Bull. Natl. Mus. Nat. Sci., Ser. B, 44(1), pp. 15–22, February 22, 2018

A new planktonic diatom, Craticula pseudocitrus sp. nov.,Naviculales, Bacillariophyceae found in Lake Kasumigaura, Japan
Akihiro Tuji1, *, Megumi Nakagawa2, Mayumi Sato2 and Haruyo Yamaguchi2
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies,
Onogawa 16–2, Tsukuba, Ibaraki 305–8506, Japan* E-mail: tuji@kahaku.go.jp(Received 16 November 2017; accepted 20 December 2017)

Abstract A new planktonic diatom, Craticula pseudocitrus Tuji, Nakagawa, Sato et Yamaguchi,
is described from Lake Kasumigaura. This taxon has been confused with C. citrus, but can be distinguished by density and pattern of striae, size of outline and form of central area. Phylogenetic
tree analysis using rbcL and 18S rRNA supports the assignment of this taxon to genus Craticula.
The seasonal pattern of this taxon is similar to total diatoms, as it appears in the spring and lateautumn.
Key words: Craticula citrus, Craticula pseudocitrus sp. nov., Lake Kasumigaura, Navicula citrus.

Introduction
Most freshwater planktonic diatoms are classiﬁed as centric or araphid diatoms. Biraphid
planktonic diatoms are very rare. Most of theappearance records of freshwater planktonic
naviculoid diatoms may not be true plankton but
rather tychoplankton. The phytoplankton of Lake
Kasumigaura has been monitored by the National
Institute for Environmental Studies (NIES) since1996 (Takamura and Nakagawa, 2012). We
found a small characteristic naviculoid diatom
from the samples of the monitoring, and identiﬁed it as Craticula citrus (Krasske) Reichardt(Tuji and Nakagawa, pers. inf.). In this paper, we
re-examined the identiﬁcation, phylogeny andseasonal variation of this taxon.

comprehensive monitoring by NIES (Takamuraand Nakagawa, 2012; National Institute for Environmental Studies, 2016). Water samples weretaken from the surface to 2.0 m depth with anacrylic column sampler at Stations 3 (36°07.302′N,140°22.652′E) and 9 (36°02.142′N, 140°24.222′E)

Materials and Methods
1. Sampling
The sampling was done as a part of monthly

Fig. 1. A map of Lake Kasumigaura showing sampling sites.

16Akihiro Tuji et al.

(Fig. 1). The water sample (100 ml) for counting
phytoplankton was ﬁxed with Lugolʼs iodine
solution (ﬁnal concentrations; 0.2–0.4%), thenplaced in a sedimentation chamber and kept therefor 24 hours. The number of each species wascounted for each sample with an inverted microscope with 40× an objective lens.
2. Establishment of a culture strain
Surface water in the St. 9 was collected using
2 l of plastic bottle on May 13, 2015. Isolation ofthe diatom cell was conducted using a ﬂow
cytometer (EPICS ALTRA; Beckman Coulter,Germany) equipped with a cell sorter and an
argon laser (excitation at a wavelength of488 nm). The established culture strain wasmaintained using CSi medium (Kasai et al.,2009) under a light intensity of 11–14 μmol photons/m2/sec with a light cycle of 10 : 14 (light :darkness). This strain is available for the public
via the Microbial culture collection of National
Institute for Environmental Studies as strain
number NIES-4015 (NIES, http://mcc.nies.go.
jp/).
3. Microscopic observations
Planktonic samples without ﬁxation wereobserved and taken photographs using a lightmicroscopy (LM, BX41, Olympus, Japan)
equipped with water immersion lenses and acamera (EOS kiss X7i, Canon, Japan). Planktonic samples and the strain were cleaned using
concentrated nitric acid as described previously(Tuji and Tanimura, 2012). Cleaned samples
were embedded using Zrax (Tuji and Tanimura,2012). Light microscopy (LM) observation andimaging were done using a microscope (Axiophoto, Zeiss, Germany) equipped with a CCDcamera (Inﬁnity 4: Lumenera, Canada). A smallpart of the sample was ﬁltered using a membraneﬁlter (RTTP02500, Merck), placed on a SEM
stub and dried at room temperature overnight.
The stub was sputter-coated with platinum andexamined with a scanning electron microscope(JEOL-6390LV, JEOL, Japan) equipped with lanthanum hexaboride cathode.

4. Molecular analysis
DNA was extracted from the cultured strain(NIES-4015) using the Chelex method (Walsh etal., 1991). Polymerase chain reaction (PCR) andsequencing of rbcL and the 18S rRNA gene werethen performed (Tuji et al., 2014). Phylogenetic
and molecular evolutionary analyses for theobtained sequences were conducted using theMEGA 7 computer program (Kumar et al.,2016). Alignments were checked manually.
Neighbor joining (NJ) and maximum likelihood(ML) trees were calculated using MEGA software with the best ﬁts model (T92＋G＋Imodel) by Bayesian information criterion scores(BIC), and the substitution nucleotide matrix
parameters were calculated by the software. Atree using 1000 bootstrap replicates was generated. All positions containing gaps and missing
data were eliminated.
Results and Discussion
Morphological differences between Craticula
citrus and new species
Morphological variation of shape and size of
this taxon is very small in Lake Kasumigaura.
One H-shaped chloroplast is observed in living
cell (Figs. 2–7). The ʻconspicuous silica granulesʼ
on the valve exterior described by Reichardt, as
well as his ﬁnding that ʻat the margins the bigger
granules are elongated into rib-like structuresʼ
(Reichardt, 1997), were corroborated using SEM(Figs. 13–15). Other morphological characteristics found by LM (Figs. 8–12) and SEM (Figs.
13–18) also agreed with Reichardtʼs descriptionof Curaticula citrus in (Reichardt, 1997).
Though our observations of the size, length
16.0–17.5 μm, breadth 5.5–7.0 μm, and striae (19per 10 μm), are more diminutive than the originaldescription (length: 22 μm, breadth: 8 μm, 15striae per 10 μm) in Krasske (1923), they agreewith the descriptions of more recent works (Hofmann et al., 2011; Burge and Bishop, 2015).
A photograph of the type material was presented by Lange-Bertalot (2001: plate 83, ﬁg. 5).
The size of this type individual (length: 19.5 μm,

Craticula pseudocitrus sp. nov.

17

Table 1. Length, breadth and density of striae (per 10 μm) of Craticula citrus in previous studies and C. pseudocitrus in this study
Taxon

Lengh

BreadthStriae per 10 μm

Reference

Navicula citrus
Navicula citrus
Navicula citrus
Craticula citrus
Craticula citrus
Craticula pseudocitrus

22
19.5
18–20
18–22
13–17
16–17.5

87
6.5
6–7
4.6–5.8
5.5–7.0

15
16
18–20
18–20
18–24
19

Krasske (1923: 199)
Holotype in Lange-Bertalot (2001)
Hustedt (1961: 780)
Lange-Bertalot (2001: 111)
Burge & Biishop (2015)
This study

Figs. 2–12. Craticula pseudocitrus sp. nov., light microscopy (LM), bar＝10 μm. 2–7. Living cells showing
H-shaped chloroplast (station 3, Lake Kasumigaura, Nov. 8, 2017). 8–12. Cleaned permanent slide, TNS-ALTNS-AL-62279 (holotype slide), 8a, 9–11, 12a. Bright ﬁeld microscopy (BF). 8b, 12b. Differential interference contrast microscopy (DIC).

breadth: 7 μm, 16 striae in 10 μm) showed dimensions slightly smaller than in the original description (Table 1).
Differences in the density of striae are important for distinguishing naviculoid diatoms. Furthermore, the size of the outline, pattern of thecentral area and striae, and form of protracted
apices showed differences between the originaldescription and type individual on the one handand Lake Kasumigaura individuals on the other.
Thus, we propose a new species for Lake
Kasumigaura individuals.

Craticula pseudocitrus Tuji, Nakagawa, Sato et
Yamaguchi, sp. nov.
(Figs. 2–18)
Synonym: Craticula citrus sensu Reichardt(1997); C. citrus sensu Burge and Bishop(2015). non: Navicula pseudocitrus Manguin
in Bourrelly and Manguin (1954).
Holotype: A slide TNS-AL-62279 in TNS(Department of Botany, National Museum ofNature and Science).
Isotype: A formalin-ﬁxed specimen of following
type strain, TNS-AL-62279 m (Ak1279) inTNS (Department of Botany, National
Museum of Nature and Science).

Akihiro Tuji et al.

18Figs. 13–18. Scanning electron microscopy (SEM) of Craticula pseudocitrus sp. nov. TNS-AL-62279 m (isotypematerial). 13–15. External views of frustules using scanning electron microscopy. 13. Whole frustule showingmany conspicuous silica granules and bars. 14 Produced subcapitate apice. 15. Central area. 16–18. Internalviews of frustules using scanning electron microscopy. 16. Whole frustule. 17, 18. Produced subcapitate apices of both side.

Craticula pseudocitrus sp. nov.

19Fig. 19. Phylogenetic tree of Craticula pseudocitrus and related taxa determined by Maximum Likelihood (ML)method using rbcL (a) and 18S rRNA (b) genes. Accession numbers are followed by taxonomic names. Numbers at branches indicate NJ (Neighbor Joining)/ML bootstrap support values (only values higher than 70 areshown).

Akihiro Tuji et al.

20Fig. 20.

Seasonal changes of the density (individuals ml−1) of diatoms (a) and Craticula pseudocitrus (b).

Fig. 21. The relationship between the density (individuals mL−1) of diatoms and Craticula pseudocitrus.

Craticula pseudocitrus sp. nov.

Type strain: NIES-4105 maintained in the NIES
collection, National Institute for Environmental Studies (NIES).
Type locality: Lake Kasumigaura, Ibaraki, Japan.
Valves are elliptical-lanceolate with protracted, rostrate apices. The axial area is straightand narrow; the central area is absent or small.
Length 16.0–17.5 μm, breadth 5.5–7.0 μm. Striae
are almost parallel throughout, 19 striae per
10 μm. Several striae are slightly curved aroundthe central area, and sometimes becomes short.
The density of these striae are coarser than otherstriae. The striae at the ends are slightly convergent. Many conspicuous silica granules and bars
exist on the outside of valves. Striae are interrupted by these silica bars. The following characteristics are different from Craticula citrus,
which it otherwise resembles. Several striae of C.
pseudocitrus at the central area has courser thanothers, but the density of striae are almost somefor C. citrus (Lange-Bertalot 2001: plate 83, ﬁg.
5). The form of the central area is asymmetrical,
butterﬂy-shaped in C. citrus, but C. pseudocitrus
has a small or invisible central area. The produced apices are wide and parallel in C. citrus,
but narrow and swollen in C. pseudocitrus. The
thickened longitudinal elements, which areformed by the aligned frets in Craticula cuspidata (Mann and Stickle, 1991), is not exist.
However, rib-like structures found in this taxon,
should be comparable with it.
Phylogeny of C. pseudocitrus
Phylogenetic trees of C. pseudocitrus andrelated taxa using rbcL and 18S rRNA are shownin Fig. 19. C. pseudocitrus occupies the sameclade as C. accomoda (Hust.) Mann et al., C.
importuna (Hustedt) K. Bruder et Hinz, C.
molestiformis (Hust.) Mayama and C. subminuscula (Manguin) Wetzel et Ector (＝ Eolimna subminuscula (Manguin) Molser et al.); all aresmaller members of the genus Craticula. A larger
Craticula member, C. cuspidata, and the clade ofsmall Craticula members, form a monophyleticgroup. Genus Stauroneis is a sister group for theCraticula clade. These results support the assign-21

ment of C. pseudocitrus to genus Craticula.
Seasonal pattern of C. pseudocitrus in Lake
Kasumigaura and its ecology
The seasonal patterns of diatoms and C. pseudocitrus are presented in Fig. 20. The two patterns are almost identical, and show a high abundance in spring and the autumn. A positive
correlation between densities of diatoms and C.
pseudocitrus was found (Fig. 21).
The optimum pH for C. citrus was reported
about 8–9, and the concentration of sodium isabout 40 mg/l in North America (Burge andBishop, 2015). Arguably, then, C. citrus sensu
Burge and Bishop (2015) should not be C. citrus,
but close to C. pseudocitrus. The pH in Lake
Kasumigaura is about 8–10, and the concentration of sodium is about 20–50 mg/L, adapted tothe optimum pH and concentration of sodium atNorth America.
The ecology of genus Craticula has beenreported as ʻEpipelic in freshwater bodiesʼ
(Lange-Bertalot, 2001). C. pseudocitrus isobserved in planktonic samples in Lake Kasumigaura. Though it can be a tychoplankton, we
have not seen many individuals of this taxon
from epipelic samples in Lake Kasumigaura, andwe found only this taxon for small naviculoid
diatoms among the planktonic samples fromLake Kasumigaura.
Acknowledgements
We would like to thank Ms. Ayako Yamaguchi
for molecular work at NMNS. The dataset of seasonal variation of phytoplankton in Lake Kasumigaura are those of the Lake Kasumigaura Longterm Environmental Monitoring Program of theNational Institute for Environmental Studies,Japan. This work was supported by JSPS
KAKENHI Grant Numbers JP15H02380 andJP15K00574 and a research grant from National
Museum of Nature and Science.

Akihiro Tuji et al.

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