Research Article |
Corresponding author: Anton Glushchenko ( gluschenkoam@studklg.ru ) Academic editor: Kalina Manoylov
© 2021 Anton Glushchenko, Evgeniy Gusev, Yevhen Maltsev, John Patrick Kociolek, Irina Kuznetsova, Maxim Kulikovskiy.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Glushchenko A, Gusev E, Maltsev Y, Kociolek JP, Kuznetsova I, Kulikovskiy M (2021) Cymbopleura natellia – a new species from Transbaikal area (Russia, Siberia) described on the basis of molecular and morphological investigation. PhytoKeys 183: 95-105. https://doi.org/10.3897/phytokeys.183.72285
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A new cymbelloid diatom species from the genus Cymbopleura (Krammer) Krammer is described on the basis of molecular and morphological investigations. Cymbopleura natellia Glushchenko, Kulikovskiy & Kociolek, sp. nov. is, on the basis of results with molecular data, close to C. naviculiformis (Auerswald ex Heiberg) Krammer. The two species differ both by molecular distance and morphological features. Morphologically, C. natellia sp. nov. is compared with several other species in the genus. This work is a pioneer investigation of cymbelloid taxa using molecular tool from Transbaikal area.
Bacillariophyceae, Cymbopleura, morphology, molecular investigation, new species, Russia
Cymbelloid diatoms have unusually high diversity in ancient Lake Baikal but we almost have no information about them in waterbodies surrounding this lake. Historically, cymbelloid diatoms have been referred to taxa with dorsiventral symmetry (
Members of the genus Cymbopleura were previously considered to be members of the genus Cymbella. Cymbopleura was separated from Cymbella based on the features of absence of apical pore fields and central raphe ends turned towards the ventral side of valve (
The aim of the present report is to begin a series of studies using molecular methods to study the identity and systematic placement of cymbelloid diatoms from Lake Baikal and surrounding watrebodies, with the description of new species from the genus Cymbopleura. We plan that this work as our pioneer investigation of dorsiventral species from this area and that this work will be continued in future with new molecular data for other known taxa in this lineage.
The sample used in the present report was collected from Eastern Siberia, Buryatia by E.S. Gusev and M.S. Kulikovskiy on 15.07.2011. It was designated as No. 11.2 and was collected from the Zagza River, periphyton, scraping from macrophytes, t = 14 °C, pH = 8.5, conductivity = 40 μS cm-1, 52°31.656'N, 107°05.114'E.
A subsample of each collection was added to WC liquid medium (
The culture was treated with 10% hydrochloric acid to remove carbonates and washed several times with deionized water for 12 h. Afterwards, the sample was boiled in concentrated hydrogen peroxide (≈ 37%) to remove organic matter. It was washed again with deionized water four times at 12 h intervals. After decanting and filling with deionized water up to 100 ml, the suspension was pipetted onto coverslips and left to dry at room temperature. Permanent diatom preparations were mounted in Naphrax. Light microscopic (LM) observations were performed with a Zeiss Axio Scope A1 microscope equipped with an oil immersion objective (× 100, n.a. 1.4, differential interference contrast [DIC]) and Axiocam ERc 5s camera (Zeiss). Valve ultrastructure was examined by means of scanning electron microscopes JEOL JSM-6510LV (JEOL Ltd., Japan) operating at 15 kV and 8 mm of working distance (
The cleaned material, sample and slide are deposited in the collection of MHA, Main Botanical Garden Russian Academy of Science, Moscow, Russia. The type slide was designated B209.
Total DNA from the studied strain was extracted using Chelex 100 Chelating Resin, molecular biology grade (Bio-Rad Laboratories, USA), according to the manufacturer’s protocol 2.2. Partial 18S rDNA (435 bp, including the highly variable V4 region) gene was amplified using primers D512for and D978rev from
Amplification was carried out using premade polymerase chain reaction (PCR) mastermixes (ScreenMix by Evrogen, Russia). Amplification conditions for 18S rDNA gene were as follows: initial denaturation for 5 min at 95 °C followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 52 °C, and 50 s extension at 72 °C, with the final extension for 10 min at 72 °C. PCR products were visualized by horizontal electrophoresis in 1.0% agarose gel stained with SYBRTM Safe (Life Technologies, USA). The products were purified with a mixture of FastAP, 10×FastAP Buffer, Exonuclease I (Thermo Fisher Scientific, USA), and water. The sequencing was performed using a Genetic Analyzer 3500 instrument (Applied Biosystems, USA).
Editing and assembling of the consensus sequences were carried out by processing the direct and reverse chromatograms in Ridom TraceEdit (ver. 1.1.0) and Mega7 software (
The nucleotide sequences of the 18S rDNA gene were aligned separately using the Mafft v7 software and the E-INS-i model (
The data set was analyzed using the Bayesian inference (BI) method implemented in Beast ver. 1.10.1 software (
Slide no B209 in collection of MHA, Main Botanical Garden Russian Academy of Science, Moscow, Russia, represented here by Fig.
Strain B209, isolated in sample No. 11.2.
Russia, Eastern Siberia, Buryatia, Zagza River, 52°31.656'N, 107°05.114'E.
LM
(Figs
SEM, external view
(Fig.
SEM, internal view
(Fig.
New species is dedicated to our friend Natella Otarovna Gabuadze.
As yet known only from the type locality.
Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number MZ503642).
The phylogenetic analyses were conducted using a single gene dataset (Fig.
Phylogenetic position of Cymbopleura natellia B209 (indicated in bold) based on Bayesian inference for the partial 18S rDNA gene. Total length of the alignment is 439 characters. Bootstrap supports from ML (constructed by RAxML) and posterior probabilities from BI (constructed by Beast) are presented on the nodes in order. Only BS and PP above 50 and 0.85 are shown. Strain numbers (if available) and GenBank numbers are indicated for all sequences.
According to the data of molecular analysis, the species Cymbopleura natellia sp. nov. is most closely related to C. naviculiformis. According to morphological data, the new species is distinguished by bluntly rostrate, not cuneate valve ends, whereas C. naviculiformis has rostrate to subcapitate, narrow-protracted valve ends. The central area of the new species is generally smaller (1/3 of the valve width) than the central area of C. naviculiformis (1/2–2/3 of the valve width). The new species differs from C. naviculiformis in a shorter valve length (17.6–23.5 µm in the new species versus 26–50 µm in C. naviculiformis), as well as, in general, a smaller valve width (8.7–9.5 µm in the new species versus 9.0–13.0 µm in C. naviculiformis). The density of striae overlaps in species (13–15 in 10 μm in the central part, 18–20 towards the ends in the new species versus 10–15 in 10 μm in the central part, 16–19 in 10 μm towards the ends in C. naviculiformis) and areolae (30–35 in 10 μm in the new species versus 27–35 in C. naviculiformis) (Table
Outline | Ends | Axial area | Central area | Valve length (μm) | Valve width (μm) | Striae in 10 μm | Areolae in 10 μm | Distribution | Reference | |
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C. natellia sp. nov. | subelliptical, dorsiventral with moderately convex dorsal margin and slightly convex ventral margin, which more often almost straight near the valve centre | bluntly rostrate, not protracted | narrow, more often weakly expands to central area, less often – almost not expands, almost the median line of the valve | more or less pronounced, rounded, 1/3 the valve breadth | 17.6–23.5 | 8.7–9.5 | 13–15 at the central part, 18–20 near the ends | 30–35 | Russia: Baikal Lake | This study |
C. naviculiformis | elliptic-lanceolate, moderately dorsiventral, dorsal margin strongly arched, ventral margin slightly convex to nearly flat | rostrate to subcapitate, narrow-protracted | narrow, linear or slightly broader towards the relatively large central area, almost in the median line of the valve | rounded, one-half to two-thirds valve width | 26–50 | 9.0–13.0 | 10–15 at the central part, 16–19 near the ends | 27–35 | Widely distributed |
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C. designata | broadly lanceolate, moderately dorsiventral with strongly arched dorsal and ventral margins | apiculate, extending laterally from valve ends | area about 1/4th to 1/5th valve width, slightly curved and narrower near apices | rounded, scarcely wider than the axial area | 26–37 | 9.0–11.6 | 12–14 at the central part, up to 18 near the ends | 26–30 | Sub-Arctic and Arctic |
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C. frequens | not or very slightly dorsiventral, subelliptical-lanceolate, dorsal an ventral margins moderately arched | protracted, apiculate to rostrate | narrow, linear, narrowing slightly towards to the ends, almost median line of the valve | irregular, asymmetric space of different extent | 14–38 | 6–9 | 11–14 at the central part, 16 near the ends | 30–36 | Holarctic |
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C. hercynica | slightly dorsiventral, broadly subelliptical to elliptical-lanceolate, dorsal margins strongly, ventral margin slightly arched | apiculate to subrostrate protracted | very narrow, almost the median line of the valve | 1/3–1/2 the valve breadth, distinctly set off, rounded, sometimes asymmetrical and then more well-developed dorsally | 16–40 | 7–10 | 12–15 at the central part, up to 20 near the ends | 32–36 | Holarctic, saline habitats or with higher electrolyte content |
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Cymbopleura natellia sp. nov. is morphologically similar to Cymbopleura designata (Krammer) Bahls (in
Cymbopleura natellia sp. nov. is also morphologically similar to C. frequens
Cymbopleura hercynica (A.W.F. Schmidt)
Publication is based on research carried out with financial support by Russian President Foundation (МД-1446.2020.4) for molecular investigation and Russian Science Foundation (21-74-00097) for LM and SEM.