Morphology and molecular phylogeny of Amphidiniopsis rotundata sp. nov. (Peridiniales, Dinophyceae), a benthic marine dinoflagellate MONA HOPPENRATH 1,3 *, MARINA SELINA 2 ,AIKA YAMAGUCHI 3 AND BRIAN LEANDER 3 1 Senckenberg Research Institute, German Centre for Marine Biodiversity Research, Su ¨dstrand 44, D-26382 Wilhelmshaven, Germany 2 A. V. Zhirmunsky Institute of Marine Biology FEB RAS, Far Eastern Federal University,Vladivostok 690041, Russia 3 University of British Columbia, Departments of Botany and Zoology, #3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada HOPPENRATH M., SELINA M., YAMAGUCHI A. AND LEANDER B. 2012. Morphology and molecular phylogeny of Amphidiniopsis rotundata sp. nov. (Peridiniales, Dinophyceae), a benthic marine dinoflagellate. Phycologia 51: 157–167. DOI: 10.2216/11-35.1 A new dinoflagellate species within the benthic, heterotrophic, and thecate genus Amphidiniopsis was discovered, independently, in sediment samples taken on opposite sides of the Pacific Ocean: (1) the Vancouver area, Canada, and (2) Vostok Bay, the Sea of Japan, Russia. The cell morphology was characterized using light and scanning electron microscopy, and the phylogenetic position of this species was inferred from small-subunit ribosomal DNA sequences. The thecal plate pattern [formula: apical pore complex 49 3a 70 5c 5(6)s 5- 2-9] and ornamentation, as well as the general cell shape without an apical hook or posterior spines, demonstrated that this taxon is different from all other described species within the genus. Amphidiniopsis rotundata sp. nov. was dorsoventrally flattened, 24.5–38.5 mm long, 22.6– 32.5 mm wide. The sulcus was characteristically curved and shifted to the left of the ventral side of the cell. This work presents the first molecular study including a representative of the genus Amphidiniopsis, and led us to propose a new combination, Amphidiniopsis dragescoi comb. nov. (formerly Thecadinium dragescoi), and also suggests a close relationship between Amphidiniopsis, Herdmania (another benthic genus), and Archaeperidinium minutum (a planktonic species). KEY WORDS: Amphidiniopsis, Archaeperidinium, Herdmania, Northeast Pacific, Sand-dwelling, Taxonomy, Sea of Japan, SSU rDNA, Thecadinium dragescoi INTRODUCTION Amphidiniopsis was introduced by Woloszyn ´ska (1928) with the type species A. kofoidii. The history of records, nomenclatural changes and classification schemes of Amphidiniopsis species were summarized in Hoppenrath (2000a) and Hoppenrath et al. (2009a). The genus includes 14 validly described species: A. aculeata Hoppenrath, Koeman & Leander, A. arenaria Hoppenrath, A. cristata Hoppenrath, A. dentata Hoppenrath, A. galericulata Hoppenrath, A. hexagona Yoshimatsu, Toriumi & Dodge, A. hirsutum (Balech) Dodge, A. kofoidii Woloszynska, A. korewalensis Murray & Patterson, A. pectinaria Toriumi, Yoshimatu & Dodge, A. sibbaldii Nicholls, A. swedmarkii (Balech) Dodge, A. urnaeformis Gail 1950, and A. uroensis Toriumi, Yoshimatu & Dodge (Woloszyn ´ska 1928; Gail 1950; Balech 1956; Dodge 1982; Nicholls 1998, 1999; Hoppenrath 2000a; Yoshimatsu et al. 2000; Murray & Patterson 2002; Toriumi et al. 2002; Hoppenrath et al. 2009a). All of these species are heterotrophic sand dwellers with diverse cell morphologies, including morphological variability within the same species (Bursa 1963; Selina & Hoppenrath 2008). Species are laterally or dorsoventrally flattened, with a complete or incomplete cingulum, and with or without an apical hook (e.g. Balech 1956; Dodge 1982; Hoppenrath 2000a; Yoshimatsu et al. 2000; Murray & Patterson 2002; Toriumi et al. 2002). Currently, Amphidiniopsis is characterized by an ascending cingulum, a distinctive curved sulcus and hypothecal plate pattern (Hoppenrath et al. 2009a). Because the genus contains a great deal of morphological heterogeneity, it is possible that Amphidiniopsis contains a pattern of subclades that represent several different ‘genus-level’ taxa. This hypoth- esis can be tested with molecular phylogenetic data, which were unavailable before this study. A revision of the genus is also needed, but to do so, the type species (A. kofoidii) needs to be defined unambiguously and reinvestigated, especially specimens from the type locality. Similarities between Amphidiniopsis and other genera and the system- atic placement of this genus within the order Peridiniales have been discussed in Hoppenrath (2000a). Comparative morphology and molecular phylogenetic analyses suggest that Thecadinium dragescoi Balech is distinct from, and only distantly related to, Thecadinium Kofoid & Skogsberg and may instead be a member of Amphidiniopsis (Hoppenrath et al. 2004). It has been shown repeatedly that patterns of thecal plates can be interpreted differently, and that this affects the plate formula and of course the discussion about species similarities (e.g. Murray & Patterson 2002; Hoppenrath et al. 2004; Hoppenrath & Selina 2006). The heterogeneity within the genus and the absence of molecular phylogenetic information made the * Corresponding author ([email protected]). Phycologia (2012) Volume 51 (2), 157–167 Published 12 March 2012 157
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Morphology and molecular phylogeny of Amphidiniopsis rotundata sp. nov.
(Peridiniales, Dinophyceae), a benthic marine dinoflagellate
MONA HOPPENRATH1,3*, MARINA SELINA
2, AIKA YAMAGUCHI3
AND BRIAN LEANDER3
1Senckenberg Research Institute, German Centre for Marine Biodiversity Research, Sudstrand 44,
D-26382 Wilhelmshaven, Germany2A. V. Zhirmunsky Institute of Marine Biology FEB RAS, Far Eastern Federal University,Vladivostok 690041, Russia3University of British Columbia, Departments of Botany and Zoology, #3529-6270 University Boulevard, Vancouver,
BC V6T 1Z4, Canada
HOPPENRATH M., SELINA M., YAMAGUCHI A. AND LEANDER B. 2012. Morphology and molecular phylogeny ofAmphidiniopsis rotundata sp. nov. (Peridiniales, Dinophyceae), a benthic marine dinoflagellate. Phycologia 51: 157–167.DOI: 10.2216/11-35.1
A new dinoflagellate species within the benthic, heterotrophic, and thecate genus Amphidiniopsis was discovered,independently, in sediment samples taken on opposite sides of the Pacific Ocean: (1) the Vancouver area, Canada, and(2) Vostok Bay, the Sea of Japan, Russia. The cell morphology was characterized using light and scanning electronmicroscopy, and the phylogenetic position of this species was inferred from small-subunit ribosomal DNA sequences.The thecal plate pattern [formula: apical pore complex 49 3a 70 5c 5(6)s 5- 2-9] and ornamentation, as well as the generalcell shape without an apical hook or posterior spines, demonstrated that this taxon is different from all other describedspecies within the genus. Amphidiniopsis rotundata sp. nov. was dorsoventrally flattened, 24.5–38.5 mm long, 22.6–32.5 mm wide. The sulcus was characteristically curved and shifted to the left of the ventral side of the cell. This workpresents the first molecular study including a representative of the genus Amphidiniopsis, and led us to propose a newcombination, Amphidiniopsis dragescoi comb. nov. (formerly Thecadinium dragescoi), and also suggests a closerelationship between Amphidiniopsis, Herdmania (another benthic genus), and Archaeperidinium minutum (a planktonicspecies).
Germany) connected to a Leica DC500 color digital camera
(Wetzlar, Germany).
Isolated fixed cells of the Russian material were
transferred onto a glass slide and observed directly with
an Olympus BX41 light microscope (Tokyo, Japan). Images
were captured using an Olympus C2020Z digital camera
Figs 1–8. Light micrographs of Amphidiniopsis rotundata sp. nov.Figs 1–4, 8. Living cells from Canada.Figs 5–7. Lugol-fixed cells from Russia.
Figs 1–4. Through focus of a single cell. Note ventral sulcus (Fig. 1), single pusule (p) (Figs 2, 3), and nucleus (Figs 3, 4).Figs 5, 6. Through focus of sulcus.Fig. 7. Large central nucleus (n).Fig. 8. A cell with two pusules (p). Scale bars 5 10 mm.
158 Phycologia, Vol. 51 (2), 2012
(Tokyo, Japan). Cell dimensions were measured for 10 cells
at 3640 magnification using an ocular micrometer.
Scanning electron microscopy
Aliquots of the agitated sand samples were fixed overnight
with two drops of acidic Lugol’s solution. Cells were
transferred onto a 5-mm polycarbonate membrane filter
(Corning Separations Div., Acton, MA), washed with
distilled water, dehydrated with a graded series of ethanol
and critical-point-dried with CO2. Filters were mounted on
stubs, sputter-coated with gold and viewed using an Hitachi
S4700 scanning electron microscope (SEM). Some SEM
images were presented on a black background using Adobe
Photoshop 6.0 (Adobe Systems, San Jose, CA). Cell sizes
were calculated from calibrated SEM images.
The SEM stubs with the natural samples used in this
study have been deposited at the Senckenberg Research
Institute and Natural History Museum in the Centre of
Excellence for Dinophyte Taxonomy, Wilhelmshaven,
Germany, with the designations CEDiT2011H11 and
CEDiT2011RM12.
Figs 9–16. Scanning electron micrographs of Amphidiniopsis rotundata sp. nov. (Figs 9–11, 14–16 stub CEDiT2011H11 and Figs 12, 13 stubCEDiT2011RM12, stored at the Centre of Excellence for Dinophyte Taxonomy, Wilhelmshaven, Germany).
Figs 9–11. Ventral views. Note the marginal listlike structure (arrow) of the Sp-plate and its generally prominent, spinelike marginal listat the border to the Ss-plate (arrowhead).Figs 12, 13. Dorsal views. Note the variable lengths of the plate border between 2a and 3a (arrow) and the beginning and end of thefourth cingular plate (4c) (arrowheads).Fig. 14. Apical view. Note the border between 2a and 3a plates (arrow).Fig. 15. Antapical view.Fig. 16. Detail of the sulcal plates. Scale bars 5 10 mm.
Hoppenrath et al.: Amphidiniopsis rotundata sp. nov. 159
DNA extraction, polymerase chain reaction (PCR) amplifi-
Thirty-four living cells were isolated by micropipetting for
DNA extraction (Canadian sample from 18 June 2007).
The cells were manually isolated and washed three times in
sterile f/2-medium. Genomic DNA was extracted using the
MasterPure Complete DNA and RNA purification kit
(EPICENTRE, Madison, WI, USA). The SSU rDNA
sequence was PCR amplified using puReTaq Ready-to-go
PCR beads (GE Healthcare, Quebec, Canada), with an
error rate of 1 per 20,000–40,000 bases, and universal
eukaryotic primers (PF1-R4, as reported previously in
Hoppenrath et al. 2009b). The PCR product of the expected
size was gel isolated and cloned into pCR2.1 vector using a
TOPO TA cloning kit (Invitrogen Corporation, CA, USA).
One clone was completely sequenced with ABI big-dye
reaction mix using both vector primers and two internal
primers oriented in both directions (acc. no. AB639343).
The sequence identity was evaluated initially by BLAST
and then by phylogenetic analyses.
The new SSU rDNA sequence was aligned with other
dinoflagellate/alveolate sequences using MacClade 4 (Mad-
dison & Maddison 2000), forming a 63-taxon alignment.
The alignment is available on request. To find the model of
evolution that fits the data set for the maximum likelihood
Figs 17–20. Scanning electron micrographs of Amphidiniopsis rotundata sp. nov. (stub CEDiT2011H11 stored at the Centre of Excellence forDinophyte Taxonomy, Wilhelmshaven, Germany).
Fig. 17. Apical view of all epithecal plates. Scale bar 5 10 mm.Fig. 18. Dorsal view of the epitheca. Scale bar 5 10 mm.Fig. 19. Outside view of the apical pore complex with ventral platelet (asterisk). Scale bar 5 1 mm.Fig. 20. Inside view of the apical pore complex with ventral platelet (asterisk) and opening of the apical pore. Scale bar 5 1 mm.
160 Phycologia, Vol. 51 (2), 2012
(ML) analysis, we used the FindModel server (http://www.
4 APC 4 3 7 x 6(7) ? 5 2This study APC 4 3 7 6(7) 4? 4 2
H. litoralis5 APC 3 2 6 x 7 3 6 1 16 APC 4 3 7 x ? 3? 6 26 APC 4 3 7 ? 5–6? 5 2This study APC 4 3 7 5? 4–5? 5 2
1 New interp., our new interpretation of the tabulation (of the images provided in the original description).2 Same tabulation as in the original description but with different interpretation in the sulcal area (see Fig. 26).3 Hoppenrath 2000c.4 Hoppenrath et al. 2004.5 Hoppenrath 2000b.6 Yamaguchi et al. 2011.
Hoppenrath et al.: Amphidiniopsis rotundata sp. nov. 161
of 15 plates (Figs 9–14, 17, 18, 21). The oval to droplike
APC was centrally located and surrounded by a flange/list
(Figs 14, 17–20). The apical pore was a tiny hole in the
centre of a slightly raised, nearly circular central area
(Figs 17, 18), and was hidden behind a cover layer (Figs 14,
19). The apical pore was open to the inside of the apical
pore plate (Fig. 20). A ventrally located darker round area
reminiscent of a huge pore at low magnification (Fig. 14)
transpired to be a deeper-set platelet (Figs 17, 19, 20). Thus,
as described in other dinoflagellate species, the APC was
composed of two platelets, the pore plate (Po) and a small
canal plate (X). The APC was encircled by four apical
plates (Figs 17, 21), with plate 19 in contact with the sulcus
(Figs 9–11, 21) and sometimes disconnected from the canal
plate (ventral platelet) of the APC (Fig. 19). There were
three anterior intercalary plates (syn. apical intercalary
plate, epithecal intercalary plate) in a series (Figs 14, 17, 18,
21). The plate border between 2a and 3a was variable in
length (Figs 12–14, 17, 18). There were seven precingular
plates (Figs 9–14, 17, 18, 21). The slightly ascending
cingulum consisted of five smooth plates (Figs 9–13, 18,
21), but the sutures were difficult to discern. The deepened
part of the sulcus was shifted markedly to the left cell side
(Figs 9–11), and the complete sulcus was covering most of
the left ventral hypotheca with its right margin in about
mid-ventral cell position. Five sulcal plates with ornamen-
tation, as described above, were identified (Figs 9–11, 15,
16, 21). The right anterior sulcal plate (Sad) was seven
sided, relatively small and slightly elongated (Figs 10, 16).
The left anterior sulcal plate (Sas) was difficult to recognize
in most cells because it was partly hidden behind a list
formed by Sad; however, Sas can be clearly seen in cells
with rudimentarily developed sulcal lists (Fig. 16). The
broad right sulcal plate (Sd) was positioned in the middle of
the sulcus, and the left margin of this plate was
characteristically curved with a large smooth list (Figs 9–
11, 21). The left sulcal plate (Ss) was elongated and
characteristically curved (Figs 9–11, 16, 21) with a list along
part of its left margin formed by the first postcingular plate.
r
Fig. 22. Maximum-likelihood tree of dinoflagellates inferred from SSU rDNA sequences. The grey box indicates the Amphidiniopsis clade.ML bootstrap values (ML) over 50 and Bayesian posterior probabilities (PP) over 0.90 are shown at the nodes (ML/PP). The scale barrepresents inferred evolutionary distance in changes/site. The DNA sequence generated in the study is indicated by a black box.
Hoppenrath et al.: Amphidiniopsis rotundata sp. nov. 163
The posterior sulcal plate (Sp) reached the antapex (Figs 9–
11, 15) and formed a marginal list at the border to the Ss
plate that could reach a prominent size and spinelike shape
(Figs 9–11, 16). A mid-sulcal plate (Sm) could not be
identified unambiguously because this plate was positioned
near the centre of the sulcus behind the Sd plate; however, a
tiny left part of Sm was probably recorded in Fig. 16. The
flagella pore(s) were obscured from view. The hypotheca
consisted of seven plates: five postcingular and two
antapical plates (Figs 9–13, 16, 21). The fifth (5-)
postcingular plate was large and covered most of the right
ventral side of the hypotheca (Figs 9–11, 21). The medium-
sized, asymmetrical, third postcingular plate (3-) was
pentagonal and only slightly pointed into a right posterior
direction (Figs 12, 13, 21). The two antapical plates were
asymmetrical (Figs 12, 13, 15, 21).
Occurrence
The new species was recorded at Centennial Beach,
Boundary Bay, BC, Canada: 16 March, 26 April, 9 May,
6 June, 12 July, 4 August, 14 October 2005; 31 May 2006;
17 and 31 May, 18 June, 31 July, and 28 August 2007. It has
been seen in a sample from Parksville, Vancouver Island,
Canada on 18 September 2005. In most samples A.
rotundata occurred in very low cell densities. No sampling
took place during the winter months in Canada.
The new species was also recorded in Vostok Bay, Sea of
Japan, Russia: 20 August 2006; 28 May, 27 June, 12 July,
21 August, 4 and 22 September and 12 December 2007.
Cells in a sand sample from the island of Sylt (List
Harbor), German Bight, North Sea taken on 27 March
2009 and in a sand sample from the island of Wangerooge,
German Bight, North Sea taken on 22 April 2010 were also
identified at the light microscope level as A. rotundata.
Molecular phylogeny
The phylogenetic analyses of the 63-taxon alignment of
SSU rDNA sequence placed A. rotundata within a clade of
the Peridiniales together with species of Protoperidinium
and Diplopsalis group. Subclade IV was strongly supported
and consisted of A. rotundata, T. dragescoi, Archaeperidi-
nium minutum, and H. litoralis (Fig. 22). The closest sister
lineage to Amphidiniopsis rotundata was T. dragescoi with
moderate to high support values (ML bootstrap: 73%;
FURTHER REFERENCE: Hoppenrath 2000, p. 101, figs 9, 10,
23–25, 61–69, as Thecadinium dragescoi.
EXCLUDED REFERENCE: Thecadinium dragescoi sensu Paul-
mier 1992, p. 60, pl. 70, figs 5, 6.
ACKNOWLEDGEMENTS
We thank Drs N. Chomerat, IFREMER Concarneau,
and A. Coute, Museum National d’Histoire Naturelle
Paris, France, for the translation of the description into
Latin. This work was partly supported by a scholarship to
MH from the Deutsche Forschungsgemeinschaft (grant
Ho3267/1-1), postdoctoral research salary to MH and AY
from the Assembling the Tree of Life grant (NSF #EF-
0629624) and operating funds to BL from the National
Science and Engineering Research Council of Canada
(NSERC 283091-09) and the Canadian Institute for
Advanced Research, Programs in Evolutionary Biology
and Integrated Microbial Biodiversity. MS got grants
from Russian Government No. 11.G34.31.0010 and FEB
RAS 09-III-A-06-211.
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Received 23 March 2011; accepted 25 July 2011
Associate editor: Stuart Sym
Hoppenrath et al.: Amphidiniopsis rotundata sp. nov. 167