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Research Article
Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses
expand article infoÉva Ács‡§, Tibor Bíró‡§, Emil Boros|, Péter Dobosy|, Mónika Duleba‡§, Angéla Földi, Keve T. Kiss‡§, Zlatko Levkov#, Péter Orgoványi‡, Orsolya Pálné Szén§‡, Zsuzsa Trábert‡§, Edit Vadkerti‡§, István Grigorszky¤
‡ University of Public Service, Baja, Hungary
§ National Laboratory for Water Science and Water Security, Budapest, Hungary
| Institute of Aquatic Ecology, Centre for Ecological Research, Budapest, Hungary
¶ Eötvös Loránd University, Budapest, Hungary
# Ss. Cyril and Methodius University in Skopje, Skopije, Republic of North Macedonia
¤ University of Debrecen, Debrecen, Hungary

Corresponding author: Éva Ács ( acs.eva@uni-nke.hu )

Academic editor: Hugo de Boer
© 2023 Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky.
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: Ács É, Bíró T, Boros E, Dobosy P, Duleba M, Földi A, Kiss KT, Levkov Z, Orgoványi P, Szén OP, Trábert Z, Vadkerti E, Grigorszky I (2023) Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses. Metabarcoding and Metagenomics 7: e111679. https://doi.org/10.3897/mbmg.7.111679
Open Access

Abstract

The research presented investigates whether DNA-based metabarcoding can replace the morphology-based identification of diatom taxa in the ecological status assessments of aquatic habitats. When comparing data obtained with microscopy and metabarcoding, significant deviations have been noticed. One of the main reasons includes the incompleteness of the reference database used for taxonomic annotation of sequences. The database library should be complemented with species inhabiting unique habitats and having specific environmental requirements representing environmental endpoints for genetic diversification. Soda pans and soda lakes are examples of an extreme habitat with the loss of sodic character as the main threat; thus, accurate identification of species and exact information on their salinity tolerance is essential for adequate ecological status assessment. In the present study, by using microscopy and metabarcoding, we investigated taxa of the genus Halamphora that are common in soda pans and soda lakes. We detected six species of which Halamphora dominici and H. veneta occurred frequently and often in high abundance (it was often dominant having relative abundance higher than 5%). Analyses of DNA data confirmed the separation of the two species; as a result, the reference database library has been supplemented with sequences of H. dominici. Furthermore, we have confirmed that this species, which is a significant indicator of sodic character, shows a positive correlation with salinity.

Key words

diatoms, Halamphora, halobity, metabarcoding, soda pans/lakes

Introduction

Diatoms are reliable bioindicators for environmental conditions in aquatic habitats and, therefore, they are used in ecological status assessments (Ector et al. 2004; Lobo et al. 2016). Benthic diatoms constitute one of the five biological elements, based on which the ecological status of surface water bodies should be assessed according to the Water Framework Directive (European Commission 2000). This process is based on diatom indices for which relative abundance data and information on pollution/trophic sensitivity and indicator values of taxa occurring in the given community are required (Berthon et al. 2011). Therefore, diatom taxa should be accurately and consistently identified. The traditional method uses microscopy to identify species, based on morphological features of the silica cell wall, the so-called frustule (Round et al. 1990). Identification of species requires deep taxonomical knowledge and expertise (Rimet and Bouchez 2012), depends on the literature used for species identification and could make identification subjective. Therefore, a more objective DNA-based method has been proposed to address taxonomic accuracy and consistency. Metabarcoding uses next generation sequencing to identify taxa in a community, based on short DNA (barcode) sequences (Hebert et al. 2003; Taberlet et al. 2012). To date, studies have compared the results of metabarcoding to morphological observations and found deviations between these two approaches (Rivera et al. 2018; Bailet et al. 2019; Kelly 2019; Mora et al. 2019; Mortágua et al. 2019; Pérez-Burillo et al. 2020; Borrego-Ramos et al. 2021; Duleba et al. 2021). One of the main reasons for the differences in the results is the incompleteness of reference databases (Rivera et al. 2018; Bailet et al. 2019; Mortágua et al. 2019; Borrego-Ramos et al. 2021; Duleba et al. 2021). It has been concluded that, currently, metabarcoding cannot replace morphology-based identification in ecological status assessments and a combination of two approaches has been proposed (Mora et al. 2019; Duleba et al. 2021).

Reference database libraries lack taxa diversity mainly from specific habitats, like soda pans (Duleba et al. 2021). Additionally, problems with misidentifications can be caused by phenotypic expression in valve morphology due to environmental stressors like changes in salinity (Håkansson and Chepurnov 1999; Trobajo et al. 2011; Leterme et al. 2013). Soda pans represent a unique habitat in the Carpathian Basin (Boros et al. 2013). These are inland alkaline soda environments providing specific living conditions within an extreme environment (Boros et al. 2017). Soda environments are characterised by high dissolved material content and frequently high trophic state and are rich in sodium and hydrogen carbonate ions (soda character) (Boros et al. 2013, 2014; Stenger-Kovács and Lengyel 2015). Therefore, soda pans are inhabited by several rare and halophilic species (Stenger-Kovács and Lengyel 2015; Ács et al. 2017, 2019). Soda pans are highly susceptible to water level changes (dilution/concentration) and poor water management when lower salinity water is channelled through or into the soda pans/lakes. Thus, monitoring the ecological status of soda pans is required. When the ecological status of soda pans is assessed, based on diatoms, accurate identification of species and information on their halobity tolerance is particularly important for correct assessment (Duleba et al. 2021).

Species within the genus Halamphora (Cleve) Levkov are common in soda pans (e.g. Stenger-Kovács and Lengyel (2015); Földi et al. (2018)). They were also commonly found in the study presented here. This genus was first recognised as a subgenus within the genus Amphora Ehrenberg ex Kützing (Cleve 1895). Levkov (2009) elevated the subgenus to genus level describing its members as having dorsoventrally linear, semi-lanceolate to semi-elliptical valves with variable, but often protracted valve ends, striae composed of round, elliptical to transversely elongate areolae, the raphe lying on a raphe ledge near the ventral margin with dorsally curved distal ends and numerous girdle bands with one to two rows of pores. This separation from Amphora sensu stricto was later confirmed with phylogenetic analyses (Stepanek and Kociolek 2014).

This study aimed to present Halamphora taxa occurring in Hungarian soda pans and soda lakes using both microscopy and metabarcoding. We have decided to study the Halamphora genus because it was the fourth most common and seventh most species-rich genus amongst the 72 genera found in the soda samples we collected and its maximum relative abundance was the eighth largest. Two species from the Halamphora genus (Halamphora dominici Ács & Levkov and Halamphora veneta (Kützing) Levkov) were also found in almost half of the samples, they are very common species in soda pans and soda lakes and their maximum relative abundance exceeded 40 and 60%, respectively. Within this genus, species in Halamphora veneta group (H. veneta, H. kevei Levkov, H. dominici and H. paraveneta (Lange-Bertalot, Cavacini, Tagliaventi & Alfinito) Levkov) are not easy to distinguish. The main distinctive features include the stria density and valve shape (Levkov 2009). We have studied this group more thoroughly, testing if the combination of microscopy and metabarcoding could help delimit the species and investigated whether distinctive features in the original description of the species are appropriate to separate them. Furthermore, we intended to identify the rbcL DNA sequence of H. dominici and investigated whether it was distinct from H. veneta. The Diat.barcode database only included H. veneta sequences from this group.

Halamphora dominici is a characteristic species in Hungarian soda pans and soda lakes, as we and others have previously found (e.g. Stenger-Kovács and Lengyel (2015); Földi et al. (2018)). It is regarded as a halophilous species along with H. kevei (Levkov 2009; Stenger-Kovács and Lengyel 2015) showing positive correlation with salinity (Földi et al. 2018). In contrast, H. veneta was widespread in waters with lower salinity, from freshwater to slightly brackish waters (Levkov 2009; Stenger-Kovács and Lengyel 2015). The distribution of H. paraveneta was described (only from Sardinia) by Levkov (2009), further supplemented by Stenger-Kovács and Lengyel (2015) and reported to be frequent in ephemeral sodic waters. Levkov also notes that probably H. paraveneta has a wider distribution in Europe than reported, but is confused with H. veneta.

Materials and methods

Sampling

Samples were taken under the framework of a monitoring project (KEHOP-1.1.0-15-2016-00002). The objective of this programme was to assess the ecological status of Hungarian surface waters, both lentic and lotic environments with various conditions, for example, trophic state, salinity based on benthic diatom assemblages. Within this assessment, 37 soda pans and soda lakes were sampled. Samples were taken in May-June and September-October 2019, as well as May-July and August-October 2020. This resulted in 69 samples that were examined with microscopy, 26 of them also being subjected to DNA sequencing. For sequencing, samples that contained Halamphora taxa in considerable amounts, based on microscopy, were selected.

Due to the unfavourable weather conditions (several soda pans were dry on sampling occasions), we were unable to collect a few samples from alkaline soda pans; thus, we used soda pans samples collected in May 2021 within the framework of another project. However, we could only investigate these samples by light microscopy (LM).

In standing waters, epiphytic samples were mostly taken from green common reed (Phragmites australis (Cav.) Trin. ex Steud.) or, if it was unavailable, lesser bulrush (Typha angustifolia Linn.) or other emergent macrophytes were sampled by choosing five randomly selected stems.

To illustrate the whole distribution of Halamphora veneta including waters with lower salinity, we used additional samples collected in the KEHOP project including 31 samples from running waters and 51 from standing waters. Sampling from running waters was carried out in the period of March-May (and two samples from June) and September-October 2019. In rivers and streams, epilithic samples were taken from five randomly chosen cobbles; if these were absent, other available substrates, mainly emergent macrophytes, rarely dead-wood or artificial substrates (e.g. brick) were sampled.

In all cases, the five random repeats were integrated into one composite sample; the biofilm was scraped with a toothbrush into tap water. The acquired slurry was homogenised and divided into two parts. For DNA analysis, 2–3 ml was pipetted into a 15 ml sterile plastic centrifuge tube that was filled with absolute ethanol (final ethanol concentration ≥ 70%), then stored at 4 °C until processing. The rest of the slurry was preserved with buffered formaldehyde (4% final concentration) for microscopy (European Committee for Standardisation 2002).

Environmental variables

Cation (Na+, K+, Ca2+, Mg2+, NH4+) and anion (Cl-, SO42-, NO3-) concentrations were determined using a Dionex ICS 5000+ dual channel ion chromatograph (Thermo Scientific, Massachusetts, USA). Total nitrogen was measured with a MULTI N/C 3100 TC/TN analyser (Analytik Jena, Jena, Germany). Total phosphorus and orthophosphate concentrations were quantified spectrophotometrically, based on the methods of Murphy and Riley (1962) and Menzel and Corwin (1965).

Silicate (Si, µg l-1; MSZ 1484-3:2006; Hungarian Standards Institution: HSI 2006), hydrogen carbonate and carbonate (HCO3- and CO32-, mg l-1, MSZ 448-11:1986, HSI 1986), chlorophyll-a concentration (CHA µg l−1, MSZ ISO 10260:1993; HSI, 1993), nitrite (NO2-, mg l−1; MSZ EN ISO 11885:2009; HSI 2009) and Secchi transparency (Secchi, cm, MSZ 260-46:1981, HSI 1981) were measured according to the national standard (see the references in Duleba et al. (2021)). The pH, electrical conductivity (µS cm-1) and dissolved oxygen (DO, mg l-1) were measured in situ with YSI EXO-2-S3 equipment.

Microscopy

For light microscopy, samples were treated with hydrochloric acid and hydrogen peroxide, then washed with distilled water. Cleaned diatom valves were mounted with Naphrax (CEN 2014) and investigated using a Zeiss Axio Imager Z2 microscope, equipped with differential interference contrast (DIC) optics at a 1600× magnification. At least 500 valves were identified to the species or genus level in lake samples. For illustrating the distribution of H. veneta, a minimum of 400 valves were counted and identified in running water samples. The relative abundance of each taxon in the sample was calculated by dividing the counted valve number of the given taxon with the total counted valve number.

For scanning electron microscopy (SEM), part of the cleaned and washed sample was filtered through a 3 µm Isopore polycarbonate membrane filter (Merck Millipore), which was then fixed on to an aluminium stub using double-sided carbon tape and coated with gold using a rotary-pumped spatter coater, Quorum Q150R S. Ultrastructural features of diatoms were observed with a Zeiss EVO MA 10 SEM operated at 10 kV and Zeiss Sigma 300 operated at 2 kV (only the images of Halamphora elongata Bennett & Kociolek). The working distance varied between 10.5 and 10.7 mm.

Metabarcoding

DNA was extracted from the samples using NucleoSpin Soil Kit (Macherey-Nagel). The protocol by Vautier et al. (2020) was used; however, a mixture of two kinds of lysis buffer from the kit in a 1:1 ratio was applied.

A 312 base pair (bp) region of the rbcL gene was amplified and sequenced on the Illumina MiSeq platform. Primer sequences, circumstances of polymerase chain reactions (PCR), library preparation and sequencing are described in Duleba et al. (2021). The workflow included the following steps: PCR with gene-specific primers; PCR product purification, concentration, quantification and dilution to equimolar concentrations; index reaction; product purification and pooling; quality and concentration control; dilution to the same concentration (4 nM); running on the Illumina MiSeq system. The rbcL-specific primers used in the first PCR were developed by Vasselon et al. (2017) and supplemented with Illumina overhang P5/P7 adapters. The PCR products were purified with 1.0× AMPure XP magnetic beads (Beckman Coulter). Concentrations were quantified by using a Qubit 4 Fluorometer (Invitrogen) with the Qubit dsDNA HS Assay Kit. In the cleaned index reactions, PCR products were provided with Nextera DNA CD Indices with P5/P7 adapters and P7/P5 tags attached. Quality assessment of the samples was performed using an Agilent TapeStation System 4150 (Agilent) with Agilent High Sensitivity D1000 ScreenTape Assay Reagents.

For the run on the Illumina MiSeq system, the starting concentration of the final library pool was 4 nM. After denaturation and dilution, sequencing was performed using the Illumina MiSeq V2(500) Reagent Kit and a 2 × 250 bp read length.

Polymerase chain reactions, library preparation and sequencing were performed by Biomi Ltd. (Hungary).

One sample (Kisteleki-Müller-szék) was subjected to long read sequencing following the description in the manual of LoopSeq PCR Amplicon Kit (Loop Genomics). This sample was chosen because microscopic analyses revealed that two taxa of the H. veneta group, Halamphora dominici and H. veneta that have different ecological preferences in terms of salinity, were dominant.

Data analysis

Sequence data analysis followed the method of Keck et al. (2019), i.e. the official DADA2 pipeline (Callahan et al. 2016) modified and applied to diatom rbcL metabarcoding (Keck et al. 2019). This included the following main steps: removing primer sequences, trimming and filtering according to read quality, dereplicating, filtering with the core sample inference algorithm of DADA2, aligning and merging paired forward and reverse reads into one contig sequence, removing chimaeras and taxonomic assignment. Diat.barcode version 9.2 (Rimet et al. 2019) was used as the reference database. Read numbers were corrected according to biovolume using the correction factor developed by Vasselon et al. (2018). For H. dominici, the correction factor of H. veneta was used. Relative abundance was calculated for each taxon in the samples dividing corrected read number of a given taxon by the total corrected read number of the sample. For more details, see Duleba et al. (2021).

All sequences that could be ranked to the genus Halamphora were assigned at species level with the DADA2 pipeline. Halamphora veneta sequences found in samples containing H. dominici, based on microscopy, were aligned to sequences in the National Center for Biotechnology Information (NCBI) GenBank database using the Basic Local Alignment Tool (BLAST, Altschul et al. (1990)). The search was performed in the Nucleotide collection database (nr/nt) using the Standard Nucleotide BLAST programme, megablast (highly similar sequences) algorithm with the default parameter settings. The short rbcL amplicon sequence variants (ASVs) were used for identifying long sequences. By using long read sequencing, we obtained sequences with different lengths (sequences identified as H. veneta ranged from 415–1548 nt in length, while sequences identified as H. dominici ranged from 379–1547 nt in length). The longest sequences per species were used in phylogeny and these sequences having been deposited in the GenBank database under accession numbers OQ588791–OQ588792.

Halamphora sequences from our samples were compared to those acquired from Diat.barcode 9.2 (Rimet et al. 2019). Alignment of sequences was performed with Clustal W (Thompson et al. 1994) implemented in MEGA7 (Kumar et al. 2016). Alignments are available as supplementary material (Suppl. materials 3, 4). The same software was used for calculating pairwise uncorrected p-distance, for selecting the best nucleotide substitution model and for performing Maximum Likelihood phylogenetic analyses; the last bootstrap test was performed in 500 replicates.

Pearson Correlation Values were calculated from data obtained by microscopy and metabarcoding using Past 4.12 (Hammer et al. 2001).

Results and discussion

Within the framework of the whole project, several environmental variables were measured and shown in Suppl. material 1. According to the international system of salinity ranges for salt content of continental waters (Hammer 1986), 22 of the studied soda pans and soda lakes were subsaline and 15 were hyposaline, 15 were natural and 22 had a disturbed status (Boros et al. 2013, 2014). Based on the dominant ions of the eight main ions, two waterbodies belonged to inland saline, 17 to alkaline soda base, two to chloride (salt) soda and two to mixed sulphate chloride soda. One lake belonged to the sulphate soda (all sample sites of Lake Fertő) and one lake to the mixed magnesium sulphate soda (all sample sites of Lake Velencei) chemical type (Boros et al. 2013, 2014). The proportion of cations and anions found during our study is presented in Fig. 1. Other physical-chemical data are provided in Suppl. material 1.

Figure 1.

The ternary diagram of cation (A) and anion (B) concentrations measured in the samples.

During this study, six Halamphora species were identified from soda pans and soda lakes. Five of these were found by both microscopy and metabarcoding. One species (Halamphora oligotraphenta (Lange-Bertalot) Levkov) was only detected by microscopy because it occurred only in samples that were not sequenced. Two of the six Halamphora species were frequent (H. dominici and H. veneta), occurring in approximately half of the samples and they were often dominant (sometimes reaching 40–60% relative abundance and H. dominici was dominant in approximately half of the samples) in soda pans and lakes. The other four Halamphora species occurred in 5–7% of the samples and were never dominant (Suppl. material 1).

Halamphora species identified by microscopy and/or metabarcoding in the studied soda pans and soda lakes

In the following, morphological description, information from sequencing and ecological preferences in terms of salinity of the Halamphora taxa detected in studied soda pans and soda lakes are provided. The morphological description is based on the descriptions by Levkov (2009), as it is the most complete guide to identify Halamphora species by microscopy. Differences between our observations and literature are indicated. Descriptions also includes the four-letter Omnidia codes to help the search in Omnidia (Lecointe et al. 1993), a software for ecological status assessment based on diatoms; thus, Omnidia has a wide database of diatom taxa along with their ecological preferences.

Halamphora coffeaeformis (Agardh) Levkov, 1903 (HACO)

Fig. 2I–J

Short morphological description. Length: 23–35 (14–55) μm, width: (3.5)5–7.2 μm, number of dorsal striae in the middle: 19–22/10 μm (Levkov 2009). Valveс narrow semi-lanceolate, dorsal margin convex, ventral margin weakly concave. Apices narrow, protracted, capitate, bent ventrally. Central area absent on both sides. Raphe slightly arcuate, very close to ventral margin. Proximal raphe end slightly deflected to dorsal valve side. Ventral striae very short, dorsal striae parallel in mid-valve becoming radiate towards apices. Puncta not visible in LM. Ultrastructural features: biseriate striae, this being the main characteristic of the species (Levkov 2009).

Figure 2.

LM images of Halamphora species detected in present study. A–D Halamphora paraveneta LM (No. of sampling site: 30 in Suppl. material 1) E–G cf. H. oligotraphenta LM (No. of sampling site: 12 in Suppl. material 1) H H. oligotraphenta LM (No. of sampling site: 12 in Suppl. material 1) I, J H. coffeaeformis LM (No. of sampling site: 36 of I and 11 of J in Suppl. material 1) K–O H. veneta LM (No. of sampling site: 12 of K, No. of sampling site: 30 of L and 29 of M–O in Suppl. material 1) P–X H. elongata LM (No. of sampling site: 21 of P, U-W, X, No. of sampling site: 30 of Q, R and No. of sampling site: 19 of S, T, Z in Suppl. material 1) (scale bar 10 µm). The description of sampling sites is provided in Suppl. material 1.

Morphologically similar taxa. Halamphora hybrida (Grunow) Levkov has a similar valve outline, but its striae are crossed by a longitudinal line close to the dorsal margin, and the dorsal striae are formed by elongated areolae.

Detection by metabarcoding. Only one short amplicon sequence variant was detected in one sample that was assigned as H. coffeaeformis. It showed 0.004 – 0.072 p-distance from the H. coffeaeformis sequences recorded in the database. The p-distance 0.072 was with the H. coffeaeformis sequence (accession number FJ002103) that showed relatively high difference (0.039 – 0.04, 51 – 54 nt difference) from the other H. coffeaeformis sequences in the database (Suppl. material 2).

Ecology, distribution. Halophilic, cosmopolitan species found in waters with high electrolyte content and in brackish and saline inland waters. It occurred in one saline pan (Sárszentágotai-sóstó) and two shallow soda lakes (Lake Fertő and Lake Szelidi) during our survey (Fig. 8A).

Halamphora dominici Ács & Levkov, 2009 (HDOM)

Fig. 3

Short morphological description. Length: 10–20 μm, width: 3.5–4 μm, number of ventral striae on the mantle: 30–34/10 μm, number of dorsal striae: 24–28/10 μm (Levkov 2009). (The ventral striae are not be visible in LM, but in SEM, we can recognise them, for example, see Fig. 3EE). Valveс semi-elliptical, strongly dorsiventral. Valve ends broadly rounded, not elongated. Axial area narrow, wider on ventral side. Central area absent on dorsal valve side. Raphe straight and proximal ends slightly bent dorsally. Dorsal striae punctate, radial throughout. Ventral striae on the mantle barely or are not visible under light microscopy. Ultrastructural features: Partial conopeum (rib adjacent to raphe) narrow, poorly developed in smaller specimens, prominent from valve surface in larger specimens. Raphe weakly to strongly curved at proximal ends. Striae uniseriate along whole length, radial and composed of elongated areolae. Areolae variable in length, generally smaller towards the valve middle and more elongated towards dorsal edge of valve and valve ends. Ventral striae not interrupted in region of central nodule. Internally, distal raphe ends terminate in weakly-developed helictoglossa. Proximal raphe ends fuse into central helictoglossa. Areolae occluded by hymens (Levkov 2009).

Figure 3.

Halamphora dominici from the studied soda lakes and soda pans. A–BB LM. CC, EE SEM internal view DD, FF SEM external view (No. of sampling site: 25 of O, P, S, T, U, W, No. of sampling site: 27 of C, N, Q, Y, AA and No. of sampling site: 28 of others in Suppl. material 1). The description of sampling sites is provided in Suppl. material 1. Scale bars: 10 µm (A–BB); 2 µm (CC, DD); 1 µm (EE, FF).

Morphologically similar taxa. It is similar to Halamphora kevei, but H. dominici has a more rounded (semi-elliptic) valve shape and broadly rounded, not protracted, not ventrally bent valve ends. It resembles H. veneta, but the latter has lower stria density (dorsal number of striae: 18–22/10 μm) with coarser punctuate striae. It is also similar to Halamphora paraveneta that also has coarser punctuate striae and lower stria density (dorsal number of striae: 18–21/10 μm).

Detection by metabarcoding. We found 23 short amplicon sequence variants that were assigned as H. veneta with DADA2, but phylogeny (Fig. 4) and BLAST search showed a closer similarity with isolate 7951-AMPH106 (accession number MG027464) that is recorded as H. veneta in Diat.barcode database (this explains assigning our sequences as H. veneta by DADA2). The corresponding long (1547 nt) rbcL sequence showed 99.77% similarity (two substitutions and one deletion) with this sequence according to BLAST search. In samples in which these ASVs were in high number, H. dominici was found in high abundance. This correlation indicated that the 23 ASVs belonged to H. dominici, suggesting that the MG027464 sequence was misidentified. The Halamphora dominici clade separated from H. veneta sequences (Fig. 4) and the long sequence showed 2% p-distance (31 nt difference, Suppl. material 2) with the long sequence of H. veneta confirming that H. dominici is a distinct species from H. veneta. The long sequences of H. dominici and H. veneta acquired in our study were inserted into the phylogenetic tree generated by Stepanek and Kociolek (2019) which involved several Halamphora taxa. The phylogeny also showed that H. dominici was located close to H. veneta, but on a distinct branch (Fig. 5). Our tree was based only on the rbcL gene, while Stepanek and Kociolek (2019) used four markers (SSU, LSU, rbcL, psbC) which caused some differences in the topology of the two trees; similarly to their tree, H. veneta and H. dominici grouped with H. oligotraphenta and H. elongata.

Figure 4.

Maximum Likelihood tree of Halamphora amplicon sequence variants (ASVs) found in soda pans and soda lakes as well as Halamphora taxa from Diat.barcode database. Pinnularia brebissoni, Fallacia pygmaea, Pseudofallacia monoculata, Caloneis lewisii, Stauroneis acuta and Craticula cuspidata were used as outgroup. The total length of the alignment was 1530 nt. Sequences from the database are provided with NCBI GenBank accession numbers (if available) or culture ID of Thonon Culture Collection. Bootstrap values are indicated at nodes. Scale bar represents 0.01 substitutions per site.

Figure 5.

Maximum Likelihood tree of Halamphora taxa from the work by Stepanek and Kociolek (2019) as well as Halamphora dominici and Halamphora veneta from our study. The total length of the alignment was 1560 nt. Sequences from NCBI GenBank are provided with accession numbers. Long rbcL sequences of H. dominici and H. veneta from our study are written in bold italics. Bootstrap values are indicated at nodes. Scale bar represents 0.02 substitutions per site.

In the case of H. dominici, metabarcoding helped delimit species boundaries. Morphologically, it is difficult to distinguish the larger specimens of H. dominici from H. kevei.

The difference between H. dominici sequence variants was below 2%, p-distance between ASVs ranged from 0 to 0.015 (0 – 4 nt difference). The H. dominici asv1 occurred in the most (10) soda pans and lakes (in 16 samples) and in highest abundances. Kisteleki Müller-szék and Madarász Lake showed the highest diversity of sequence variants.

Ecology, distribution. Halophilic species. Typical, characteristic, often strongly dominant species in Hungarian soda pans. Present in one soda lake (Lake Fertő) as well, but was never dominant during our survey (Fig. 8B).

Halamphora elongata Bennett & Kociolek, 2014 (HEGT)

Figs 2P–X, 6A–C

Short morphological description. Length: 22–57 μm, width: 7–11 μm, number of dorsal striae: 19–23/10 μm, number of ventral striae: 26–30/10 μm (Kociolek et al. 2014; Stepanek and Kociolek 2018). In our study: length: 22–40 μm (mean: 30 μm, n = 13), width: 5–11 μm (mean: 7.7 μm, n = 13), number of dorsal striae: 20–23/10 μm. Valveс semi-elliptical and strongly dorsiventral. Ventral margin usually weakly concave or sometimes slightly convex. Valve ends rostrate to capitate ventrally curved and slightly protracted. Axial area narrow, often curved or with semi-circular hyaline area (it can be significantly broad in large valves) on dorsal side of the central node. Raphe located near ventral margin. Proximal raphe endings expanded and dorsally curved; distal raphe endings dorsally curved. Dorsal striae punctate, with dash-like areolae, radiating throughout and more widely spaced at mid-valve. Ventral striae short and difficult to observe in LM because of the very narrow ventral valve. Ultrastructural features: proximal raphe endings curved in dorsal direction, dorsal striae consisting of several elongated areolae. Area of central striae thickened and striae of this area coarser than others. In SEM, short ventral striae visible, composed of short dashes. This is a halophilic species (Kociolek et al. 2014; Stepanek and Kociolek 2018).

Morphologically similar taxa. It is very similar to Halamphora subcapitata (Kisselev) Levkov and their morphometric features strongly overlap, especially in LM. According to Kociolek et al. (2014), H. elongata differs from H. subcapitata in that the former is relatively narrower (7.5 – 12 µm, Levkov (2009)) with more capitate ends and its areola has external foramen areolar occlusions. We measured the width and length of 20 H. subcapitata individuals (nine from Stenger-Kovács and Lengyel (2015), 11 from Levkov (2009)) and 21 H. elongata individuals (10 from Kociolek et al. (2014) and Stepanek and Kociolek (2018); and 11 from the present study) and calculated the length/width ratio (L/W). We found no significant difference in L/W of the two species (Fig. 7); thus, it does not confirm the claim that H. elongata is relatively narrower. Stenger-Kovács and Lengyel (2015) presented two SEM images of H. subcapitata, where the external foramen areolar occlusions are clearly visible, while on our specimens, they are not (Fig. 6B, C). Several experiments correlated salinity and the structure or size of the frustule (Tuchman et al. 1984; Håkansson and Chepurnov 1999; Trobajo et al. 2011); therefore, we think that H. elongata is the same species as H. subcapitata, but to synonymise it, a detailed analysis of the type materials is required.

Figure 6.

SEM images of two Halamphora species from the studied sites. A–C Halamphora elongata D, E H. veneta A, E internal view B, D external view C detail of B (No. of sampling site: 21 of A–C and No. of sampling site: 30 of D, E). The description of sampling sites is provided in Suppl. material 1. Scale bars: 3 µm (A); 2 µm (B, D, E); 500 nm (C).

Figure 7.

The length/width ratio of measured H. subcapitata (HSCA) and H. elongata (HEGT) specimens observed in our study.

Detection by metabarcoding. We detected one ASV that was assigned as H. elongata (in three samples) that differed only in one nucleotide (p-distance = 0.004) from the sequence in the database (the same record is present in Diat.barcode and GenBank). Unfortunately, neither Diat.barcode nor GenBank contain sequences data for H. subcapitata; therefore, we did not have the possibility to compare their DNA sequences, but a single nucleotide difference from the database sequence of H. elongata suggests that H. elongata was present in our samples.

Ecology, distribution. Halophilic species. This taxon occurred in two soda pans and one shallow soda lake (Lake Velencei) during our survey (Fig. 8C).

Figure 8.

The occurrence of Halamphora coffeaeformis (A), H. dominici (B), H. elongata (C), H. oligotraphenta (D), H. paraveneta (E) and H. veneta (F) in Hungarian soda pans and soda lakes, based on microscopic and molecular investigations carried out in present study. In the case of H. veneta, we plotted all occurrences during our survey (light grey circle: based on microscopy, dark grey circle: based on metabarcoding, grey diamond: running waters based on microscopy, light grey triangular: based on microscopy and metabarcoding). This approach was applied because the distribution of H. veneta pointed beyond soda lakes and soda pans.

Halamphora oligotraphenta (Lange-Bertalot) Levkov, 2009 (HOLI)

Fig. 2E–H

Short morphological description. Length: 17–39 μm, width: 3.2–4.5 μm, number of dorsal striae in the middle: 26–30/10 μm (Levkov 2009).

Valveс semi-lanceolate, dorsiventral with arched dorsal margin and straight or slightly tumid ventral margin. Valve ends shortly protracted, capitate and slightly ventrally bent. Axial area narrow and wider on the ventral side. Central area absent on dorsal side, on ventral side not differentiated from axial area. Raphe slightly arched, proximal endings distantly spaced and slightly dorsally deflected. Dorsal striae punctuate and radiating throughout. Ventral striae hard to resolve in LM. Ultrastructural features: distal raphe endings prolonged and dorsally curved. On ventral side, central area extends to valve margin as a fascia. Dorsal striae uniseriate and composed of areolae with various shapes. Ventral striae also uniseriate and comprised of longitudinally elongated areolae. Ventral striae interrupted in the region of central nodule (Levkov 2009).

Morphologically similar taxa. This species resembles Halamphora coraensis Foged (Levkov), but the latter species is significantly wider (4.5–6.5 µm) and has a lower stria density (24–26 in 10 µm).

Detection by metabarcoding was not possible because this species was only observed under the microscope in samples that were not sequenced.

Ecology, distribution. Freshwater, oligotrophic species (Levkov 2009). Unfortunately, soda pan and soda lake samples in which H. oligotraphenta occurred were not sequenced; therefore, our DNA data could not confirm the distribution of the species in soda pans and soda lakes. This species was recorded in one soda pan and two shallow soda lakes (Lake Velencei and Lake Fertő) during our survey (Fig. 8D).

Halamphora paraveneta (Lange-Bertalot, Cavacini, Tagliaventi & Alfinito) Levkov, 2009 (HPVE)

Fig. 2A–D

Short morphological description. Length: 20–75 μm, width: 4.7–5 μm (measurement in this study), number of dorsal striae: 18–21/10 μm (Levkov 2009).

Valveс semi-elliptical or semi-lanceolate and slightly dorsiventral. Valve ends rounded and slightly ventrally bent. Axial area moderately narrow, more expressed on ventral valve side. Central area absent on dorsal side and not differentiated from axial area on ventral valve side. Dorsal striae radiating throughout, coarsely punctuate. Proximal raphe ends distantly spaced. Ultrastructural features: Striae uniseriate and consisting of elongate areolae. Ventral striae on mantle not interrupted in region of central nodule and composed of round areolae. Proximal raphe ends slightly dorsally curved, distal ends strongly dorsally deflected (Levkov 2009).

Morphologically similar taxa. This species is very similar to H. veneta, differing from it in stria density (stria density of H. veneta: 24–30 in 10 µm) and valve end shape (more broadly rounded in H. paraveneta).

Detection by metabarcoding. Sequence data for H. paraveneta is not available either in Diat.barcode (the reference database we used for taxonomic assignment) or in NCBI GenBank database; therefore, metabarcoding could not detect it as an assigned species in our samples. However, there was one ASV (Halamphora veneta asv 10) that was assigned as H. veneta, but showed higher p-distance (Suppl. material 2) from other H. veneta ASVs. We assume this is the sequence of H. paraveneta, but it requires further investigations.

Ecology, distribution. Halophilic species. During our survey, H. veneta was recorded in three soda pans and one shallow soda lake (Lake Velencei) (Fig. 8E).

Halamphora veneta (Kützing) Levkov, 2009 (HVEN)

Figs 2K–O, 6D, E

Short morphological description. Length: (8)17–35 μm, width: (3.5)4–6.5 μm, number of ventral striae: 24–30/10 μm, number of dorsal striae: 18–22/10 μm (Levkov 2009; numbers in parentheses Lange-Bertalot et al. (2017)).

Valve shape semi-lanceolate with strongly convex dorsal margin and slightly concave ventral margin. Valve ends narrowly rounded, slightly protracted and ventrally bent. Axial area narrow, wider on ventral side. Central area absent on dorsal valve side. Raphe filiform, proximal ends straight or dorsally curved, distantly spaced (it is important to emphasise that this feature is expressed especially on large specimens; however, proximal endings can be more closely spaced and this character is variable). Dorsal striae can be observed with distinct punctuations in LM, radiating throughout. Ventral striae on mantle hard to resolve in LM, not interrupted at the central nodule. Ultrastructural features: Partial conopeum is moderately wide, gradually narrowing towards valve ends. Sometimes has ornamentation (small, round depressions) in region of the central nodule. Raphe arched, proximal ends straight and terminating in slightly expanded central pores. Distal raphe ends dorsally deflected. Striae uniseriate throughout, radiating and consisting of elongate areolae. Ventral striae not interrupted in region of central nodule and composed of round or elongate areolae. Girdle bands open, each with large round or oval poroids located in two rows. Internally central internal costae thickened inwards and elevated from valve plane. Distal raphe ends ventrally deflected and terminating with poorly-developed helictoglossae. Proximal raphe endings fuse into a central helictoglossa. Areolae occluded by hymens (Levkov 2009).

Morphologically similar taxa. It is similar to H. oligotraphenta, but the latter has more capitate valve ends and live in oligotrophic freshwater habitats rich in calcium bicarbonate. It resembles H. kevei that has a higher stria density (number of dorsal striae: 24–30/10 μm) and more finely punctuated striae. It is most similar to H. paraveneta; however, the latter is usually larger (20–75 μm), although the size ranges of the smaller cells overlap. The frustule width of H. paraveneta (11–20 μm) and H. veneta (9–17 μm) also overlaps. The valve width of H. paraveneta is 4–9 μm also overlapping with H. veneta in the case of smaller specimens. Valve ends of H. paraveneta are bluntly and more broadly rounded. Dorsal striae are less radiate in the central region, number of dorsal striae is 18–21/10 μm, becoming more radiate and denser (22–23/10 μm) towards the valve ends. This is the case also with smaller specimens. The two species can occur together.

Detection by metabarcoding. Overall, 25 ASVs that were assigned as H. veneta could be accepted as H. veneta, showing more similarity to other H. veneta sequences than to the MG027464 sequence (Suppl. material 2; Fig. 4). Except for the above mentioned Halamphora veneta asv 10 (that could be H. paraveneta), they showed p-distances ranging from 0 to 0.015 (0 – 4 nt difference). The corresponding long (1548 nt) sequence showed 100% identity with H. veneta according to BLAST that was confirmed by phylogeny (Fig. 5).

The most widely distributed ASV was H. veneta asv1 occurring in 14 soda pans and lakes (19 samples). Interestingly, similarly to H. dominici, the highest number of H. veneta sequence variants was found in Kisteleki Müller-szék and Madarász Lake.

Ecology, distribution. A cosmopolitan species, common in freshwater and slightly brackish waters, often detected in waters with high nutrient content. It can tolerate drying (aerophytic species) and waters with high organic content. H. veneta was frequent and often a dominant species in several standing and running waters during our survey (Fig. 8F). (In the case of this species, the occurrences experienced during the entire survey are indicated on the map, showing that it was also found in many non-soda habitats, since the non-soda occurrences were also taken into account when calculating the halobity value).

Comparison of results, based on microscopy and metabarcoding

Amongst the six Halamphora species detected in our samples, H. dominici and H. veneta were dominant (relative abundance higher than 5%) in several samples, based on microscopy and/or metabarcoding. H. dominici showed corresponding occurrences, based on morphological and molecular data in 15 samples analysed by both methods; for H. veneta corresponding occurrences were shown in 13 samples. For H. dominici, a significant positive correlation was found between relative abundances, based on microscopy and metabarcoding (Pearson correlation r = 0.73, p < 0.05). H. veneta and H. dominici were only detected, based on sequencing in six and five samples, respectively.

The observation that a taxon can be detected in more samples analysed by metabarcoding than by microscopy is understandable, considering that DNA was extracted from more cells than the number of cells counted under the microscope. In two samples that were investigated by both microscopy and metabarcoding, H. veneta was found only under the microscope. A potential explanation includes that the observed valves belonged to dead cells.

The other four species did not reach 5% (the maximum value was 2.9% for H. oligotraphenta, based on microscopy) either based on morphology or DNA sequences. They occurred only in a few samples compared to the dominant two species and we could not find correspondence between microscopy- and metabarcoding-detected occurrences (except for one sample in the case of H. elongata). Moreover, H. oligotraphenta was not detected by metabarcoding at all. Reasons to explain why a taxon can only be found either by microscopy or metabarcoding may include the observation of dead cells under the microscope, incomplete DNA extraction, failure of primer annealing, detection of extracellular DNA or resting stages and unequal distribution of individuals between subsamples (Duleba et al. 2021).

Salinity demands of the two dominant Halamphora species

Based on the salinity system of Van Dam et al. (1994), Halamphora dominici belongs to category 4 (mesohalobous) (the average salinity of the waters in which the species was found was 3.1‰, while in the case of H. veneta, it was 1.6‰). It prefers waters with higher salinity compared to H. veneta as demonstrated by the total salinity violin plot (Fig. 9).

Figure 9.

Violin plot of salinity (‰) in those samples where H. dominici (HDOM) and H. veneta (HVEN) occurred.

Given that the ecological status of Hungarian soda pans and shallow soda lakes depends on salinity (Földi et al. 2018), it is a major characterising environmental factor for these environments. The exact identification of species along with information on their salinity tolerance have great importance, especially for dominant species.

Summary

During our study, we have obtained very similar results, both by morphological and DNA analyses for prominent species. However, our results also highlight that, during sequence matching, it is of great importance that sequences are entered into the database library only after a precise morpho-taxonomic investigation of the species. This is also crucial, as there is an increasing pressure to use environmental DNA-based methods in ecological status assessments and if a dominant species is misidentified in the database, it can distort the results of ecological status assessments.

Acknowledgements

We give thanks to Dr. Krisztina Buczkó for helping our taxonomic works in project KEHOP, to Dr. Patrik Kociolek for his professional help with Halamphora elongata, to Judit Knisz for the correction of the English of the manuscript, to the Biomi Ltd. for the DNA work and to the SEM Laboratory of H-ION Ltd. for making the SEM images of H. elongata.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

The research presented in the article was carried out within the framework of the Széchenyi Plan Plus programme with the support of the RRF 2.3.1 21 2022 00008 project and was supported by KEHOP-1.1.0-15-2016-00002 project as well.

Author contributions

Conceptualization: ÉÁ. Data curation: MD, OPS. Funding acquisition: TB. Investigation: ZT, PD, AF. Project administration: EV. Resources: EB. Visualization: PO. Writing - original draft: ÉÁ, ZL, KTK, MD. Writing - review and editing: IG.

Author ORCIDs

Tibor Bíró https://orcid.org/0000-0002-5553-9850

Orsolya Pálné Szén https://orcid.org/0000-0002-6610-3681

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

Relative abundances of Halamphora species based on microscopy and metabarcodin

Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky

Data type: xlsx

Explanation note: Relative abundances of Halamphora species, based on microscopy and metabarcoding (this latter is indicated with “DNA” after the code of the species), as well as environmental variables measured at each sampling location and time. (See abbreviation of the species code in the text). Relative abundances indicating dominancy (> 5%) are written in bold. Abbreviations of physical-chemical variables: Cond = conductivity; DO = dissolved oxygen concentration; CHA = chlorophyll a concentration; Secchi = Secchi transparency; TN = total nitrogen concentration; TP = total phosphorus concentration. “<” stands for under detection limit. For more details, see Material and methods.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (33.37 kb)
Supplementary material 2 

Pairwise p-distance values and number of differences

Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky

Data type: xlsx

Explanation note: Pairwise p-distance values and number of differences between Halamphora amplicon sequence variants found in soda pan and soda lake samples and sequences of Halamphora taxa from Diat.barcode database.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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Supplementary material 3 

Supplementary Alignment 1

Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky

Data type: fas

Explanation note: Alignment of Halamphora amplicon sequence variants (ASVs) found in soda pans and soda lakes as well as Halamphora sequences from Diat.barcode database for Fig. 4.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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Supplementary material 4 

Supplementary Alignment 2

Éva Ács, Tibor Bíró, Emil Boros, Péter Dobosy, Mónika Duleba, Angéla Földi, Keve T. Kiss, Zlatko Levkov, Péter Orgoványi, Orsolya Pálné Szén, Zsuzsa Trábert, Edit Vadkerti, István Grigorszky

Data type: fas

Explanation note: Alignment of long rbcL sequences of Halamphora dominici and H. veneta acquired in this study and Halamphora taxa from the work by Stepanek and Kociolek (2019) for Fig. 5.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (170.83 kb)
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