original description). 1973 Blepharisma lateritium var. bimicronucleata – Hirschfield et al., In: Giese AC (ed): Blepharisma. 313: Fig. 10 (brief description). 1980 Blepharisma lateritium – Foissner, Ber. Nat. -Med. Ver. Salzburg., 5: 75, Fig. 3a, b (short description of an Austrian population and the author misidentified as B. lateritium). 1989 Blepharisma bimicronucleatum – Foissner, Sber. Akad. Wiss. Wien, 196: 228, Fig. 31 g, h (brief description of an Austrian population and detailed review of this species). 2002 Blepharisma bimicronucleatum – Foissner et al., Denisia, 5: 872, Fig. 189 a–e (detailed description of Namibia population). Blepharima bimicronucleatum was originally described by Villeneuve-Brachon (1940) and subsequently redescribed several times. An improved diagnosis based on previous populations and on present population of Korea. Improved diagnosis. Cells usually about 60–220 × 20–50 ␮m in vivo, lanceolate in outline, with blunt rounded posterior end and beak-shaped anterior end; coloration changing from pale pink to purple red, even blue; buccal area dominant and extending to 50–66% of body length; 28–45 adoral membranelles; paroral membrane conspicuous in protargol-impregnated specimen, posterior one-fifth including zig-zag arrangement of dikinetids; single contractile vacuole positioned at posterior end; single macronucleus positioned near middle part; two micronuclei; 14–36 somatic kineties. Voucher slide. One voucher slide (accession number NIBRPR0000107884) with protargol impregnated speci-

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Fig. 3. A–J. Photomicrographs of Blepharisma bimicronucleatum from life (A–C, F, G) and after protargol staining (D, E, H–J). A–C: General right-lateral views of various individuals in vivo, arrows indicate contractile vacuoles. D: Ventral view of typical individual. E: Left side view of ciliature. F: Cortical granulation and cell surface, arrowheads show cortical granules. G: Ventral view to show oral cavity (arrow) and convex left side. H, I: Two micronuclei (arrowheads) attached to the macronucleus. J: Oral ciliature and part of somatic ciliature, arrow indicates paroral membrane, arrowhead denotes zigzag arrangement of the dikinetidal part of the paroral membrane. Scale bars 50 ␮m (A–G, J), 10 ␮m (H, I). Table 4. Comparison of five populations of Blepharisma bimicronucleatum. Population

Body size in vivo (␮m)

Cell colour

No. PGa

No. SK

No. AZM

No. Mi

Ref.

Korea France Austria 1 Austria 2 Namibia

100–220 × 35–50 100–150 × 30–40 70–90 in length 60–110 × 30–50 60–110 × 20–35

Light pink or purple red Pale pink Weak to strongly red Pale pink to brick red Blue-grey to blue-green

6–8 NA ca. 6 ca. 8 ca.10

15–36 17–18 ca. 20 18–23 14–19

28–38 ca. 40 30–35 31–42 28–36

2 2 1 or 2 2 2

[1] [2] [3] [4] [5]

Abbreviations: AZM, adoral zone of membranelles; Mi, micronucleus; NA, not available; PG, pigment granules; SK, somatic kineties. References : [1] present work; [2] Villeneuve-Brachon (1940); [3] Foissner (1980); [4] Foissner (1989); [5] Foissner et al. (2002). a Number of pigment granules lines between two ciliary rows.

mens is deposited in the National Institute of Biological Resources (NIBR), Incheon, South Korea. Description of Korean population. Cell size about 100–220 × 35–50 ␮m in vivo. Body shape lanceolate, with beak-shaped anterior end that curved leftwards (Figs 1F, 3A–C). In general, starved individuals compressed bilaterally with width to thickness ratio about 3:2, while fed individuals droplet-shaped with rounded posterior end. Coloration varying from light pink to purple red at low magnification in bright-field microscopy (3 A–C, G). Buccal area occupied about half of body length (Figs 1F, 3A–C, G). Macronucleus ovoid and positioned in middle of body

(Figs 1F, H, 3D, E). Two spherical micronuclei attached to macronucleus, and each about 1.3–2.3 ␮m in diameter (Figs 1F, H, 3H, I). Contractile vacuole located at posterior end without canal, about 14–18 ␮m across (Figs 1F, 3A, B). Several food vacuoles located at posterior part of body. Pale pink to purple spherical cortical granules 0.2–0.3 ␮m in diameter, irregularly embedded on surface of thin cortex (Fig. 3F). Locomotion mainly by swimming with rotation in clockwise direction. Adoral zone composed of 28–38 membranelles and extending to 50–66% of body length (Figs 1F, G, 3D, E). Paroral membrane not easily observed in vivo, but conspicu-

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ous after protargol-impregnation and on right side of adoral zone of membranelles, posterior fifth with zig-zag arrangement of dikinetids (Figs 1E, 3J). Fifteen to 36 longitudinal somatic kineties, with cilia about 10 ␮m in length (Figs 1F–H, 3D–F). The SSU rRNA gene sequence of Blepharisma bimicronucleatum has a length of 1623 bp, a GC content 47.8%, and Genbank accession number KX119522. Distribution. France (Villeneuve-Brachon 1940); Austria (Foissner 1980, 1989); Namibia (Foissner et al. 2002); Korea (present work). Remarks. Although Blepharisma bimicronucleatum Villeneuve-Brachon, 1940 is commonly found in terrestrial and freshwater habitats, populations have been described as Blepharisma lateritium or B. steini when micronuclei were not apparent (Foissner 1980; Kahl 1932). Hirschfield et al. (1973) described this species as B. lateritium var.bimicronucleata. Foissner (1989) mentioned that previous descriptions of B. bimicronucleatum were not sufficient, it remains difficult to identify this species. We agree with the points raised by Foissner (1989) and provide SSU rRNA gene sequences that could aid in future studies. The South Korean population of Blepharisma bimicronucleatum corresponds well with the original description and redescriptions with regard to body shape, number of adoral membranelles, somatic kineties, and micronuclei, therefore, its identity is not in doubt (Table 4; Foissner 1980, 1989; Villeneuve-Brachon 1940). In addition, the pigment granules of the Namibia population are blue-grey to blue-green and about 0.5 ␮m across. Foissner et al. (2002) considered that it was characteristics of the population, because the colour of the pigment can be changed from red to blue by light and oxygen (Foissner et al. 2002). Family Spirostomidae Stein, 1867 Genus Spirostomum Ehrenberg, 1834

Spirostomum yagiui Shigenaka, 1959 (Figs 4A–H, 5A–N; Tables 2 and 5) 1959 Spirostomum yagiui n. spec. – Shigenaka, Zool. Mag. (Tokyo), 68: 368, 370, Figs 1, 2 (original description in Japanese and morphological description provided in English abstract). 2014 Spirostomum yagiui – Boscaro et al., Protist, 165: 530, Fig. 1B, H, N (morphologic description of living cell and gene sequence analysis of four populations from Mediterranean Sea islands, northern Europe, Russia). Prior to the current investigation, this species was reported two times and the ciliature has never been provided. Based on both previous and present studies, an improved diagnosis is supplied. Improved diagnosis. Small, brackish Spirostomum, extended living cell with rod-like body shape, about

9

250–500 × 25–40 ␮m in size, contracted body lanceolate in outline; peristome length variable, about 33–50% of body length; adoral membranelles 85–124; 12–24 somatic kineties; cortical granules grayish, relatively regularly arranged in two or three rows in-between somatic kineties; elongated macronucleus exhibiting different shapes from rod to tortuous band (length: width ratio ≥ 5); two micronuclei closely attached to macronucleus; posteriorly positioned contractile vacuole less than 20% of body length with a long canal extending anteriorly. Voucher slides. One voucher slide (accession number NIBRPR0000107885) of Jeju population, and one voucher slide (accession number NIBRPR0000107889) of Gangwondo population with protargol impregnated specimens are deposited in the National Institute of Biological Resources (NIBR), Incheon, South Korea. Description of Korean populations. Body flexible and very contractile, about 260–410 × 30–40 ␮m in vivo. Slender and extended body rod-like with anterior end slightly pointed and posterior end widely blunted, length to width ratio about 10:1 (Figs 4A, 5A–C); contracted cell lanceolate in shape with length to width ratio about 3:1 (Figs 5D, E). Large peristome extending about half of body length (Figs 4A, 5A–C). Pellicle soft and transparent with spherical, grayish cortical granules (about 0.8 × 0.6 ␮m), densely distributed in-between somatic ciliary rows forming two or three relatively regular rows (Figs 5F, G). Cytoplasm opaque at low magnification due to mass of dark-gray inclusions (ellipsoidal droplets and granules, 1–3 ␮m across) and several large food vacuoles filled with bacteria and organic debris (10–15 ␮m across). Macronucleus rod-shaped in extended cells, about 100 × 15 ␮m in size, while tortuous in contracted individuals, and usually located in middle portion of body (Figs 4A, C, 5E, H–M). Two or three globular micronuclei not easy observed in vivo, but conspicuous and attached to tortuous macronucleus in protargol-impregnated cells (Figs 4C, F–H, 5J, N). Contractile vacuole prominent and terminally positioned, with a collecting canal on dorsal side extending nearly to anterior end of body (Figs 4A, 5C). Cells relatively active, always crawling slowly on or through water plants, occasionally swimming leisurely by rotating about main body axis. Adoral zone of membranelles spirally turns around 1.33 times from apical end to mid-body in contracted body after protargol impregnation (Figs 4B, C, 5H, I). Eighty-five to 124 adoral membranelles, each composed of three rows of basal bodies, two long and one short (Figs 4D, 5N). Two or three membranelles at proximal end with three basal rows almost equal in length (Fig. 4D). Circumoral kinety commencing at apical end, parallel to right margin of adoral zone of membranelles, finally connected with paroral membrane (Fig. 4D, arrow). Paroral membrane located near proximal portion of adoral zone (Figs 4B, D, 5N). Fifteen to 24 longitudinal somatic kineties spirally twisted in contracted cells, most of them commencing at apical end

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Fig. 4. A–H. Spirostomum yagiui from life (A, E) and after protargol impregnation (B–D, F–H). A: Right-lateral view of typical specimen. B, C: Ventral and dorsal views to show the somatic and oral ciliature. D: Detail of rear portion of oral apparatus around the cytopharynx, arrow indicates circumoral kinety connected with paroral membrane. E: Spherical cortical granules arranged between somatic kineties. F–H: Various macronuclear shapes, arrowheads indicate micronuclei. Scale bars 100 ␮m (A–C), 15 ␮m (D), 10 ␮m (F–H), 5 ␮m (E).

of adoral zone of membranelles, and terminating at posterior end of cell; several of them starting from left margin of adoral zone of membranelles or below rear end of adoral zone (Figs 4B, C, 5H, I). The SSU rRNA gene sequence of Spirostomum yagiui population-1 (Jeju) is 1682 bp in length, GC content 48.0% (GenBank accession number KU848227; reported in Shazib et al. 2016); population-2 (Gangwon-do) is 1668 bp in length, GC content 48.1% (GenBank accession number KX119519). Distribution. Hiroshima Bay, Japan (Shigenaka 1959); Mediterranean Sea islands, northern Europe, Russia (Boscaro et al. 2014); Korea (present work). Remarks. Spirostomum yagiui is a brackish ciliate that was

originally isolated from a ditch near Hiroshima Bay, Japan and briefly described based on live observations (Shigenaka 1959). More than 50 years later, it was redescribed by Boscaro et al. (2014) based on four populations collected from Mediterranean Sea islands, northern Europe, and Russia, which allowed a concise morphological description and convincing phylogenetic analysis. According to those former studies, S. yagiui is a small to medium-sized (250–500 ␮m) brackish Spirostomum characterized, inter alia, by an elongated macronucleus (rod or tortuous-shaped with length to width ratio > 5:1). Prior to the present study, this species has never been investigated using silver staining and its infraciliature was unknown. Our isolates correspond perfectly with the previous descriptions in terms of the brackish habitat and

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Fig. 5. A–N. Photomicrographs of Spirostomum yagiui from life (A–G) and after protargol impregnation (H–N). A–C: Right-lateral side views to show the different ratios of oral length to body length, arrows indicate the oral apparatus ends. D: Right-lateral side view of a contracted cell. E: Rod-shaped macronucleus in contracted cell. F: Arrangement of cortical granules. G: Lateral side view of cortical granules (arrows). H: Ventral view of a typical impregnated individual to show the torsional somatic kineties, adoral zone of membranelles, and the rod-shaped macronucleus. I: Detail of adoral zone of membranelles and anterior somatic kineties. J–M: Various shapes of the macronucleus, arrowhead indicates the micronucleus. N: Arrow denotes the paroral membrane, arrowhead marks the lenticular micronucleus. Scale bars 100 ␮m (A–D), 50 ␮m (E, H), 20 ␮m (F, I, J–M), 10 ␮m (G, N).

general morphology, especially the elongated macronucleus. Hence, the identity of this organism is not in doubt (Boscaro et al. 2014; Shigenaka 1959). Spirostomid ciliates have been studied for more than two centuries since Spirostomum ambiguum (Müller, 1786) Ehrenberg, 1834 was first reported. To date, about twenty nominal species have been recorded from various habitats

worldwide. Members of the genus Spirostomum Ehrenberg, 1834 have a large, elongated, often worm-like, cylindrical (seldom laterally flattened) body and a terminally positioned contractile vacuole system. In most species, the anterior end is rounded and the posterior end is truncated. After Kahl (1932), three further reviews of this genus have been published, viz., Repak and Isquith (1974), Foissner et al. (1992),

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Table 5. Morphometric characterisation of Spirostomum yagiui and comparison with ten congeners. S. yagiui (S. yag.), S. ambiguum (S. amb.), S. caudatum (S. cau.), S. dharwarensis (S. dha.), S. inflatum (S. inf.), S. intermedium (S. int.), S. loxodes (S. lox.), S. minus (S. min.), S. semivirescens (S. sem.), S. subtilis (S. sub.), and S. teres (S. ter.). Species

BL in vivo (␮m)

BL/BW ratio

OL/BL ratio (%)

No. SK

Ma shape

Habitat (S, ‰)

Ref.

S. yag.

260–410 320–480 1000–4000 200–700 300–500 300–400 ca. 600 300–600 300–800 600–2000 700–1000 150–600

ca. 10 12–18 10–17 10–20 8–14 10–12 ca. 10 6–7 10–20 17–40 14–24 10–15

ca. 50 ca. 50 65–70 20–30 50–65 50–60a ca. 50 ca. 30 35–50 ca. 50 ca. 50 25–30

15–24 ca. 20 70–90 28–32 14–26 NA 25–60 NA 20–30 14–15 18–24 40–50

Rod-shaped Rod-shaped Moniliform Oval Filiform Moniliform Moniliform Moniliform Moniliform Moniliform Moniliform Ellipsoidal

Brackish, (1–6) Brackish, (13–17) Freshwater to brackish (4–10) Freshwater Freshwater Marine Freshwater Freshwater Freshwater to brackish (2–8.5) Freshwater Freshwater Freshwater

[1] [2] [3] [3] [4] [5] [5,6] [7] [3] [4] [4] [3]

S. amb. S. cau. S. dha. S. inf. S. int. S. lox. S. min. S. sem. S. sub. S. ter.

Abbreviations: BL, body length; BW, body width; Ma, macronucleus; NA, not available; OL, oral length; SK, somatic kineties. References: [1] present work; [2] Shigenaka (1959); [3] Foissner et al. (1992); [4] Boscaro (2014); [5] Kahl (1932); [6] Eberhardt (1962); [7] Stokes (1885). a Data from drawing.

and Boscaro et al. (2014). Based on historic data, we recognize 11 valid Spirostomum morphospecies in the present work: S. ambiguum (Müller, 1786) Ehrenberg, 1834 [Syn.: Trichoda ambigua Müller, 1786; S. ambiguum var. major Roux, 1901]; S. caudatum (Müller, 1786) Delphy, 1939 [Syn.: Enchelis caudata Müller, 1786; Uroleptus filum Ehrenberg, 1833; S. filum (Ehrenberg, 1833) Dujardin, 1841; S. teres var. caudatum Zacharias, 1903]; S. dharwarensis Desai, 1966; S. inflatum Kahl, 1927 [Syn.: S. ambiguum var. inflatum Kahl, 1927]; S. intermedium Kahl, 1932; S. loxodes Stokes, 1885 [Syn.: S. ambiguum sensu Dujardin, 1841; S. ambiguum sensu Stein, 1867]; S. minus Roux, 1901 [S. ambiguum var. minor, Roux, 1901]; S. semivirescens Perty, 1852; S. subtilis Bouscaro et al., 2014; S. teres Claparède and Lachmann, 1858; S. yagiui Shigenaka, 1959. Morphological characteristics are compiled in Table 5. According to the obvious difference in body shape, Spirostomum caudatum has a long tapered tail, allowing it to be easily distinguished from S. yagiui. Similarly, S. loxodes differs from S. yagiui in its anterior end with a beak-like projection (Table 5; Foissner et al. 1992; Jang et al. 2012; Kahl 1932; Repak and Isquith 1974; Stokes 1885). Boscaro et al. (2014) found that macronuclear features are quite stable in Spirostomum species. Some species, such as S. ambiguum, S. intermedium, S. minus, S. semivirescens, S. subtilis, and S. inflatum possess a moniliform macronucleus against a rod-shaped macronucleus in S. yagiui. Another three species, namely S. caudatum, S. dharwarensis and S. teres, which do not have a moniliform macronucleus, should be compared with S. yagiui. As discussed above, S. caudatum can be clearly separated from S. yagiui by the tapered tail. Spirostomum teres can be distinguished from S. yagiui by the shape of the macronucleus (ellipsoid vs. rod-shaped), the number of somatic kineties (40–50 vs. 15–24), and the SSU rRNA gene sequences have a similarity of 99.55–98.58%

(with 7–22 nucleotides differences). However, S. dharwarensis and S. yagiui were very similar to each other and can only be morphologically distinguished by the shape of macronucleus (filiform vs. rod-shaped). Moreover, the SSU rRNA gene sequence of S. dharwarensis (HG939537) is differed from that of S. yagiui in only two nucleotides (Boscaro et al. 2014; Claparède and Lachmann 1858; Desai 1966; Dragesco and Dragesco-Kernéis 1986; Foissner et al. 1992; Kahl 1932; Repak and Isquith 1974). Actually, based on the minor differences of macronucleus shape and SSU rRNA gene sequence, we cannot give a final conclusion whether these two forms are different species or not. It is likely that the SSU rRNA gene might be too conserved to discriminate relationships at lower level (i.e. intra- or interspecies). Thus, further studies with more evidences (i. e. infraciliature from S. dharwarensis, multi-gene analyses) will allow these issues to be disentangled (Kher et al. 2011; Liu et al. 2016).

Phylogenetic analyses based on SSU rRNA gene sequences (Fig. 6) Phylogenetic trees inferred from ML and BI analyses had very similar topologies and were congruent with the trees reported by Shazib et al. (2014) and Yan et al. (2016). All heterotrichean families sensu Shazib et al. (2014) had strong nodal support, but the majority of deeper nodes were not resolved, which is also consistent with previous studies (Fernandes et al. 2016; Schmidt et al. 2007; Shazib et al. 2014; Thamm et al. 2010; Yan et al. 2015, 2016). The genus Blepharisma is monophyletic (ML 100%, BI 1.00) and forms a clade that is sister to the Stentor assemblage. The newly sequenced Korean population of Blepharisma bimicronucleatum (KX119522) clusters with its congeners and not clearly separated from other Blepharisma species

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Fig. 6. Maximum likelihood (ML) tree based on SSU rRNA gene sequences of heterotrichous ciliates. Nodal support is indicated as follows: Maximum likelihood (ML) bootstraps/Bayesian inference posterior probabilities (PP). A dash indicates a mismatch in the branching pattern in the Bayesian analysis. Species newly sequenced in this study are shown in bold font. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

in the ML tree (Fig. 6). However, B. bimicronucleatum is the sister taxon of all other Blepharisma species in the Bayesian tree (data not shown). SSU rRNA gene sequences could not resolve phylogenetic relationships of the genus Blepharisma. Because many sequences are nearly identical, there is a lack of phylogenetic informative sites in Blepharisma (Fernandes et al. 2013; Yan et al. 2016), even though the corresponding species show morphological differences.

Interestingly, two populations of B. steini from the Dominican Republic (AM713187) (Schmidt et al. 2007) and Brazil (KP970224) (Fernandes et al. 2016) had low sequence similarities (97.08%) and 45 nucleotide differences in their SSU rRNA gene sequences. Moreover, monophyly of these two B. steini populations were weakly supported from both trees (ML 53%, BI 0.51). We suppose that these two B. steini populations may be different species and could not justify them

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as valid B. steini species due to not providing morphological data by Fernandes et al. (2016) and Schmidt et al. (2007). We agree with the suggestion of Fernandes et al. (2013) that macronuclear shape is not a good taxonomic character for classification of Blepharisma at the species level because it does not form monophyletic clade according to the shape of macronucleus or this discrepancy might be due to misidentification. Our findings also suggest that the classification of Blepharisma species based on morphological data has to be revised using other taxonomic characters. Furthermore, more gene sequence information is required, including information from fast evolving gene sequences e.g. the D1D2 domain of the large rRNA gene, ribosomal internal transcribed spacer regions (ITS1 and ITS2), or protein coding genes such as mitochondrial cytochrome oxidase I (CO1), to clarify phylogenetic relationships within the genus Blepharisma in the future (Dong et al. 2011; Zhao et al. 2016). Two Korean populations of Spirostomum yagiui (KX119519, KU848227) were positioned with four other populations of S. yagiui (HG939533, HG939534, HG939535, HG939536) with strong support (Fig. 6; ML 99%, BI 0.99). SSU rRNA gene sequences confirmed the morphological identification of two populations of Spirostomum yagiui collected from South Korea. In this study, Spirostomum species were clearly divided into two major clades (see also Shazib et al. 2016). The first major clade (CLADE I) was poorly supported in both phylogenetic analyses (ML 46%, BI 0.52) and comprised three Spirostomum species (S. ambiguum, S. minus, and S. subtilis). However, the second clade (CLADE II) was more strongly supported (ML 85%, BI 1.00) and contained S. caudatum, S. dharwarensis, S. teres, and S. yagiui. The species of Spirostomum can be distinguished in two major clades based on macronuclear shape, which is an important taxonomic character for separating Spirostomum morphospecies (Boscaro et al. 2014; Shazib et al. 2016). CLADE I species have a nodulated (moniliform) macronucleus, while CLADE II species have compact (ellipsoid and/or elongated) macronucleus. Interrelationships within these two major clades were complex and conflicted with the morphology-based taxonomy. Spirostomum teres was splited into several different clades in CLADE II, and S. minus formed a clade that was divided into two subclades within CLADE I (Fig. 6), suggesting the presence of species complexes within S. teres and S. minus (Boscaro et al. 2014; Fernandes and da Silva Neto 2013; Shazib et al. 2016). Anigsteinia paraclarissima spec. nov., which was sequenced and deposited in the GenBank under the name of Anigsteinia clarissima (accession number HM140405) without morphological description previously (Shazib et al. 2016), is newly named and described morphologically here, and is sister to A. clarissima (Fig. 6; ML 99%, BI 1.00). The Anigsteinia clade is the sister group of the Spirostomum lineage and together they form the monophyletic family Spirostomidae.

Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) and National Institute of Biological Resources (NIBR) from the Ministry of Education, Ministry of Science, ICT and Future Planning, and Ministry of Environment of Korean government (NRF-2015R1D1A1A09058911, NRF2016R1D1A2B03933285, NRF-2015M1A5A1041804, NRF-2015R1C1A1A02037803, and NIBR201701201), and grants from the Natural Science Foundation of China (project number: 31572230), as well as the K. C. Wong Magna Fund in Ningbo University.

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