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Protistology 13 (4), 215-235 (2019)
Protistology
Diatom assemblages of the brackish Bolshaya Samoroda River (Russia) studied via light microscopy and DNA metabarcoding
Elena A. Selivanova, Marina E. Ignatenko, Tatyana N. Yatsenko-Stepanova and Andrey O. Plotnikov
Institute for Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences, Orenburg 460000, Russia
| Submitted October 15, 2019 | Accepted December 10, 2019 | Summary
Diatoms are highly diverse and widely spread aquatic photosynthetic protists. Studies of regional patterns of diatom diversity are substantial for understanding taxonomy and biogeography of diatoms, as well as for ecological perspectives and applied purposes. DNA barcoding is a modern approach, which can resolve many problems of diatoms identification and can provide valuable information about their diversity in different ecosystems. However, only few studies focused on diatom assemblages of brackish rivers and none of them applied the genetic tools. Herein, we analyzed taxonomic composition and abundance of diatom assemblages in the brackish mixohaline Bolshaya Samoroda River flowing into the Elton Lake (Volgograd region, Russia) using light microscopy and high-throughput sequencing of the V4 region of the 18S rDNA gene amplicons. In total, light microscopy of the samples taken in 2011-2014 and 2018 allowed to distinguish 39 diatom genera, represented by 76 species and infraspecies taxa. Twenty three species of diatoms were recorded in the river for the first time. Next-generation sequencing revealed a larger number of diatom taxa (26 genera and 47 OTUs in two samples vs. 20 genera and 37 species estimated by light microscopy). As a result, sequences of Haslea, Fistulifera, Gedaniella were recorded in the river for the first time. Significant differences in the data obtained with molecular and light microscopy approaches are discussed. Some V4 18S rDNA sequences were characterized by a low similarity with homologues from the reference database. We revealed high spatial-temporal heterogeneity of the diatom assemblages, occurrence of freshwater species together with brackish and marine ones, and predominance of benthic and plankto-benthic species. Thus, investigations of diatoms in brackish rivers based on both morphological and molecular approaches provide a good chance of improving an understanding of diversity, ecology and biogeography of Bacillariophyta.
Key words: Bacillariophyta, brackish river, diatoms, diversity, metabarcoding, NGS, 18S rDNA
doi:10.21685/1680-0826-2019-13-4-5 © 2019 The Author(s)
Protistology © 2019 Protozoological Society Affiliated with RAS
Introduction
Diatoms (Bacillariophyta) are numerous, highly diverse and ubiquitous photosynthetic protists. The number of diatom species varies from 20,000 to 200,000 (Yi et al., 2017). Bacillariophyta inhabit fresh, brackish and saline inland water bodies, seas and oceans, soils, and wet substrates (Guo et al., 2015). They serve as the base of food webs in the water ecosystems and are responsible for most part of primary production in reservoirs of various types (Siqueiros-Beltrones et al., 2017; An et al., 2018). In addition, diatoms are considered to be environmentally and economically significant microorganisms (Pniewski et al., 2010).
Species diversity of Bacillariophyta is greatly influenced by environmental conditions. For this reason, diatoms are used as suitable bioindicators in ecological studies and water monitoring assessments (Barinova et al., 2006; Zimmermann et al., 2011, 2015; Pinseel et al., 2019). Diatoms are widely used in paleoecological reconstruction, forensic science, as well as oil and gas exploration, due to long-term preservation of their siliceous frustules in marine, lake and peat sediments (Bertrand, 2010; Kulikovskiy, 2016; Pinseel et al., 2019). Many diatom species produce carotenoids and polyunsaturated fatty acids, therefore, they are promising objects for biotechnology (Bertrand, 2010; Shishlyannikov et al., 2014; Petrushkina et al., 2017; Yi et al., 2017).
Diatoms attract an increased interest of researchers (Krivosheia and Vlasiuk, 2016; Siqueiros-Bel-trones et al., 2017; An et al., 2018; Komulaynen, 2018). A large number of new species and genera of diatoms have been described during the last 30 years (Kulikovskiy, 2016). Diatoms distribution studied with molecular-based methods in different regions of the world demonstrates that many diatom genera and species, which were considered ubiquitous previously, consist of a number of cryptic or pseudo-cryptic species (Stepanek and Kociolek, 2014; Pinseel et al., 2019). There is a need for studies of regional diatom diversity and compilation of species lists including rare and endemic taxa, to develop a better understanding of taxonomy and biogeography of diatoms and to ensure the use of this knowledge for applied purposes including environmental management. Nevertheless, diversity of diatoms has not yet been studied in many regions of Russia and only few reports on the floristic di-
versity and distribution of Bacillariophyta contain microphotographs and genetic data (Kulikovskiy, 2016).
The hypersaline lake Elton with the inflowing saline rivers is one ofthe most unique natural aquatic systems of Russia (Kalyuzhnaya, 2007; Kalyuzh-naya et al., 2011). The Elton Nature Park including the Elton Lake with saline rivers was created in 2001 to preserve the unique saline ecosystems. In 2019, the Elton Nature Park was added to the World Network of Biosphere Reserves by UNESCO's Man and the Biosphere (MAB) programme. Unlike other brackish and saline habitats, rivers with elevated salinity are scarce on the Earth. Some of them are characterized by a wide salinity gradient and a variable hydrological regime both serving as the structure-forming factors for communities of the saline rivers (Zinchenko et al., 2017). Since 2006, researchers have been studying the ecological status and biological diversity of the saline rivers in the Elton region (Zinchenko et al., 2010; Kalyuzhnaya et al., 2011; Nomokonova et al., 2013; Zinchenko and Golovatyuk, 2013; Yatsenko-Stepanova et al., 2015; Burkova, 2016; Gorokhova and Zinchenko, 2016; Gusakov, 2019). One of the longest Elton rivers, the brackish Bolshaya Samoroda River, contains rich and unique biota, and plays a crucial role in stabilizing the natural environment and forming a biodiversity hotspot (Shubin et al., 2000). All previous studies of algal diversity in the Elton region have been carried out using only morphology-based approaches without genetic tools (Yatsenko-Stepanova et al., 2015; Burkova, 2016; Gorokhova and Zinchenko, 2016). Besides, identifications of diatoms in the previous studies of the Elton rivers have not been supported by microphotographs. DNA barcoding is a modern approach, which can resolve many problems of diatoms' identification (Mann et al., 2010; Guo et al., 2015; Rivera et al., 2018). Molecular-based methods that use the techniques of next-generation sequencing (NGS) provide a much more comprehensive insight into the taxonomic diversity of diatoms in the environmental samples (Zimmermann et al., 2015). Therefore, in this study we aimed to characterize the taxonomic composition and abundance of diatom assemblages in the brackish mixohaline Bolshaya Samoroda River flowing into the Elton Lake, using light microscopy (LM) and high-throughput sequencing of the 18S rRNA gene amplicons.
Fig. 1. A — Scheme of Elton region with sampling sites marked with red dots. B, C — Photos of the Bolshaya Samoroda River (B — middle course, C — mouth).
Material and methods
Water sampling
The Bolshaya Samoroda River is located in the Elton Nature Park (Fig. 1). The river flows through a wide valley with gentle slopes. It has a meandering channel and slow current (less than 0.2 m/s). The total length of the river is 21-24 km; the catchment area is 130 km2. The channel is 6—35 m wide, and the depth is 0.1—0.7 m (Gusakov, 2019). The river is fed mainly by groundwater and precipitation (Brylev and Pryakhin, 2011; Burkova, 2015). The Bolshaya Samoroda River is mixohaline according to the Venice system (1958), with salinity ranging from 6.5 g/L in the middle course to 19 g/L in the mouth. A single observation of 118.8 g/L salinity in the mouth of this river as a result of a brine influx from the Elton Lake was recorded in May 2012. A wide range of salinity is formed due to salt and carbonates sedimentary rocks, salt marshes, and mineral springs in the floodplain terrace, including the Smorogdinsky mineral spring with sulfate-chloride-sodium water.
Water samples were taken in the middle course (49.208889°N, 46.941111°E) and in the river mo-
uth (49.283333°N, 47.036944°E) during vegetative seasons of 2011-2014 and 2018. Salinity was measured using a Master S-28a portable refrac-tometer (Atago, Japan).
Light microscopy observation
Water samples of 0.5 L were fixed with 4% formaldehyde immediately after sampling. Algae were concentrated by sedimentation method. Algal cells were counted in a Nageotte Counting Chamber (Assistent, Germany) at 400 * magnification. organic content of diatom cells was destroyed by the method of cold burning (Balonov, 1975). Then empty diatom frustules were embedded in the Canada balsam. Permanent slides were examined by phase contrast microscopy under an «Axioskop» microscope, equipped with 60* objective, 100* oil objective, and an «Axiocam» digital camera (Carl Zeiss, Germany). For diatom species identification the qualifiers Süßwasserflora von Mitteleuropa were used (Krammer and Lange-Bertalot, 1986, 1988, 1991a, 1991b). Taxonomy and nomenclature of Bacillariophyta is given according to the on-line AlgaeBase database https://www.algaebase.org/ (Guiry and Guiry, 2019).
Ecological analysis
Salinity and habitat preferences of the revealed diatom species were assessed according to Barinova et al. (2006). Thus, all species were referred to one of four salinity indicator groups: oligohalobes (0—5 g/L), mesohalobes (5—20 g/L), euhalobes (20—40 g/L), and polyhalobes (40-300 g/L). Oligohalobes included oligohalobes-halophobes (typically freshwater avoiding brackish waters), oligohalobes-indif-ferent (typically freshwater, sometimes found in slightly brackish waters), oligohalobes-halophiles (mostly freshwater, also common in brackish waters). Comparison of the species composition was performed using Sorensen similarity coefficient (Sorensen, 1948). The similarity coefficient is 1 when the compared species sets are completely identical; it decreases when their differences increase; it is 0 when the species sets are completely different. In terms of their occurrence, diatom species were referred to constant (more than 70%), additional (20-70%) and rare (less than 20%), according to Kosolapova, 2005.
DNA extraction
Water samples of 500 mL were taken and filtered through membranes with 0.45 pm pore size. Total genomic DNA was isolated from the filters by a combined method, including mechanical homogenization and chemical extraction (Liu et al., 2009) in modification of Bel'kova et al. (2008). A lysing matrix E (MP Biomedicals, USA) and 400 pL of Tris-salt buffer (20 mM EDTA, 750 mM NaCl, 100 mM Tris-HCl, pH 8.0) were added in every sample. The samples were homogenized in Tissue Lyser LT (QIAGEN, Germany) for 1 min at 50 Hz. Then 50 pL of a sterile lysis buffer with lysozyme (50 pg/mL) were added and the samples were incubated for 60 min at 37 °C, followed by addition of 10 pL proteinase K (10 mg/mL) and 10% sodium dodecyl sulfate up to 1% in a final volume. The mixtures were incubated for 60 min at 60 °C. After extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1), DNA in the aqueous phase was precipitated overnight at -20 °C with threefold volume of anhydrous ethanol and 10 M ammonium acetate added up to 10% of a final volume. After centrifuging and double washing with 80% ethanol, DNA was dried on air and dissolved in autoclaved MQ water. To assess contamination during DNA extracting, a negative control containing 100 pL of autoclaved MQ water
was subjected to the same procedure. The quality of extracted DNA was checked with electrophoresis in 1.5% agarose gel. The DNA concentration was quantified using Qubit 2.0 Fluorometer (Life Technologies, USA) with dsDNA High Sensitivity Assay (Life Technologies, USA).
Preparing of DNA libraries and NGS
DNA libraries were prepared according to the Illumina workflow (Illumina protocol, part no. 15044223, Rev. B) (https://support.illumina.com/ documents/documentation/chemistry_documen-tation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). DNA amplification was performed using primers targeting the hypervariable V4 region of the 18S rRNA gene: forward TAReuk454FWD1 and reverse TAReukRev3 (Stoeck et al., 2010), producing amplicon with length about 500 bp. The polymerase chain reaction (PCR) mixture of volume 30 pL contained 0.25 mM of each primer, 0.125 mM of dNTP, PCR buffer and 0.15 U of Q5 DNA polymerase (New England Biolabs, Ipswich, MA, USA). Amplification was performed according to a PCR protocol applied by Stoeck et al. (2010). Size of the obtained amplicons was verified using electrophoresis in 1% agarose gel. Neither procedures to reduce artificial dominance of some PCR products nor mock communities were used. The following steps of the DNA-library preparation were carried out in full accordance with the Illumina workflow (Illumina protocol, part no. 15044223, Rev. B) and included clean-up ofthe amplicons obtained, index PCR, clean-up ofthe DNA libraries obtained, their quantification, normalization and pooling. Cleanup of amplicons and indexed DNA-libraries was performed with Agencourt AMPure XP magnetic beads (Beckman Coulter, USA). Index PCR with amplicons was carried out according to the Illumina protocol (part no. 15044223, Rev. B, p. 10-12) using dual indices from Nextera XT Index kit (Illumina, USA) and Q5 DNA polymerase (New England Biolabs, Ipswich, MA, USA). DNA libraries were quantified using Qubit 2.0 Fluorometer (Life Technologies, USA) with dsDNA High Sensitivity Assay (Life Technologies, USA). The DNA libraries were normalized by dilution up to 10 nM and pooled. The concentrated pooled library was diluted finally to 4 nM.
Sequencing was performed on a MiSeq sequencer (Illumina, USA) using MiSeq Reagent Kit v3 (600 cycle) (Illumina, USA) for paired-end sequencing 2x300 bp in the Center of Shared Scientific Equipment "Persistence of Microorganisms" of the
Fig. 2. Absolute and relative abundances of Bacillariophyta in the Bolshaya Samoroda River. A — middle course, B — mouth.
Institute for Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences.
deposited to the GenBank (NCBI) under accession numbers MK626723 - MK626753 and MK656291 - MK656308.
Bloinformatic analysis
Bioinformatic analyses were conducted using several tools. Paired-end reads were merged with PEAR v.0.9.10 (Zhang et al., 2014). Evaluation of the filtering quality was carried out with FastQC v.0.11.3. Quality filtering and amplicon size selection (350 bp minimal size) were conducted using USEARCH 10.0.240_i86linux32 (Edgar, 2013). During the filtering reads with Ns or an overall mean, Q-score <15 were discarded. As a result of dereplication and clustering with USEARCH, operational taxonomic units (OTUs) were formed at 97% level of similarity, while singletons were removed. The most common sequence was selected as representative in each OTU. Each OTU was formed by 2 to 18,295 reads. The similarity of reads with the most common sequence was in the range of 97-100%, but more than 90% of sequences had similarity of 99.5-100%. Chimera detection and removal was conducted via UCHIME (Edgar et al., 2011) using USEARCH 10.0.240_i86linux32.
For taxonomic classification all OTUs that belonged to diatoms were aligned using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the nr/nt database ofnucleotide sequences of the National Center for Biotechnology Information (NCBI). The OTUs obtained in this study were
Results and discussion
Light microscopy data
The phytoplankton of the Bolshaya Samoroda River is composed of the phyla Bacillariophyta, Chlorophyta, Euglenophyta, Cryptohpyta, Dino-phyta, and Chrysophyta. Diatoms were present in all samples unlike other algal phyla. Relative abundances of Bacillariophyta in the algal communities ranged greatly from 0.1 to 99.8%, and their absolute abundances varied from 8*103 to 3.7* 107 cells/L (Fig. 2). Richness of diatom species and infraspecies taxa ranged from 4 to 30 per sample which drastically exceeded richness of other algal phyla. Similar high levels of diatoms relative abundance and diversity in the phytoplankton have been previously reported in many other inland brackish and saline lakes (Naumenko, 2001; Ovchinnikov et al., 2015; Makeeva and Naumenko, 2016), as well as lagoons (Siqueiros-Beltrones et al., 2017), bays (An et al., 2018), and estuaries (Begyn, 2017).
In this study, a total of 76 diatom species and infraspecies taxa belonging to 39 genera, 26 families, 14 orders, 4 subclasses, and 2 classes, were revealed by light microscopy in the middle course and in the mouth of the Bolshaya Samoroda River
Table 1. List of Bacillariophyta species and infraspecies taxa found in the Bolshaya Samoroda River via light microscopy.
to to o
Site (m. - mouth; m.c. - middle course) m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. s.t. G.e.
Month V V VIII VIII V V VIII VIII VIII VIII VIII VIII VIII VIII
Year 11 11 11 11 12 12 12 12 13 13 14 14 18 18
Salinity, g/L 6.5 19 9.6 8.9 8.4 119 11.5 14.3 13 16 10 19 10 14
Bacillariophyceae, Bacillariophycidae
Mastogloiales, Mastogloiaceae
1 Mastogloia pumila (Grunow) Cleve* + + mh m
Mastogloiales, Achnanthaceae
2 Achnanthes armillaris (O.F. Muller) Guiry + + + hi m
3 A. brevipes C. Agardh var. brevipes + + + + + + hi
4 A. brevipes var. intermedia (Kutzing) Cleve + + + + + + mh b
5 A. pan/ula Kutzing* + hi m/f
6 Platessa salinarum (Grunow) Lange-Bertalot + + + + mh f
Cocconeidales, Achnanthidiaceae
7 Planothidium delicatulum (Kutzing) Round & Bukhtiyarova + hi u
Cocconeidales, Cocconeidaceae
8 Cocconeis placentula Ehrenberg + + + i f
9 Cocconeis lineata Ehrenberg + + i f
Cymbellales, Anomoeoneidaceae
10 Anomoeoneis sphaerophora (Ehrenberg) Pfitzer + + + hi m/f
11 A. sphaerophora var. sculpta (Ehrenberg) 0. Muller* + + mh f
Cymbellales, Cymbellaceae
12 Cymbella sp. + -
Cymbellales, Gomphonemataceae
13 Encyortema silesiacum (Bleisch) D.G. Mann* + i f
14 Gomphonema pan/ulum (Kutzing) Kutzing + + i f
Cymbellales, Rhoicospheniaceae
15 Rhoicosphenia abbreviata (C. Agardh) Lange-Bertalot + + i f
Bacillariales, Bacillariaceae
16 Bacillaria paxillifera (O.F. Muller) T. Marsson + + mh b
17 Cylindrotheca closterium (Ehrenberg) Reimann & J.C. Lewin + + mh m
m
o* 3
ia
o §
§ ia
ia
tn
№3
P 8.
o g-
o
o P3
Site (m. - mouth; m.c. - middle course) m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. S.t. G.e.
Month V V VIII VIII V V VIII VIII VIII VIII VIII VIII VIII VIII
Year 11 11 11 11 12 12 12 12 13 13 14 14 18 18
Salinity, g/L 6.5 19 9.6 8.9 8.4 119 11.5 14.3 13 16 10 19 10 14
18 Nitzschia acicularis (Kutzing) W. Smith + + + i m/f
19 N. communis Rabenhorst + + + + + + + i f
20 N. obtusa W. Smith* + + mh m
21 N. reversa W. Smith* + hl f
22 N. scalpelliformis Grunow* + hl b
23 N. sigma (Kutzing) W. Smith* + mh b
24 Tryblionella apiculata W. Gregory + + + + + mh m
25 T. compressa (Bailey) Poulin + + + mh m
26 T. hungarica (Grunow) Frenguelli + + + + + + mh b
27 T. hantzschiana Grunow* + i m
Naviculales, Naviculineae, Naviculaceae
28 Caloñéis amphisbaena (Bory) Cleve* + hl f
29 Gyrosigma acuminatum (Kützing) Rabenhorst + i f
30 Gyrosigma sp. + -
31 Hippodonta capitata (Ehrenberg) Lange-Bertalot, Metzeltin & Witkowski + + + ¡ f
32 Navícula capitatoradiata Germain + + i b
33 N. cryptocephala Kützing + - m/f
34 N. radiosa Kützing + + + + i f
35 N. subrhynchocephala Hustedt + i f
36 N. veneta Kützing + + + hl b
37 Navícula sp. + + + + + + + -
Naviculales, Naviculineae, Pleurosigmataceae
38 Pleurosigma elongatum W. Smith* + + + + + mh m
39 Pleurosigma sp. + -
Naviculales, Naviculineae, Stauroneidaceae
40 Stauroneis anceps Ehrenberg + i f
41 Stauroneis sp. + -
to to to
Site (m. - mouth; m.c. - middle course) m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. S.t. G.e.
Month V V VIII VIII V V VIII VIII VIII VIII VIII VIII VIII VIII
Year 11 11 11 11 12 12 12 12 13 13 14 14 18 18
Salinity, g/L 6.5 19 9.6 8.9 8.4 119 11.5 14.3 13 16 10 19 10 14
Naviculales, Neidiineae, Amphipleuraceae
42 Halamphora coffeiformis (C.Agardh) Mereschkowsky + + + + + + + + + + mh b
43 H. holsatica (Hustedt) Levkov + + mh b
Naviculales, Sellaphorineae, Sellaphoraceae
44 Fallacia pygmaea (Kützing) Stickle & D.G. Mann + + + + + + + mh f
Naviculales, Sellaphorineae, Pinnulariaceae
45 Pinnularia viridis (Nitzsch) Ehrenberg + i f
Rhopalodiales, Rhopalodiaceae
46 Epithemia adnata (Kützing) Brebisson* + i f
47 E. operculata (C. Agardh) Ruck & Nakov* + - f
48 Rhopalodia gibberula (Ehrenberg) 0. Müller* + mh m/f
49 R. musculus (Kützing) 0. Müller * + + mh b
Surirellales, Entomoneidaceae
50 Entomoneis paludosa var. subsalina (Cleve) Krammer* + + hi m
Surirellales, Surirellaceae
51 Campylodiscus clypeus (Ehrenberg) Ehrenberg ex Kutzing + + + mh m/f
52 C.bicostatus W. Smith ex Roper* + hi f
53 Surirella brebissonii Krammer and Lange-Bertalot + + i b
54 S. ovalis Brebisson* + + + mh m/f
55 S. striatula Turpin + + + + + + + + mh m
Thalassiophysales, Catenulaceae
56 Amphora commutata Grunow + + + + + mh b
57 A. libyca Ehrenberg* + + hi m
A. ovalis (Kutzing) Kutzing + i m/f
m
o* 3
ja
00
o §
ja
ja tri
№3 O
o
o P3
Site (m. - mouth; m.c. - middle course) m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. m.c. m. S.t. G.e.
Month V V VIII VIII V V VIII VIII VIII VIII VIII VIII VIII VIII
Year 11 11 11 11 12 12 12 12 13 13 14 14 18 18
Salinity, g/L 6.5 19 9.6 8.9 8.4 119 11.5 14.3 13 16 10 19 10 14
Bacillariophyceae, Fragilariophycidae
Fragilariales, Fragilariaceae
59 Fraguaría mesolepta Rabenhorst* + i f
60 F. crotonensis Kitton + hl m/f
61 Fragilaria sp. + -
Fragilariales, Staurosiraceae
62 Opephora mutabilis (Grunow) Sabbe & Wyverman + - m
63 Staurosira construens Ehrenberg + + + i f
Licmophorales, Ulnariaceae
64 Ctenophora pulchella (Ralfs ex Kützing) D.M.Williams & Round + + + + hl f
65 Tabularía fasclculata (C.Agardh) D.M. Williams & Round + + + + + + + + + + + hl m
Tabellariales, Tabellariaceae
66 Diatoma moniliformis (Kützing) D.M. Williams* + - f
67 D. vulgaris Bory* + i f
Mediophyceae, Chaetocerotophycidae
Chaetocerotales, Chaetocerotaceae
68 Chaetoceros mulleri Lemmermann + hl m/f
69 Chaetoceros sp. 1 + + -
70 Chaetoceros sp. 2 + -
Mediophyceae, Thalassiosirophycidae
Stephanodiscales, Stephanodiscaceae
71 Cyclostephanos dubius (Hustedt) Round + + - f
72 Cyclotella distinguenda Hustedt* + + hl f
73 Cyclotella meneghiniana Kützing + + + + + + + + + + + hl m/f
5
«
O
M
O
ö
£
to to
OJ
c o U
A (D I-
G.e. Thalassiosirales, Thalassiosiraceae
S.t.
E VIII CO i—i i—i CO i—i
m.c. VIII CO i—i o i—i o ro
E VIII i—i c^ 1—1
m.c. VIII i—i o 1—1 <N
E VIII ro 1—1 UD i—1 + O <N
m.c. VIII ro 1—1 ro 1—1 + O <N
E VIII <N i—1 14.3 + i—1 <N
m.c. VIII <N i—1 11.5 LO
E > <N i—1 i—i i—i + <N i—1
m.c. > <N i—1 CO CO
E VIII i—1 i—1 c^ CO i—1
m.c. VIII i—1 i—1 UD cK + UD
E > i—1 i—1 i—1 + C^ i—1
m.c. > i—1 i—1 LO UD + 4
Site (m. - mouth; m.c. - middle course) Month Year Salinity, g/L Cyclotella sp. Stephanodiscus sp. Thallasiosira sp. Species number
LO r-v r-v
(Table 1). The orders Naviculales (18 species), Bacillariales (12), and Surirellales (6 species and infraspecies taxa) were the most diverse. The most species-rich families were Bacillariaceae (12 species), Naviculaceae (10 species), Surirellaceae (5 species), Stephanodiscaceae (5 species), Ach-nanthaceae (5 species and infraspecies taxa), and Rhopalodiaceae (4 species). These families included 41 species and infraspecies taxa, which corresponded to 53.9% of the total Bacillariophyta species and infraspecies taxa. Most families comprised of 2-3 species only, whereas eight families were represented by only one species, such as Mastogloiaceae, Achnanthidiaceae, Cymbellaceae, Rhoicospheniaceae, Sellaphoraceaea, Pinnula-riaceaea, Entomoneidaceae, and Thalassiosiraceae.
Compared to the previously reported data (Yatsenko-Stepanova et al., 2015; Burkova, 2016; Gorokhova and Zinchenko, 2016) our study revealed 23 new species and infraspecies taxa of Bacil-lariophyta, which have never been recorded in the Bolshaya Samoroda River before, such as Surirella ovalis Brebisson, Mastogloia pumila (Grunow) Cleve, Epithemia adnata (Kutzing) Brebisson, Cam-pylodiscus bicostatus W. Smith, Diatoma moniliformis Kutzing, Entomoneis paludosa var. subsalina (Cleve) Krammer, etc. (Figs 3, 4; Table 1).
The analysis of diatoms occurrence showed that only three diatom species were estimated to be permanent in all samples studied, namely Halam-phora coffeiformis (C. Agardh) Mereschkowsky, Tabularia fasciculata (C. Agardh) D.M. Williams et e ® Round, Cyclotella meneghiniana Kutzing. Twenty
g r four species and infraspecies taxa (31.6%) were re-
^ « ferred to additional, whereas 49 species and infra-
™ | species taxa (64.5%) were revealed to be rare. Such
CT
g a variability of community composition may be rela-
Fig. 3. Light and phase-contrast microphotographs of diatom valves recorded in the Bolshaya Samoroda River in August 2014. A, B — Achnanthes brevipes var. intermedia, C — Amphora commutata, D — Gomphonema parvulum, E — Hippodonta capitata, F, I — Tabularía fasciculata, G — Cteno-phora pulchella, H — Rhoicosphenia abbreviata, J — Campylodiscus bicostatus, K — Cocconeis line-ata, L (a) — Platessa salinarum, L (b) — Cocconeis ü E placentula, M — Navicula radiosa, N — Halamphora
I Is coffeiformis, O — Rhopalodia gibberula, P — Cyc-
&"§ lotella meneghiniana, Q — Surirella ovalis, R —
Epithemia operculata, S — Tryblionella hungarica, T — Surirella striatula, U — Tryblionella hantzschiana, V — Tryblionella apiculata.
F -i= -n
u ro
o S fa jz
. E
(U
Q <
Fig. 4. Light and phase-contrast microphotographs of diatom valves newly recorded in the Bolshaya Samoroda River. A — Diatoma moniliformis, B — Epithemia adnata, C — Mastogloia pumila, D — Entomoneispaludosa var. subsalina.
ted to salinity fluctuations (Table 1) or instability of other hydrochemical parameters in the river (Zinchenko et al., 2017). Significant ecological plasticity of H. coffeiformis, T. fasciculata, and C. meneghiniana most likely determines their survival in a wide range of salinity proved by their presence in most samples. This observation is in a good agreement with other reports that noted these species to have cosmopolitan distribution at different salinities including both fresh and marine waters (Krammer and Lange-Bertalot, 1986, 1991a). H. coffeiformis and C. meneghiniana are considered typical inhabitants of freshwater bo-dies in the "Roztocze" International Biosphere Reserve, Ukraine (Krivosheia and Vlasiuk, 2016). C. meneghiniana was reported in the Lake Baikal (Genkal et al., 2013). H. coffeiformis and C. meneghiniana were registered in the Ubsu-Nur Lake (Tyva, Russia) at salinity of 18.7 g/L (Naumenko, 2001) and the Ulugkol Lake (Khakassia, Russia) at salinity range of 18.7-21.7 g/L (Makeeva and Naumenko, 2016). Litvinenko et al. (2013) indicated the constant presence of H. coffeiformis in the algal assemblages of meso- and hypersaline lakes in the south of Western Siberia, where salinity ranged from 28 to 417 g/L. T. fasciculata and H. coffeiformis dominated in the benthic samples of saline lakes in the Republic of Kalmykia (Russia), where salinity varied from 156 to 252 g/L (Ovchinnikov et al., 2015).
A comparison of the species lists in the middle course and the mouth of the river showed that 31 species and infraspecies taxa were common for both sampling points, which corresponded to 40.8% of
total diatom species revealed. Twenty two (28.9%) species and infraspecies taxa were specific for the middle course while 23 (30.3%) species were specific for the mouth ofthe river. Sorensen coefficients (SC) were rather low for diatom assemblages revealed in different sampling sites and periods oftime. The SCs varied between the diatom assemblages sampled in different years from 0 to 0.63 (mean — 0.23) in the middle course; and from 0.08 to 0.61 (mean — 0.34) in the mouth. The SCs between the diatoms sampled at the same time in the middle course and the mouth were also low ranging from 0.07 to 0.65 (mean — 0.24). Thus, the obtained results demonstrate high spatial-temporal heterogeneity and specificity of the diatom assemblages in the Bolshaya Samoroda River.
The analysis of the taxonomic composition of the diatom species lists in terms of salinity tolerance revealed the presence ofmesohalobes, oligohalobes-halophiles, oligohalobe-indifferent taxa, and the absence of oligohalobes-halophobes (Fig. 5, A). In terms of general environments from the Algaebase database (Fig. 5, B) the identified diatom taxa belonged to brackish, marine, freshwater, and ubiquitous groups. Interestingly, the proportion of freshwater taxa in the species lists was rather large reaching almost 50% in the middle river course (Fig. 5, B). These findings show high adaptive ability of many diatom taxa to the conditions of varying salinity. Unquestionably, in further studies traditional preferences of many diatom taxa to salinity should be revised. Proportion of oligohalobes-halophiles together with mesohalobes increased from 60% in the middle course to 74%
Fig. 5. Number of diatom species with different salinity preferences in the species lists according to Barinova et al., 2006 (A) and Algaebase (B).
in the river mouth vs. proportion of oligohalobe-indifferent taxa (Fig. 5, A). These data corresponded to a smaller proportion (1.6 times) of freshwater species in the mouth compared to the middle course of the river (Fig. 5, B). The observed shifts in the diatom species composition are in good agreement with elevated level of salinity in the river mouth vs. the middle course.
The analysis of the habitat preferences showed that most of the diatom species (34) were benthic, 22 species were plankto-benthic, 1 species was soil and plankto-benthic, and 5 species were planktonic. Probably, the predominance of benthic and plankto-benthic diatoms is determined by a shallow depth of the river. These observations are in good agreement with the similarity and close linkage of plankton and bottom communities ofinvertebrates described recently in the Elton saline rivers (Zinchenko et al., 2018).
NGS data and their comparison with LM data
Samples taken in August 2014 were analyzed by NGS and LM. A library from the mouth sample of the Bolshaya Samoroda River contained 1,541 V4 SSU rDNA assembled reads of diatoms that were equal to 15% of total microalgae reads. A library from the middle course sample included 22,691 reads (80.3% oftotal microalgae reads). The assembled reads had the length of410-446 bp and an average overlap of 182 bp. The relative abundances of diatoms estimated by LM were in similar ratio between the river mouth (0.1%) and the middle course (96.2%) samples.
The overall genetic diversity of diatoms found in our study was higher than morphological diversity. In total, 47 different OTUs referred to 26 genera were revealed in two samples in contrast to 37 species from 20 genera estimated using light microscopy. The genera Haslea, Fistulifera, and Gedaniella were detected in the Bolshaya Samoroda River by NGS for the first time. In addition, more OTUs that belonged to the genera Halamphora, Navicula, Nitzschia, and Cyclotella were found with NGS compared to the number of morphologically identified species. For example in the middle course sample only one representative of the genus Halamphora, H. coffeiformis, was recorded using LM (Table 2). In the same sample NGS revealed 3 OTUs that belonged to the Halamphora genus. The closest homologues of these OTUs in the GenBank database were the sequences deposited: Halamphora americana MG027295, Halamphora coffeiformis (deposited as coffeaeformis) KX257363, and Halamphora terroris KC222330 (Table 3). Besides, Nitzschia communis and Navicula radiosa were detected by LM vs. three Nitzschia and two Navicula OTUs recorded with NGS (Table 3). The reason of the observed differences may be a greater sensitivity ofmetabarcoding allowing detection of rare and not numerous species (Groendahl et al., 2017). Perhaps, our NGS data underestimate genetic diversity of diatoms, due to a low resolution of the 18S rDNA gene insufficient for species discrimination of diatoms (An et al., 2017). In our study many OTUs were aligned at high similarity level (more than 99.0%) to several closely related sequences belonging to different species, e.g. representatives
Table 2. Heatmap of diatom taxa revealed by LM (number of species) and NGS (number of OTUs) in plankton samples of the Bolshaya Samoroda River.
Class Order Family Genus Middle course Mouth
LM NGS LM NGS
Bacillariales Bacillariaceae Cylindrotheca 0 1 1 1
Nitzschia 1 3 1 1
Tryblionella 3 1 0 1
Cocconeidales Cocconeidaceae Cocconeis 2 0 0 0
UI Cocconeidaceae 0 1 0 0
Cymbellales Anomoeoneidaceae Anomoeoneis 0 0 1 0
UI Anomoeoneidaceae 0 0 0 1
Gomphonemataceae Encyonema 1 0 0 0
Gomphonema 1 1 0 0
Rhoicospheniaceae Rhoicosphenia 1 1 0 0
Mastogloiales Achnanthaceae Achnanthes 1 0 0 0
Platessa 1 0 0 0
Naviculales Naviculaceae Hippodonta 1 1 0 0
e CÖ e Navicula 1 2 1 0
> JZ Haslea 0 0 0 1
o lar UI Naviculaceae 0 0 0 1
'Ö a CÛ Pleurosigmataceae Pleurosigma 1 1 0 0
Stauroneidaceae UI Stauroneidaceae 0 0 0 1
Amphipleuraceae Halamphora 1 3 0 2
Stauroneidaceae Fistulifera 0 1 0 0
Rhopalodiales Rhopalodiaceae Rhopalodia 2 0 0 0
Surirellales Surirellaceae Campylodiscus 1 0 0 0
Surirella 2 2 1 1
UI Bacillariophycidae 0 2 0 0
Thalassiophysales Catenulaceae Amphora 1 1 0 1
Fragilariales Fragilariaceae Gedaniella 0 0 0 1
UI Fragilariophycidae 0 0 0 1
Licmophorales Ulnariaceae Ctenophora 1 0 0 0
Tabularia 1 1 0 0
UI Bacillariophyceae 0 1 0 0
Chaetocerotales Chaetocerotaceae Chaetoceros 0 0 0 3
ae aj c Stephanodiscales Stephanodiscaceae Cyclotella 1 2 1 1
JZ d o Thalassiosirales Thalassiosiraceae Thalassiosira 0 2 0 1
Tl (V Z UI Thalassiosirales 0 1 0 0
UI Mediophyceae 0 1 0 0
UI Bacillariophyta 0 2 0 0
Designation: UI - unidentified member of certain taxon.
of the genera Thalassiosira, Cyclotella, Fistulifera, Gomphonema, Nitzschia, Tabularia, Surirella, Navicula, Halamphora, Cylindrotheca, Gedaniella, and Chaetoceros (Table 3). That is why identification of most diatom OTUs was possible at the genus level only.
All OTUs of diatoms were represented by 2 classes, 13 orders, 16 families, and 18 genera. Thirteen OTUs could not be identified at the genus level.
Cylindrotheca, Nitzschia, Tryblionella, Halamphora, Surirella, Amphora, Cyclotella, and Thalassiosira were shared genera for both river mouth and middle course samples. Nevertheless, major part of diatom genera was specific for each sampling site (Table 2). In the middle course sample 31 OTUs were represented by 15 genera while 8 OTUs remained unidentified at the genus level. At the same time, in this sample 24 species belonging to 19 genera
Table 3. Closest homologues of 18S rRNA gene diatom sequences from the NCBI GenBank database.
OTU (accession no.) Identified as Closest homologue (accession no.) Similarity (%) Query Cover " (%)
ID-19-1 (MK626723) Thalassiosira sp. Thalassiosira weissflogii (HM991702) 99.76 100
ID-19-7 (MK626724) Cyclotella sp. Cyclotella meneghiniana (KT3S6323) 99.76 100
ID-19-23 (MK626725) Halamphora coffeiformis Halamphora coffeaeformis (KX257363) 99.76 100
ID-19-25 (MK626726) Hippodonta capitata Hippodonta capitata (AM501966) 99.76 100
ID-19-26 (MK626727) Tryblionella apiculata Tryblionella apiculata (HQ912600) 99.57 100
ID-19-31 (MK62672S) Bacillariophyceae sp. Mayamaea fossalis var. fossalis (KF959655) 91.41 100
ID-19-36 (MK626729) Surirella striatula Surirella striatula (KX120757) 99.52 100
ID-19-40 (MK626730) Fistulifera sp. Fistulifera saprophila (AB76995S) 99.2S 100
ID-19-45 (MK626731) Pleurosigma sp. Pleurosigma intermedium (AY485489) 9S.34 100
ID-19-4S (MK626732) UI Cocconeidaceae Cocconeis placentula (AM502013) 95.63 100
ID-19-49 (MK626733) Halamphora sp. Halamphora americana (MG027295) 9S.7S 100
ID-19-57 (MK626734) Gomphonema sp. Gomphonema parvulum ( KF959660) 99.76 100
ID-19-79 (MK626735) Nitzschia sp. Nitzschia microcephala (KC759159) 99.76 100
ID-19-S9 (MK626736) Tabularla sp. Tabularía fasciculata (EF423417) 99.76 100
ID-19-9S (MK626737) Nitzschia sp. Nitzschia sp. (FJ546709) 99.53 100
ID-19-106 (MK62673S) Nitzschia sp. Nitzschia supralitorea (AJS67019) 99.76 100
ID-19-112 (MK626739) UI Mediophyceae Minutocellus polymorphus (KY9S0146) S2.62 100
ID-19-119 (MK626740) Amphora commutata Amphora commutata (KX120667) 99.52 100
ID-19-13S (MK626741) Rhoicosphenia abbreviata Rhoicosphenia cf. abbreviata (KU965565) 100 100
ID-19-139 (MK626742) Surirella sp. Surirella minuta (KX120726) 99.76 100
ID-19-151 (MK626743) Navicula sp. Navicula perminuta (KY320361) 99.04 100
ID-19-15S (MK626744) Thalassiosira sp. Thalassiosira weissflogii (HM991702) 99.74 100
ID-19-160 (MK626745) Halamphora sp. Amphora terroris (KC222330) 99.04 100
ID-19-210 (MK626746) UI Bacillariophyta Thalassiosira weissflogii (HM991702) 96.71 72
ID-19-214 (MK626747) UI Thalassiosirales Thalassiosira pseudonana (KU90021S) 96.92 100
ID-19-24S (MK62674S) UI Bacillariophycidae Nitzschia supralitorea (KU341756) 95.04 100
ID-19-254 (MK626749) Cylindrotheca sp. Cylindrotheca closterium (KY045S4S) 99.52 100
ID-19-262 (MK626750) UI Bacillariophyta Thalassiosira weissflogii (HM991702) 93.3S 100
ID-19-317 (MK626751) Navicula sp. Navicula phyllepta (FJ624231) 99.05 100
ID-19-320 (MK626752) Cyclotella sp. Cyclotella meneghiniana (KY364696) 97.S7 100
ID-19-334 (MK626753) UI Bacillariophycidae Achnanthidium daonense (KJ65S413) 96.37 100
ID-20-19 (MK656293) Cylindrotheca closterium Cylindrotheca closterium (GQ46S535) 99.76 100
ID-20-55 (MK656301) Nitzschia sp. Nitzschia microcephala (KC759159) 100 100
ID-20-95 (MK656305) Tryblionella sp. Tryblionella apiculata (HQ912600) 9S.33 100
ID-20-S9 (MK656304) UI Anomoeoneidaceae Dickieia ulvacea (AY4S5462) 97.S1 100
ID-20-54 (MK656300) Halamphora sp. Halamphora subtropica (KY054941) 99.76 100
ID-20-137 (MK656307) Halamphora sp. Halamphora aponina (MG027296) 99.51 100
ID-20-27 (MK656294) Haslea spicula Haslea spicula (HMS05034) 99.52 100
Table 3. Continuation.
OTU (accession no.) Identified as Closest homologue (accession no.) Similarity (%) Query Cover " (%)
ID-20-136 (MK656306) UI Naviculaceae Navicula sp. (KF791556) 97.60 100
ID-20-49 (MK656299) UI Stauroneidaceae Stauroneis latistauros (KM116114) 97.12 100
ID-20-74 (MK656303) Surirella sp. Surirella striatula (KX120757) 98.52 100
ID-20-138 (MK656308) Amphora sp. Amphora commutata (KX120667) 98.80 100
ID-20-44 (MK656297) Gedaniella sp. Gedaniella boltonii (MF093083) 99.76 100
ID-20-47 (MK656298) UI Fragilariophycidae Tabularia sp. (KT860991) 97.86 100
ID-20-14 (MK656292) Chaetoceros sp. Chaetoceros muellerii (KX609786) 99.76 100
ID-20-7 (MK656291) Chaetoceros sp. Chaetoceros sp. (EF473734) 99.76 100
ID-20-34 (MK656296) Chaetoceros sp. Chaetoceros sp. (HM106503) 98.57 100
ID-20-59 (MK656302) Cyclotella meneghiniana Cyclotella meneghiniana (KY364696) 99.76 100
ID-20-32 (MK656295) Thalassiosira sp. Thalassiosira weissflogii (HM991702) 99.76 100
Designation: UI - unidentified member of certain taxon.
were revealed by LM (Fig. 4). Among 18 diatom OTUs from the river mouth sample 14 OTUs were attributed to 11 genera, whereas 4 OTUs remained unidentified at the genus level. Only 6 species belonging to 6 genera were revealed by LM there. Thus, taxonomic richness of diatoms in the middle course of the river was higher than in the mouth based on the results of both NGS and LM methods.
The Venn diagrams were created to compare common diatom genera identified by LM and NGS simultaneously, as well as specific genera found by each method separately. More than a half of all identified diatom genera were found by both LM and NGS in the middle course sample, whereas in the river mouth sample only 30.8% of those were shared (Fig. 6).
Representatives of only few genera were found simultaneously under light microscope and with NGS, such as Nitzschia, Gomphonema, Rhoicosphenia, Hippodonta, Pleurosigma, Surirella, Tabularia, and Cyclotella. The genera Cylindrotheca, Tryblionella, Halamphora, and Amphora were revealed in one of two samples by both NGS and LM (Table 2). At the same time their cells were not found with LM in another sample, whereas their sequences were distinguished. Furthermore, some other genera were recognized only with NGS, e.g. Fistulifera, Gedaniella, Thalassiosira, Chaetoceros, and Haslea. This fact suggests that NGS approach is more sensitive than LM. Sometimes light-microscopic identification of diatoms may be doubtful because of too small size of a cell or slight morphological
differences between species (Rivera et al., 2018). Another reason may be detection of free DNA recovered from dead diatom cells and transported by the river flow.
The genera Cocconeis, Anomoeoneis, Encyonema, Achnanthes, Platessa, Rhopalodia, Campylodiscus, and Ctenophora were not supported by NGS and were revealed with LM only (Table 2). These genera might be identified at too low level as unidentified representatives of the appropriate families, orders, subclasses and even classes, due to insufficient data on the mentioned genera or their misidentification in the GenBank database. Also universal primers were shown to reveal only half of OTUs due to insufficient coverage compared to more selective primer pairs (Lentendu et al., 2014). In addition, some diatoms found with LM and not confirmed by the NGS may represent frustules of dead diatoms, which are able to retain for a long time due to highly resistant and strong siliceous composition. At last, erroneous morphological classification, misclassification of OTUs due to low variability within the metabarcoding marker could take place (Groendahl et al., 2017). Therefore, only eight diatom species identified by morphology were robustly supported by 18S rDNA metabarcoding, e.g. Cyclotella meneghiniana Kützing, Surirella striatula Turpin, Cylindrotheca closterium (Ehrenberg) Reimann & J.C. Lewin, Amphora commutata Gru-now, Halamphora coffeiformis, Hippodonta capitata (Ehrenberg) Lange-Bertalot, Metzeltin & Wit-kowski, Tryblionella apiculata W. Gregory, and Rho-
LM NGS LM NGS
Fig. 6. Venn's diagrams showing common and specific diatom genera revealed with LM and NGS in the Bolshaya Samoroda River. A — middle course, B — mouth.
icosphenia abbreviata (C. Agardh) Lange-Bertalot (Table 3).
Alignment of the OTUs against nr database of GenBank (NCBI) (Table 3) retrieved some OTUs closely related to those diatom genera and species, for which LM identification had not been confirmed by NGS. These OTUs were identified at taxonomic levels of family or order. For example, Cocconeis placentula Ehrenberg was detected in the middle course sample by LM only, whereas an OTU of Cocconeidaceae sp. was found with NGS. The sequence closest to the OTU in GenBank is assigned to C. placentula (AM502013) with low similarity (95.63%), which is insufficient for diatom identification even at the genus level (An et al., 2017). Then, the sequence of Anomoeoneis was not found, whereas Anomoeoneidaceae sp. phylotype was identified. A phylotype of Naviculaceae sp. was similar at 97.60% or less with sequences of known Navicula strains. Such morphologically identified diatoms, which were not supported with respective 18S signatures, may belong to novel species and genera.
Study of diatom biodiversity in brackish rivers could provide valuable information about pseu-docryptic or cryptic species, which are still unde-scribed, and have slight, or even do not have any morphological distinctions from the existing diatom species, respectively. For example, Vanelslander et al. (2009) revealed three pseudocryptic species of the widespread benthic diatom Navicula phyllepta in an estuary in the Netherlands, and described their distinct ecological niches characterized by different salinity tolerance ranges, preferred sediment types, and optimal ammonium concentrations. Estimation
of diatom pseudocryptic or cryptic species in brackish rivers using metabarcoding will be successful, if it is supplemented by culture isolation and evaluation of their additional genetic markers, e.g. ITS2 secondary structure, as well as phenotypic properties, such as sexual compatibility, chemotaxonomic markers, and ecological features (Amato et al., 2019).
Conclusions
The Bacillariophyta was revealed to be a permanent, taxonomically diverse, and often the most abundant component of algal communities in the brackish mixohaline Bolshaya Samoroda River located in the Elton Nature Park. In total, light microscopy of the samples taken in 2011-2014 and 2018 allowed to distinguish 39 diatom genera, represented by 76 species and infraspecies taxa. Twenty three species of diatoms were recorded in the river for the first time.
The diatom assemblages showed high spatial-temporal heterogeneity in the Bolshaya Samoroda River, probably due to the influence of dynamically changing abiotic factors. Thus, differences in salinity influence the species composition and proportions of freshwater, marine and brackish species of diatoms. Detection of species known as freshwater together with brackish and marine ones suggests that adaptive capacities of diatoms to high salinity are underestimated.
Our study demonstrated the absence of a direct relationship of diatoms species richness and abundance with salinity in the river. We suggest that
drastic fluctuations in abundance and richness of diatoms in the river cannot be justified by only salinity fluctuations. Nevertheless, they may be determined by the combined influence of environmental factors including biotic interactions.
NGS was applied for the first time to characterize the taxonomic diversity of Bacillariophyta in the Bolshaya Samoroda River. As a result, sequences of the genera Haslea, Fistulifera, and Gedaniella have been recorded in the river for the first time. The data obtained with NGS and LM demonstrated pronounced differences. The diatoms taxonomic richness revealed with NGS was higher compared to that estimated by LM. Next-generation sequencing revealed 26 genera and 47 OTUs in two samples vs. 20 genera and 37 species estimated by light microscopy. However, DNA barcoding based on the V4 marker region of 18S rRNA gene did not allow distinguishing most diatom species reliably. In our study we discovered high genetic richness of diatoms and some V4 18S rDNA sequences characterized by a low similarity with homologues from the reference database. That is why we can expect that a large number of novel for science diatom taxa might be described in further studies of the saline Elton rivers. For this, future investigations of diatoms will require isolation ofpure cultures and their thorough study. Results of our investigations of diatoms in the brackish Bolshaya Samoroda River using both morphology-based and molecular techniques open new perspectives for the in-depth understanding of the diversity patterns, ecology and biogeography of Bacillariophyta.
Acknowledgments
The authors are grateful to Prof. T.D. Zinchenko (Institute of Ecology of the Volga River Basin of the Russian Academy of Sciences) for arrangement of the field research expedition, as well as to Dr. Sci. Yu.A. Khlopko for bioinformatics treatment. The English language check and manuscript proofreading was completed by Effective Language Tutoring Services. This work was financially supported by the Russian Foundation for Basic Research (project 17-04-00135).
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Address for correspondence: Elena A. Selivanova. Institute for Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences, Pionerskaya St. 11, Orenburg 460000, Russia; e-mail: selivanova-81@mail.ru.
