# Hidden aliens: Application of digital PCR to track an exotic foraminifer across the Skagerrak (North Sea) correlates well with traditional morphospecies analysis

Filip Morin, Marina Antonina Zoe Panova, Magali Schweizer, Marlene Wiechmann, Nicole Eliassen, Per Sundberg, Irina Polovodova Asteman

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## ► To cite this version:

Filip Morin, Marina Antonina Zoe Panova, Magali Schweizer, Marlene Wiechmann, Nicole Eliassen, et al.. Hidden aliens: Application of digital PCR to track an exotic foraminifer across the Skagerrak (North Sea) correlates well with traditional morphospecies analysis. Environmental Microbiology, 2023, 155, pp.101726. 10.1111/1462-2920.16458. hal-04241107

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Submitted on 13 Oct 2023

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RESEARCH ARTICLE

# Hidden aliens: Application of digital PCR to track an exotic foraminifer across the Skagerrak (North Sea) correlates well with traditional morphospecies analysis

Filip Morin<fnref n="1" /> | Marina Antonina Zoe Panova<fnref n="1" />,<fnref n="2" /> ID | Magali Schweizer<fnref n="3" />,<fnref n="4" />,<fnref n="5" /> ID |
Marlene Wiechmann<fnref n="1" /> | Nicole Eliassen<fnref n="1" /> | Per Sundberg<fnref n="1" />,<fnref n="2" /> ID |
Lili Cluzel-Burgalat<fnref n="2" /> | Irina Polovodova Asteman<fnref n="1" />,<fnref n="6" /> ID

**Correspondence**

Irina Polovodova Asteman, Department of Marine Science, University of Gothenburg, Gothenburg, Sweden.

Email: irina.polovodova@marine.gu.se

**Present address**

Marlene Wiechmann, Jördnära Miljökonsult AB, Lidköping, Sweden.

**Funding information**

Swedish Environmental Protection Agency and Swedish Agency for Marine and Water Management, Grant/Award Number: 802-0107-18

**Abstract**

The problem of invasive species is a well-studied one, but knowledge of free-living unicellular eukaryotic invasive species is lacking. A potentially invasive foraminifer (Rhizaria), *Nonionella* sp. T1, was recently discovered in the Skagerrak and its fjords. Digital polymerase chain reaction (dPCR) was applied to track the spread of this non-indigenous species using a new dPCR assay (T1-1). The use of dPCR appears highly complementary to traditional hand picking of foraminiferal shells from the sediment, and is far less time-consuming. This study indicates that *Nonionella* sp. T1 has bypassed the outer Skagerrak strait, instead becoming established in Swedish west coast fjords, constituting up to half of the living foraminiferal community in fjord mouth areas. The ecology of *Nonionella* sp. T1 and its potential invasive impacts are still largely unknown, but it appears to be an opportunist using several energy sources such as nitrate respiration and kleptoplasty along with a possibly more efficient reproductive strategy to gain an advantage over the native foraminiferal species. Future ecological studies of *Nonionella* sp. T1 could be aided by dPCR and the novel *Nonionella* sp. T1-specific T1-1 assay.

INTRODUCTION

Background and aims of the study

As anthropogenic effects on the natural environment continue to escalate, so does one of its more subtle and unpredictable consequences; the proliferation of invasive species across the globe. The establishment

rates of invasive species are steadily increasing, driven by both increasing temperatures (Huang et al., 2011; Mainka & Howard, 2010) and intensification of global trade (Hulme, 2021). The impact of invasive species on marine environments is complex and difficult to predict, generally having a negative effect on native biodiversity and ecosystems (Katsanevakis et al., 2014).

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Environmental Microbiology* published by Applied Microbiology International and John Wiley & Sons Ltd.

<footnote n="1">¹Department of Marine Science, University of Gothenburg, Gothenburg, Sweden</footnote>

<footnote n="2">²SeAnalytics AB, Gothenburg, Sweden</footnote>

<footnote n="3">³Laboratory of Planetology and Geosciences (LPG) UMR6112, University of Angers, CNRS, Angers, France</footnote>

<footnote n="4">⁴Laboratory of Planetology and Geosciences (LPG), Nantes University, CNRS, Nantes, France</footnote>

<footnote n="5">⁵Laboratory of Planetology and Geosciences (LPG), Le Mans University, CNRS, Le Mans, France</footnote>

<footnote n="6">⁶Gothenburg Global Biodiversity Centre, University of Gothenburg, Gothenburg, Sweden</footnote>
With ever increasing marine traffic, ships travelling across the oceans unintentionally carry with them various passenger species. Whether bored into the wooden hull or hidden in anchor mud, a result of these travels was the propagation of species, such as the clam *Teredo navalis* Linnaeus, 1758, whose now cosmopolitan distribution obscures their original native range. In a modern context, in part due to hull antifouling technology, potential invaders are instead transported mainly in ballast tanks. The immense amount of ballast water (and its associated sediment) carried by modern ships is likely responsible for transporting up to 10,000 species beyond their natural extents, every single day (Carlton, 1999).

The Scandinavian Skagerrak strait (North Sea) is heavily exposed to major shipping traffic and the associated with it risk of invasion. Indeed, just over the last two decades, several well-known today non-indigenous species (NIS) have established a predominant position in the ecosystems of the Skagerrak. Species such as the pacific oyster, *Crassostrea gigas* Thunberg, 1793 are known to create ecosystem-altering oyster reefs (Laugen et al., 2015) and the American ctenophore *Mnemiopsis leydi* A. Agassiz, 1865 reduces zooplankton biomass by predation (Riisgård, 2017).

At the same time, there is a severe lack of knowledge concerning invasive non-pathogenic eukaryotic microorganisms, despite their high abundance, widespread occurrence worldwide (Litchman, 2010), and documented presence in ballast tanks (Drake et al., 2005; Galil & Hülsmann, 1997). These types of invasions have been linked to degradation of benthic macroorganism communities (Gillis & Chalifour, 2009) and changes in marine biogeochemical cycles (Litchman, 2010). NIS foraminifera have primarily been reported in the eastern Mediterranean Sea (Deldicq et al., 2019), where the dramatic increase in alien species is explained by Lessepsian migration driven by increasing temperatures and shipping (Guastella et al., 2019; Zenetos et al., 2012). These foraminiferal invasions, particularly the genus *Amphistegina*, have altered the composition of benthic substrates and reduced native biodiversity (Mouanga & Langer, 2014). Another invasive foraminifer is the Japanese species *Trochammina hadai* Uchio, 1962 whose establishment has negatively impacted native foraminiferal diversity in North America (McGann et al., 2000) and Brazil (Eichler et al., 2018) and may have also arrived in western Swedish fjords (Brinkmann et al., 2023).

Research on invasive microorganisms in the Skagerrak is mainly concerned with those whose presence has a visible effect on macroscopic species, such as NIS phytoplankton with potential for dramatic blooms, for example, *Prorocentrum minimum* (Pavillard) J. Schiller, 1933 (Olenina et al., 2010), or economically detrimental pathogens like the parasitic protist *Marteilia*

*refringens* Grizel, Comps, Bonami, Cousserans, Duthoit, and Le Pennec, 1974, causing disease in farmed mussels and oysters (Swedish Agency for Marine and Water Management, 2011).

The ability to spread over large distances is aided in many microorganism groups by means of propagules, in the form of hardy resting stages, which can be transported over long distances and periods of time by ocean currents or in ballast water and sediments. This ability has been described in foraminifera (Alve & Goldstein, 2002, 2003), and large amounts of dormant forms of both native and several NIS foraminifera have been found in the deepest Skagerrak basin, likely deposited there by the north-easterly Jutland Current (Alve & Goldstein, 2010).

In 2013, living adult individuals of a non-indigenous foraminifer were discovered in surface sediment samples collected from the Skagerrak-Kattegat front area and the Gullmar fjord on the west coast of Sweden (Polovodova Asteman & Schönfeld, 2015). These individuals were initially identified based on shell morphology (see Figure 1A,B) as the eastern Pacific species *Nonionella stella* Cushman & Moyer, 1930. However, phylogenetic analyses using the foraminiferal barcoding marker (ribosomal DNA small subunit [SSU] 3' region, from 37F helix to V9, see Pawlowski & Holzmann (2014)) found a significant difference between the Pacific *N. stella* and the Skagerrak phylotype, which was subsequently given the provisional name *Nonionella* sp. T1 (Deldicq et al., 2019).

In addition to the discovery of *Nonionella* sp. T1 in the Gullmar fjord, several observations (identified as *N. stella* prior to 2019) have been made across the Skagerrak coasts; in the Oslo fjord and Kosterhavet Marine National Park in 2012 (Deldicq et al., 2019; Polovodova Asteman et al., 2021), in Dynekilen fjord in 2014 (Polovodova Asteman & Schönfeld, 2015), and further south in the Kattegat all the way to Öresund in 2013 (Charrieau et al., 2018). The first known occurrence of *Nonionella* sp. T1 in the Skagerrak was found re-examining a Gullmar fjord sediment core collected near the Alsbäck Deep in 2009, at a sediment core depth of 10–11 cm approximately corresponding to year 1985, seemingly following a hypoxic event in 1984 (Polovodova Asteman & Schönfeld, 2015).

Despite its now significant presence on the Swedish west coast (Charrieau et al., 2018; Choquel et al., 2021), little is known about the ecology of *Nonionella* sp. T1 and the larger implications of its apparent invasion of Scandinavian waters. The species has been shown to be infaunal, dwelling down to 6–10 cm below the sediment surface (Choquel et al., 2021; Polovodova Asteman & Schönfeld, 2015) and is capable of complete denitrification, even acting as the dominant benthic denitrifier in some areas of the Gullmar fjord (Choquel et al., 2021). Foraminifera are the first known marine eukaryotes to perform complete denitrification
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Scanning electron microscopy (SEM) micrographs of *Nonionella* sp. T1 from the Skagerrak, showing umbilical (A) and spiral (B) sides (Polovodova Asteman et al., 2021). Clearly seen in image (A) is the large, hand-like extension of the final shell chamber, a morphological feature that led to its initial identification as *Nonionella stella*, but is not sufficiently discriminant as it is shared by several nonionellid species, including *N. stella*, *Nonionella* sp. T1 and *Nonionella* sp. T4. Also shown is related species *Nonionellina labradorica* from the Gullmar fjord, with side (C) and aperture (D) views.</figcaption></figure>

(Risgaard-Petersen et al., 2006). *Nonionella* sp. T1 has also demonstrated the ability of kleptoplasty, with its cytoplasm containing isolated algal chloroplasts of yet unknown origin and utility (LeKieffre et al., 2018). The closely related species *Nonionellina labradorica* Dawson, 1860 (Figure 1C,D) has been shown to collect kleptoplasts from planktonic diatoms. The kleptoplasts in *N. labradorica* are not used for photosynthesis, but they may be important for aphotic foraminiferal metabolism of ammonium and sulphate (Jauffrais et al., 2019).

Recent advances in the field of molecular biology might provide more powerful tools for tracking invasive species. One such method is digital polymerase chain reaction (dPCR), a tool that is especially useful in the case of smaller and less visible invaders, like protists. These dPCR techniques can be used to find environmental DNA (eDNA) derived from living cells of microbial and tiny meiofaunal species (Pawlowski et al., 2020), to target specific species with known

sequences, and does not require complicated bioinformatic analyses. Previous studies have shown that, applied correctly, dPCR outperforms traditional techniques and previous real-time PCR methods in the detection of invasive organisms (Doi et al., 2015).

Taking advantage of this new technology and applying it to the problem of tracking unicellular eukaryotic NIS, the aim of this study is to design a dPCR assay targeting the eDNA traces left by the potentially invasive *Nonionella* sp. T1. This will allow mapping the presence of this NIS across the Skagerrak and its fjords, and investigating the possibility for the species natural transport pathways to the area with currents. Furthermore, the *Nonionella* sp. T1 populations in the Skagerrak and three Swedish fjords with different environments were analysed using both eDNA and manual morphospecies identification to further investigate *Nonionella* sp. T1 spread, ecology, and level of establishment in benthic areas with variable oxygen conditions.

## Study area

### Skagerrak

The investigated central part of the Skagerrak strait (Figure 2) is an area with heavy shipping traffic and three dominant water masses. The outflowing Norwegian Coastal Current transports coastal brackish water influenced by Swedish and Norwegian river runoff and outflow from the Baltic Sea. The inflowing Jutland Current brings North Sea water mixed with runoff from major European rivers into the Skagerrak and Kattegat. Finally, at depth, there is dense saline and nutrient-rich Atlantic water flowing in from the west. The interaction between these three currents causes a large-scale cyclonic circulation (Christensen et al., 2018).

## Fjords on the west coast of Sweden

The studied fjords included the Gullmar fjord, By fjord, and Hake fjord (Figure 3), which differ in their bottom water and sediment oxygenation. The Gullmar fjord is the deepest of the three with a maximum depth of 120 m, and a sill depth of 43 m. It is stratified, with seasonally hypoxic bottom water, relying on annual renewals of oxygenated deep water cascading over the fjord sill to avoid severe bottom water hypoxia with (O₂) < 1 mL/L (Filipsson & Nordberg, 2004).

The By fjord lies much further inland and has an almost permanently anoxic benthic environment. It is heavily stratified with a halocline near the sill depth of 13 m and has a maximum depth of 50 m (Stigebrandt et al., 2015).

The permanently oxic Hake fjord is not a true silled fjord, but the first in a series of straits connecting the
<figure><img src="image_4.png" /><figcaption>FIG URE 2 Map of the Hirtshals-Torungen transect study area showing dominant currents in the area. Sediment samples were taken at the eight stations labelled HT2–HT10. Bathymetric data by GEBCO Compilation Group (2022).</figcaption></figure>

Skagerrak-Kattegat region to the innermost By fjord. The sill in the Hake fjord is not well defined due to its great width and its depth of 25 m (Björk et al., 2000), thus favouring the regular water exchange in the fjord (Ahlfors, 1980). The dominant current along the coast, flowing past the sampled fjords, is the northward Baltic Current, which mixes with the Skagerrak and North Sea water and becomes the Norwegian Coastal Current, leaving the Skagerrak along the southern coast of Norway.

## EXPERIMENTAL PROCEDURES

### Sediment samples

Sediment samples were collected in October 2020 in the Skagerrak along a transect with stations stretching between Hirtshals in Denmark and Torungen in Norway (Figure 2). This transect crosses the Norwegian Trench, the deepest part of the Skagerrak, with a maximum depth of 700 m. The sediment at the sampled stations mostly consisted of oxygenated clay with a soft, soupy texture, and macroscopic infauna consisting of polychaetes and brittle stars.

Sediments in three fjords on the Swedish west coast were also sampled for this study in September and October 2021 onboard the R/V Skagerak. Surface sediment collected from the Gullmar fjord was light brown oxygenated clay containing polychaetes and brittle stars, with darker coloured clay at the deepest sampled station at the Alsbäck deep (Figure 3, see GC). Surface sediment samples collected from the By fjord were black and anoxic, although having an oxidized surface at the station furthest into the fjord at the mouth of the Bäveån river. In the permanently oxic Hake fjord, collected sediment consisted of olive-grey oxygenated clay, containing brittle stars, sea urchins, crustaceans, and shell fragments.

Surface sediment (0–3 cm) samples were collected for eDNA extraction and foraminiferal morphospecies analysis, by using Gemini corer, a modified version of Niemistö corer (Niemistö, 1974) with 8 cm inner diameter, onboard of the R/V Skagerak. The first samples for eDNA analysis were collected in October 2020 from a transect made up of eight stations between Hirtshals in Denmark and Torungen in Norway (Figure 2). The other sampling areas were three fjords on the Swedish west coast; the seasonally hypoxic Gullmar fjord, the permanently anoxic By fjord, and the permanently oxic
<figure><img src="image_5.png" /><figcaption>FIGURE 3 Map of the Swedish west coast study area, with its fjords and the sampling stations therein. From north to south; The Gullmar fjord (G-stations), the By FJORD (B-stations), and the Hake fjord (H-stations). The sediment samples were taken at the mouth (M), centre (C), and head (H) of each fjord. Bathymetric data by GEBCO Compilation Group (2022).</figcaption></figure>

Hake fjord. Surface sediment (0–3 cm) samples were taken in September and October 2021 by using a Gemini corer onboard of the R/V Skagerak from the mouth, centre, and head of each of these fjords, constituting another nine stations (Figure 3).

For eDNA analysis at all stations, 50 mL of surface sediment (0–3 cm) was collected in sterile Falcon tubes using sterile spoons and kept frozen at –20°C. The frozen samples were sent to the Laboratory of Planetology and Geosciences (University of Angers) in France, where DNA extraction was performed using a QIAGEN DNeasy PowerMax Soil Kit, according to manufacturer's instructions. In addition, at all stations, two to three replicates of the surface sediment (0–3 cm) per station were collected for foraminiferal morphospecies analysis, and stained with a Rose Bengal-ethanol solution (2 mg/L) in order to distinguish individuals alive at the moment of sampling from empty (sub-fossil) shells, according to protocol of Schönfeld et al. (2012).

Additionally, single-cell DNA extractions from *Nonionella* sp. T1 (OF002, OF004, and OF005, previously published in Deldicq et al. (2019)) were used as positive controls in eDNA analyses. To assess the

specificity of the dPCR assay, single-cell DNA extractions of the related species *N. labradorica* sampled from the Gullmar fjord in September 2011 at 113 m depth were also used as negative controls. Prior to dPCR both single cell and sediment DNA extractions were tested using foraminiferan primers s14F1 and J2 (Pawlowski et al., 2020) and bands were observed on the gel electrophoresis.

## dPCR assay design

A novel dPCR assay specific for *Nonionella* sp. T1 was designed in this study (Table 1). Partial sequences of SSU ribosomal RNA (rRNA) for *Nonionella* sp. T1 and four other nonionellids from the phylogenetic study by Deldicq et al. (2019) were downloaded from GenBank, 68 sequences in total, and aligned using Geneious software. Forward primer, reverse primer, and a fluorescent probe were designed with a perfect match to all *Nonionella* sp. T1 sequences, as many mismatches as possible to the other clades, and following dPCR primer and probe requirements according to Bio-Rad Droplet
<table><caption>TABLE 1 List of primer sequences and probe constituting the novel T1-1 assay.</caption><thead><tr><td>Type</td><td>Designation</td><td>Sequence (5'-3')</td></tr></thead><tbody><tr><td>Forward primer</td><td>T1-44F</td><td>CTTTATGCTTCGGTATAAAGCTGC</td></tr><tr><td>Reverse primer</td><td>T1-181R</td><td>CGTAAGCAAAACCTAGTTAAAGGC</td></tr><tr><td>Fluorescent probe</td><td>T1-P1</td><td>FAM-CGTCAAACCTTGCTATCTGCGTATA-BHQ1</td></tr></tbody></table>

Note: These primers target a 115 bfragment of the *Nonionella* sp. T1 SSU rRNA gene. The probe is labelled with FAM dye at the 5' end and has the Black Hole Quencher modification at the 3' end.

Digital PCR Applications Guide (Bio-Rad Laboratories, Inc., n.d.). These primers (listed in Table 1) amplify a 115 bp-long fragment of the SSU rRNA gene, with the probe being complementary to this fragment.

To ascertain the specificity of the T1-1 assay, single-cell DNA extractions were tested, including three samples of *Nonionella* sp. T1 and three samples of *N. labradorica* (mentioned above). A trial run of the dPCR method yielded positive results for all *Nonionella* sp. T1 samples, and zero positives for *N. labradorica*. These results are shown in Figure S1 as an example of dPCR output.

## Foraminiferal eDNA analysis by dPCR

The measurements were performed for sediment-extracted eDNA samples from eight Skagerrak Hirtshals-Torungen transect stations and the nine fjord stations. Each sample was measured twice using the QIAGEN QIAcuity One Digital PCR System and QIA-GEN QIAcuity Nanoplate 26,000-partition 24-well microfluidic dPCR plate; 2 plates in total. In addition to the samples, both plates contained a positive control (single-cell DNA extraction OF004 for *Nonionella* sp. T1) and 2–3 negative (non-template) controls.

Total DNA concentrations in the sediment-extracted eDNA samples were measured by using the Invitrogen Qubit fluorometer dsDNA assays. Following concentration measurements, dPCR was performed according to the following protocols for dPCR mixture in a well volume of 40 µL; for sample wells: 10 µL QIAGEN QIAcuity Probe (4×) Master Mix, 2 µL T1-1 (20×) assay, 18 µL nuclease-free water, and 10 µL of sediment-extracted eDNA sample. For positive control wells: 10 µL QIAGEN QIAcuity Probe (4×) Master Mix, 2 µL T1-1 (20×) assay, 27 µL nuclease-free water, and 1 µL of single cell-extracted *Nonionella* sp. T1 DNA sample. For negative control wells: 10 µL QIAGEN QIAcuity Probe (4×) Master Mix, 2 µL T1-1 (20×) assay, and 28 µL of nuclease-free water.

The PCR cycling consisted of an initial denaturation phase of 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 s, and annealing and extension at 58°C for 1 min. After PCR, images of the plate were obtained for the green colour channel for the FAM dye, using 300 ms of exposure with a gain of six.

## Foraminiferal morphospecies analysis

The sediment level of the Rose Bengal-ethanol stained surface sediment (0–3 cm) samples was marked for estimation of the sediment volume prior to sieving. The samples were then thoroughly shaken and thereafter washed through 1000- and 63-µm sieves for the fjord samples and 1000-, 125-, and 63-µm sieves for the Skagerrak transect samples. The residual sediment fractions >1000, >63, and > 125 µm were collected from the sieves onto separate Ahlstrom-Munksjö grade 1F filter paper and dried overnight at 50°C. Original sediment volumes in the sample bottles were measured by filling the bottles with water up to the marker lines, then pouring it into a measuring cylinder. After drying, fraction >1000 µm was screened for occasional occurrence of foraminifera and if present those were added to the total foraminiferal numbers.

The dried >63 and >125 µm residue was split into aliquots (see Table 3), transferred into glass vials, and labelled accordingly. Sample aliquots were examined by dry picking, with their contents evenly distributed onto a micropaleontological picking tray with a 5 × 9 grid. Using a stereomicroscope, randomly chosen squares were picked clean for living (stained) foraminifers until at least 300 individuals per aliquot (where possible) had been collected. For fjord samples, the 63–1000 µm fraction was analysed for foraminiferal morphospecies, whereas for the Skagerrak transect samples only the larger fraction 125–1000 µm was analysed and fraction 63–125 µm was kept as an archive.

Only *Nonionella* sp. T1 individuals were identified to the species level and all other foraminiferal species were noted as ‘others’. Absolute numbers (ind./10 mL sediment) of total living foraminifers and of *Nonionella* sp. T1, as well as relative (% of total living foraminifera) abundances of *Nonionella* sp. T1 were quantified. In the samples with highly abundant *Nonionella* sp. T1 (those included mainly fjord samples), its individuals were also sorted by ontogenetic stages defined by number of shell chambers, with adults having ≥10 chambers and juveniles with 6–9 chambers. As juvenile nonionellids are difficult to identify at the species level, the smallest individuals (with ≤5 chambers) were only noted as *Nonionella* sp. Results of foraminiferal morphospecies analysis are shown in Table 3.
# RESULTS

## dPCR analysis

The sampled sediment from the Skagerrak transect stations showed the highest total DNA concentration; 3.5–7.7 ng/µL along the Danish slope (HT2–HT4), very low concentration; 0.2–2.2 ng/µL in the central Norwegian Trench (HT5 and HT6), and slightly higher concentration; 1.1–3.5 ng/µL along the Norwegian slope (HT7.5–HT10; Table 2).

In the dPCR results for the *Nonionella* sp. T1 SSU rDNA, a single positive partition was observed in one of the non-template controls in each plate. Therefore, all samples with only one single positive partition were considered negative. *Nonionella* sp. T1 SSU rDNA was only detected at stations HT2 and HT5, with exceptionally low dPCR-measured concentrations at these stations, 0.10–0.15 copies/µL (Figure 4A and Table 2).

Sediment samples taken from the fjords on the Swedish west coast showed high total DNA concentration in both the Gullmar fjord; 6.1–15.0 ng/µL and in the By fjord; 10.4–14.4 ng/µL, with far lower values in

the Hake fjord; 0.06–0.16 ng/µL (Table 2). For the dPCR-measured *Nonionella* sp. T1-specific SSU rDNA, the highest average values were found in the Gullmar fjord; 0.75–34.89 copies/µL, with the exceptionally high value of 34.89 copies/µL at the centre station. In the By fjord sediments, *Nonionella* sp. T1 SSU rDNA was only found in extremely low concentration at the centre station; 0.10 copies/µL. *Nonionella* sp. T1 SSU rDNA values were higher in the Hake fjord; 0.26–2.14 copies/µL, despite being the location with the lowest overall DNA concentration (Figure 4A and Table 2).

The values for fluorometer-measured total DNA concentration show no significant correlation with the results of the dPCR-measured DNA concentrations (Figure 5), with a calculated Pearson's correlation coefficient between the results of the two methods of; *r* (14) = 0.087 (*p* > 0.7). This is likely due to the total DNA extracted from sediment samples being mainly represented by bacterial DNA.

The three single-cell extractions of *Nonionella* sp. T1 used for positive control in dPCR runs had total DNA concentrations of 0.17, 0.56, and 0.34 ng/µL. The number of target DNA copies per microlitre reaction as

<table><caption>TABLE 2 List of samples and sampling areas, with fluorometer-measured DNA concentrations for each sample, as well as calculated original concentrations of *Nonionella* sp. T1 DNA, averaged between replicate wells.</caption><thead><tr><th>Sample name</th><th>Sampling area</th><th>Longitude</th><th>Latitude</th><th>Sampling date</th><th>Water depth (m)</th><th>DNA concentration (ng/µL)</th><th>Nonionella sp. T1 DNA concentration (copies/µL)</th></tr></thead><tbody><tr><td>HT2</td><td>Hirtshals-Torungen transect</td><td>57° 50,55'N</td><td>009° 34,79' E</td><td>19 October 2020</td><td>67</td><td>7.24</td><td>0.10</td></tr><tr><td>HT3</td><td>Hirtshals-Torungen transect</td><td>57° 56,20'N</td><td>009° 27,03' E</td><td>19 October 2020</td><td>192</td><td>3.55</td><td>0</td></tr><tr><td>HT4</td><td>Hirtshals-Torungen transect</td><td>57° 59,45'N</td><td>009° 22,69' E</td><td>19 October 2020</td><td>374</td><td>7.72</td><td>0</td></tr><tr><td>HT5</td><td>Hirtshals-Torungen transect</td><td>58° 03,11'N</td><td>009° 17,26' E</td><td>20 October 2020</td><td>542</td><td>0.25</td><td>0.15</td></tr><tr><td>HT6</td><td>Hirtshals-Torungen transect</td><td>58° 4564'N</td><td>009° 15,277' E</td><td>21 October 2020</td><td>603</td><td>2.24</td><td>0</td></tr><tr><td>HT7,5</td><td>Hirtshals-Torungen transect</td><td>58° 13, 57'N</td><td>009° 02,46' E</td><td>21 October 2020</td><td>441</td><td>3.53</td><td>0</td></tr><tr><td>HT9</td><td>Hirtshals-Torungen transect</td><td>58° 18,938'N</td><td>008° 54,786' E</td><td>21 October 2020</td><td>302</td><td>1.13</td><td>0</td></tr><tr><td>HT10</td><td>Hirtshals-Torungen transect</td><td>58° 21,460'N</td><td>008° 51,149' E</td><td>21 October 2020</td><td>188</td><td>2.70</td><td>0</td></tr><tr><td>GM</td><td>Gullmar fjord, mouth</td><td>58° 15,750'N</td><td>11° 27.447' E</td><td>28 September 2021</td><td>59</td><td>15.00</td><td>2.97</td></tr><tr><td>GC</td><td>Gullmar fjord, centre</td><td>58° 19.399'N</td><td>11° 32.772' E</td><td>29 September 2021</td><td>118</td><td>6.68</td><td>34.89</td></tr><tr><td>GH</td><td>Gullmar fjord, head</td><td>58° 25.816'N</td><td>11° 40.494' E</td><td>29 September 2021</td><td>28</td><td>6.08</td><td>0.75</td></tr><tr><td>BM</td><td>By fjord, mouth</td><td>58° 19.645'N</td><td>11° 51.525' E</td><td>28 September 2021</td><td>10</td><td>10.4</td><td>0</td></tr><tr><td>BC</td><td>By fjord, centre</td><td>58° 19.901'N</td><td>11° 52.531' E</td><td>27 September 2021</td><td>47</td><td>14.40</td><td>0.10</td></tr><tr><td>BH</td><td>By fjord, head</td><td>58° 20,666'N</td><td>11° 54,467' E</td><td>27 September 2021</td><td>11</td><td>13.00</td><td>0</td></tr><tr><td>HM</td><td>Hake fjord, mouth</td><td>57° 55.815'N</td><td>11° 40,709' E</td><td>1 October 2021</td><td>42</td><td>0.16</td><td>1.80</td></tr><tr><td>HC</td><td>Hake fjord, centre</td><td>57° 57.821'N</td><td>11° 43,257' E</td><td>30 September 2021</td><td>31</td><td>0.06</td><td>2.14*</td></tr><tr><td>HH</td><td>Hake fjord, head</td><td>58° 02.843'N</td><td>11° 47,315' E</td><td>30 September 2021</td><td>21</td><td>0.15</td><td>0.26</td></tr></tbody></table>

Note: Value marked * had only one valid replicate, due to incomplete filling of well partitions.
<figure><img src="image_6.png" /><figcaption>FIGURE 4 (A) Results of dPCR analysis of sediment-extracted eDNA samples from both the fjord stations and the Hirtshals-Torungen transect stations. All stations are listed on the X-axis. On the Y-axis are the calculated original *Nonionella* sp. T1 DNA concentrations in number of copies per microlitre. A logarithmic scale was chosen to visualize very low concentrations. (B) Results of morphospecies-based analysis of *Nonionella* sp. T1 foraminiferal concentrations (ind./10 mL sediment) across all stations as listed on the X-axis. On the Y-axis are the average values for concentration of living *Nonionella* sp. T1 cells at every sampling station, in number of individuals/10 mL of sampled sediment.</figcaption></figure>

measured by dPCR was 31.8, 122.4, and 0.83 correspondingly. Considering the reaction volume (40 µL) and the total DNA extraction volume (50 µL), this gives the estimates of 63,600, 244,700, and 1705 SSU rDNA copies per cell.

## Morphospecies analysis

For the morphospecies analysis of the fjord sediment samples, the highest total concentration of living foraminifera in the Gullmar fjord was 473–3589 (average value 1757) individuals/10 mL sediment, as well as the highest concentration of *Nonionella* sp. T1 individuals; 15–1372 (average value 351) individuals/10 mL sediment. The highest average values for both measurements were found at the mouth of the fjord, and the lowest at the head. In the By fjord, extremely few living

foraminifers were found at the mouth and centre stations; 0.3–1.3 (average value 1) individuals/10 mL sediment, with a higher concentration at the head of the fjord; 499–897 (average value 672) individuals/10 mL sediment. There were no *Nonionella* sp. T1 individuals found in the By fjord samples. Finally, in the Hake fjord, total concentrations of living foraminifera were 309–1167 (average value 630) individuals/10 mL sediment, and *Nonionella* sp. T1-specific concentrations showed a range of 42–493 (average value 235) individuals/10 mL sediment, with highest average values for both measurements at the centre of the fjord, and the lowest at the head (Figure 4B and Table 3).

Of all sampled areas, the location with the largest proportion of *Nonionella* sp. T1 in foraminiferal population was the Hake fjord, where 12%–63% (average value 36%) of all found foraminifers were *Nonionella* sp. T1. The Gullmar fjord had a larger absolute number
<figure><img src="image_7.png" /><figcaption>FIGURE 5 Relationship between fluorometer-measured total DNA concentration in nanogram per microlitre on the X-axis, versus digital polymerase chain reaction-measured *Nonionella* sp. T1-specific DNA concentration on the Y-axis. Each circle represents a sampling station. The values show no significant correlation; Pearson's *r*(14) = 0.087, *n* = 16, *p* > 0.7. Station GC represents an outlier (z-score = +3.858) and has been omitted in this figure, due to a measured DNA concentration an order of magnitude greater than all other stations.</figcaption></figure>

of *Nonionella* sp. T1, but they only made up 3%–38% (average value 15%) of the total foraminiferal population (Figure 6 and Table 3). The majority (86%) of all found *Nonionella* sp. T1 individuals were juvenile.

The results of morphospecies-based analysis of foraminiferal concentrations (ind./10 mL sediment) correlated well with dPCR-measured *Nonionella* sp. T1 SSU rDNA concentration results for the fjord stations (Figure 7), with a calculated Pearson's correlation coefficient of *r*(6) = 0.998 (*p* < 0.001) with an exception for one outlier, the GC station from the centre of the Gullmar fjord. This sample had a very high target DNA concentration measured by dPCR (34.89 copies/µL) but only an average of 387 *Nonionella* sp. T1 cells per millilitre sediment found in the morphological analysis.

Using the number of *Nonionella* sp. T1 cells identified in the morphological analyses, the average number of SSU rDNA copies per cell can also be estimated for the sediment samples. The average number of SSU rDNA copies in five sediment samples from the Gullmar fjord and Hake fjord (excluding the deviating GC sample) varied between 102 and 139 copies per cell.

In the Skagerrak transect samples, no living *Nonionella* sp. T1 individuals were found. Out of 9083 total living and dead foraminifera counted along the transect, only one single, dead (non-stained) *Nonionella* sp. T1 individual was found at station HT2 (depth 67 m). However, eDNA of *Nonionella* sp. T1 was detected in HT2 and HT5, suggesting the presence of propagules or DNA of dead specimens picked up by dPCR analysis.
<table><caption>TABLE 3 List of sediment samples prepared for foraminiferal morphospecies analysis showing foraminiferal concentrations (individuals/10 mL sediment) and proportions (% of the assemblages).</caption><thead><tr><th>Sample name</th><th>Sample depth (cm)</th><th>Stain</th><th>Sediment volume (mL)</th><th>Split</th><th>Foram. Concentration (ind./10 mL sediment)</th><th>Nonionella sp. T1 (ind./10 mL sediment)</th><th>Nonionella sp. T1 (%)</th><th>T1 juvenile (% of T1)</th><th>N. labradorica (ind./10 mL sediment)</th><th>N. labradorica (%)</th></tr></thead><tbody><tr><td>GM-A</td><td>0-3</td><td>RB</td><td>37</td><td>½</td><td>2019</td><td>170</td><td>8</td><td>100</td><td>55</td><td>3</td></tr><tr><td>GM-B</td><td>0-3</td><td>RB</td><td>41</td><td>½</td><td>3589</td><td>1372</td><td>38</td><td>98</td><td>55</td><td>2</td></tr><tr><td>GM-C</td><td>0-3</td><td>RB</td><td>49</td><td>½</td><td>2173</td><td>122</td><td>6</td><td>100</td><td>61</td><td>4</td></tr><tr><td>GC-A*</td><td>0-3</td><td>RB</td><td>44</td><td>ns</td><td>3498</td><td>1043</td><td>30</td><td>—</td><td>0</td><td>0</td></tr><tr><td>GC-B*</td><td>0-3</td><td>RB</td><td>45</td><td>½</td><td>473</td><td>15</td><td>3</td><td>—</td><td>0</td><td>0</td></tr><tr><td>GC-C*</td><td>0-3</td><td>RB</td><td>34</td><td>ns</td><td>1171</td><td>103</td><td>9</td><td>—</td><td>0</td><td>0</td></tr><tr><td>GH-A</td><td>0-3</td><td>RB</td><td>48</td><td>¼</td><td>1477</td><td>117</td><td>8</td><td>92</td><td>9</td><td>1</td></tr><tr><td>GH-B</td><td>0-3</td><td>RB</td><td>36</td><td>¼</td><td>707</td><td>102</td><td>14</td><td>91</td><td>7</td><td>1</td></tr><tr><td>GH-C</td><td>0-3</td><td>RB</td><td>37</td><td>¼</td><td>704</td><td>116</td><td>16</td><td>88</td><td>7</td><td>1</td></tr><tr><td>BM-A</td><td>0-3</td><td>RB</td><td>33</td><td>ns</td><td>0.6</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BM-B</td><td>0-3</td><td>RB</td><td>30</td><td>ns</td><td>0.3</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BM-C</td><td>0-3</td><td>RB</td><td>32</td><td>ns</td><td>1.3</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BC-A</td><td>0-3</td><td>RB</td><td>54</td><td>ns</td><td>0.7</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BC-B</td><td>0-3</td><td>RB</td><td>45</td><td>ns</td><td>0.4</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BC-C</td><td>0-3</td><td>RB</td><td>46</td><td>ns</td><td>0.7</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BH-A</td><td>0-3</td><td>RB</td><td>51</td><td>½</td><td>897</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BH-B</td><td>0-3</td><td>RB</td><td>42</td><td>½</td><td>499</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>BH-C</td><td>0-3</td><td>RB</td><td>28</td><td>½</td><td>621</td><td>0</td><td>0</td><td>—</td><td>0</td><td>0</td></tr><tr><td>HM-A</td><td>0-3</td><td>RB</td><td>48</td><td>½</td><td>523</td><td>274</td><td>52</td><td>71</td><td>0</td><td>0</td></tr><tr><td>HM-B</td><td>0-3</td><td>RB</td><td>60</td><td>½</td><td>598</td><td>377</td><td>63</td><td>88</td><td>0</td><td>0</td></tr><tr><td>HM-C</td><td>0-3</td><td>RB</td><td>46</td><td>½</td><td>562</td><td>318</td><td>57</td><td>91</td><td>0</td><td>0</td></tr><tr><td>HC-A*†</td><td>0-3</td><td>RB</td><td>66</td><td>—</td><td>—</td><td>—</td><td>41</td><td>—</td><td>0</td><td>0</td></tr><tr><td>HC-B*</td><td>0-3</td><td>RB</td><td>56</td><td>½</td><td>1167</td><td>267</td><td>23</td><td>—</td><td>0</td><td>0</td></tr><tr><td>HC-C*</td><td>0-3</td><td>RB</td><td>73</td><td>¼</td><td>1159</td><td>493</td><td>43</td><td>—</td><td>0</td><td>0</td></tr><tr><td>HH-A</td><td>0-3</td><td>RB</td><td>66</td><td>½</td><td>309</td><td>58</td><td>19</td><td>80</td><td>0</td><td>0</td></tr><tr><td>HH-B</td><td>0-3</td><td>RB</td><td>49</td><td>½</td><td>365</td><td>54</td><td>15</td><td>73</td><td>1</td><td>0,4</td></tr><tr><td>HH-C</td><td>0-3</td><td>RB</td><td>62</td><td>½</td><td>354</td><td>42</td><td>12</td><td>69</td><td>0</td><td>0</td></tr></tbody></table>

Note: Samples marked “*” were not split. In samples marked “*”, juvenile and adult individuals were not counted separately. Sample marked “†” was analysed with a wet-picking in a partition of unknown size, preventing foraminifer concentration calculations.
<figure><img src="image_8.png" /><figcaption>FIGURE 6 Proportions of *Nonionella* sp. T1 in the living foraminiferal assemblages of the sampled fjord stations listed on the X-axis. On the Y-axis is the proportion of *Nonionella* sp. T1, expressed as a percentage of the total number of found living foraminifers.</figcaption></figure>

# DISCUSSION

## Distribution and ecology of *Nonionella* sp. T1

The lack of *Nonionella* sp. T1 DNA in the dPCR results from the Skagerrak Hirtshals-Torungen transect is supported by the foraminiferal morphospecies analysis. Charrieau et al. (2018) did not find any *Nonionella* sp. T1 either, at a similar location in the open Skagerrak. This suggests that the few positive indications of *Nonionella* sp. T1 DNA along the Hirtshals-Torungen transect might come from dead individuals or dormant propagules transported to these deeper waters by the currents of the Skagerrak, highlighting high sensitivity of dPCR method.

Absence of *Nonionella* sp. T1 in the open Skagerrak also fits well with the ballast water transport hypothesis. Since shipping vessels generally only discharge or take on ballast water while loading or unloading cargo, respectively, transport of NIS is likely to occur to or from industrial harbours such as those on the Swedish west coast; for example, in Lysekil, Uddevalla, and Gothenburg, which all lie in the vicinity of the investigated fjords. Lysekil harbour, specifically, lies near the earliest known occurrence of *Nonionella* sp. T1 in the Gullmar fjord and stands out as a possible origin of their subsequent spread to the Skagerrak, which could have been aided by continued shipping traffic and propagule dispersion by currents.

The almost complete absence of foraminifera observed by morphospecies analysis at the By fjord centre and mouth stations is to be expected due to the long-lasting and widespread benthic anoxia creating a

hostile environment at this location (Stigebrandt et al., 2015). Neither oxygen respiration nor denitrification by foraminifera is viable here, since nitrification and therefore availability of nitrate is also limited due to lack of oxygen. Despite this, the By fjord has remarkably high total concentrations of sediment-extracted DNA, likely due to (1) the anoxic conditions preserving DNA of dead organisms in the sediment (Corinaldesi et al., 2011) and (2) presence of abundant bacteria in anoxic sediments (Mori et al., 2021).

The foraminiferal morphological assemblage at the By fjord head station consisted mostly of highly tolerant intertidal *Ammonia* species, some of which are able to metabolize under anoxia (Nardelli et al., 2014). The lack of foraminifera at the other By fjord stations suggests that the foraminiferal community at the shallow head station is sustained by supplied oxygen from the adjacent Bäveån river.

In the seasonally depleted oxygen conditions of the Gullmar fjord, sediment cores show that *Nonionella* sp. T1 appears in its largest abundances at depths corresponding to periods of severe winter hypoxic events (Polovodova Asteman & Schönfeld, 2015). Their establishment at this location suggests an opportunistic, r-selection-driven ecology taking advantage of this fluctuating environment. *Nonionella* sp. T1 contributes the majority of the total denitrification in areas of the Gullmar fjord (Choquel et al., 2021). This, in combination with heterotrophy and kleptoplasty (LeKieffre et al., 2018), suggests that the species is an effective mixotroph taking advantage of several sources of energy, constituting a competitive advantage.

The location where *Nonionella* sp. T1 constituted the largest proportion of the foraminiferal community
<figure><img src="image_9.png" /><figcaption>FIG URE 7 Relationship between results of fjord station digital polymerase chain reaction-measured *Nonionella* sp. T1 DNA concentrations in copies per microlitre on the X-axis versus concentration of *Nonionella* sp. T1 cells found by morphospecies analysis, in individuals/10 mL sediment, on the Y-axis. Each circle represents a fjord station. The values show a significant positive correlation; Pearson's *r*(6) = 0.998, *n* = 8, *p* < 0.001. Station GC represents an outlier (z-score = +2.655) and has been omitted in this figure, due to a measured DNA concentration an order of magnitude greater than the other fjord stations.</figcaption></figure>

was the comparatively hospitable Hake fjord with well-oxygenated bottoms, also demonstrating a capacity of the species to prosper under benign conditions. The oxygen-rich sediments of the Hake fjord contained extremely little total DNA, which is likely caused, in part, by the presence of abundant dissolved oxygen causing the breakdown of DNA from dead organisms (Corinaldesi et al., 2008, 2011) or by lower bacterial abundances (Mori et al., 2021).

Contributing ecological factors for the apparent lack of success for *Nonionella* sp. T1 establishing itself in the open Skagerrak, especially in the Norwegian Trench, are likely the greater water depth and lack of labile organic carbon. In the Norwegian Trench, ~90% of

sedimentation-deposited organic carbon is allochthonous and likely refractory (de Haas & van Weering, 1997; Diesing et al., 2021), making it difficult to utilize for heterotrophic foraminifera (Fontanier et al., 2008). However, the Skagerrak-Kattegat front with recurrent algal blooms, and fjord basins exposed to eutrophication (Boesch et al., 2006; Håkansson, 2003), would readily provide useful organic carbon for benthic meiofauna. Hence, the shallower Kattegat and Swedish west coast fjords might also provide a more dynamic and fluctuating environment for the potentially opportunistic *Nonionella* sp. T1 than the deep, stable environment of the Norwegian Trench, where well-established native open ocean species might be too much competition for the newcomer.
Finally, the high proportion of juvenile *Nonionella* sp. T1 observed in fjord samples suggests a recent reproduction event in late September, an autumn reproduction peak possibly taking place earlier than those of competing local foraminifera such as *Stainforthia fusiformis* Williamson, 1858 and *Elphidium* spp. (Gustafsson & Nordberg, 1999, 2000). With other apparent reproduction events having taken place in late May and June (I. Polovodova Asteman, personal communication), multiple early reproduction events per year, especially occurring during phytoplankton blooms supplying organic carbon in shallow areas, might be a more efficient reproductive strategy and another competitive advantage for *Nonionella* sp. T1. Yet another advantage allowing quick reproduction and growth of *Nonionella* sp. T1 can be related to its thin shells, which similarly to the opportunistic *S. fusiformis* likely require less time and energy to precipitate (Alve, 1994), contributing to rapid proliferation.

## Evaluation of dPCR to detect invasive foraminiferal species

In this study, dPCR using the *Nonionella* sp. T1-specific assay proved itself to be a quick and practical technique for detecting the presence of a non-indigenous foraminifer across large marine areas. The method detected even very low ($<$1 copy/µL) concentrations of the target DNA. Further, the measured *Nonionella* sp. T1 DNA concentrations did not correlate with total DNA concentrations in the samples (Figure 5), showing that the method is not sensitive to potential variation in quantity and quality of DNA extracted from sediment samples.

The initial comparison with a traditional hand-picking technique seems to show an astonishing degree of accordance (Figure 7), with a puzzling exception at GC, the centre station of the Gullmar fjord. This sampling station had a measured *Nonionella* sp. T1 SSU rDNA concentration of 34.89 copies/µL, a value more than 10 times larger than the second highest concentration (2.97 copies/µL, at the GM station), while the morphological analysis found fewer *Nonionella* sp. T1 individuals at station GC (~387 ind./10 mL) than at the GM station (~555 ind./10 mL). An explanation for this discrepancy could be that adult *Nonionella* sp. T1 were about to reproduce, therefore they were full of gametes and DNA, which were still present in single cell. This would correspond with an early fall reproduction event hypothesis. Also, a higher abundance of *Nonionella* sp. T1 observed at GM site is consistent with the results of Choquel et al. (2021), who sampled in September and November and also reported higher *Nonionella* sp. T1 morphospecies abundance proximal to the fjord mouth as compared to its deepest basin.

The GC station stands out as the deepest fjord station, but despite the sampled sediment having a darker colour, it was still oxygenated, making anoxic accumulation of eDNA similar to the By fjord unlikely. This exceptionally dense cluster of *Nonionella* sp. T1 DNA likely consists of dormant propagules 'raining down' from shallower fjord slopes and accumulating in the deepest basin (Brinkmann et al., 2023).

Depending on the goals of a dPCR-assisted study on protist distribution, the ability to detect propagules could be either positive or negative. A study interested only in mature or developing individuals might get skewed results in locations with unfavourable conditions where high amounts of dormant propagules accumulate. To determine whether a positive dPCR result is caused by the presence of developed individuals or propagules, manual examination for morphospecies analysis would still be necessary. On the other hand, analysing areas with significant propagule accumulation might provide insight into biodiversity of sediment propagule banks, as well as species transport by currents and future distribution patterns. For example, a study by Brinkmann et al. (2023) has analysed Swedish fjord sediments and found eDNA of several exotic foraminiferal species, which have not been described to date from the Swedish west coast as morphospecies (e.g., *Nonionella* sp T4 from off Namibian coast, *N. stella* from off California and *T. hadai*, an invasive Japanese foraminifer).

The use of dPCR for absolute quantification of the number of individual foraminifera in a sampled area, similar to manual morphospecies analysis, is not straightforward. In general, the number of copies of the SSU rDNA varies immensely between different groups of protists, and even within the same species it may scale exponentially with cell volume (Fu & Gong, 2017). Foraminifera are likely to represent an extreme case of this variation due to their complex life cycle involving endoreplication (Goetz et al., 2022). Endoreplication occurs both at haploid and diploid stages and may involve duplications of the whole genome or its parts. As a result, DNA content in foraminiferal cells greatly depends on the life stage and cell size (Goetz et al., 2022).

Using the three single-cell samples of *Nonionella* sp. T1, estimates were obtained ranging from 1700 to 244,000 copies of the SSU rDNA per cell, confirming a large variation between individuals. This upper range might appear quite high, but similar estimates have been reported for other species. Recently, Milivojević et al. (2021) found that SSU rDNA gene copy numbers in planktonic foraminifera could vary between ~300 and 370,000 copies in different individuals of the same species. Previously, Weber and Pawlowski (2013) estimated the number of SSU rDNA copies in three foraminiferal species to be 10,000–30,000 in specimens of average size. The high end of these SSU rDNA copy
number estimates are comparable to those measured
in ciliates, which are believed to be the protist group
with the largest known numbers of rDNA copies (Gong
et al., 2013), with average values above 560,000 cop-
ies/individual in some cases (Wang et al., 2017). Since
only three individuals were analysed in the present
study the results should be treated with caution; future
studies on more individuals at different metabolic
stages are required to determine the SSU rDNA copy
number range in *Nonionella* sp. T1. A combination of
dPCR with other methods such as staining nuclei, is a
promising tool to study genome dynamics during the life
cycle in *Nonionella* and other foraminifera.

Interestingly, the average number of SSU rDNA
copies estimated in the sediment samples was much
lower and showed much less variation than the single-
cell samples: 102–139 copies/cell. These low copy
numbers likely reflect the population structure of *Nonio-
nella* sp. T1 at the moment of sampling. The low varia-
tion suggests that the population structure might be
stable, with the majority of cells being juveniles
(as observed in the morphospecies analysis) or propa-
gules. The number of *Nonionella* sp. T1 SSU rDNA
copies obtained by dPCR in the sediment samples can
potentially be calibrated, and translated to number of
individuals using only a few morphospecies analyses.

Future prospects

Even as a clearer picture of the distribution of *Nonio-
nella* sp. T1 is emerging, there is still very little known
about its ecology. Predicting the potential invasive
impacts of *Nonionella* sp. T1 in the Skagerrak-Kattegat
area and its fjords requires further insight into its life
cycle, seasonality and environmental influence, and
into the general characteristics of invasive non-
pathogenic protists. Future studies on *Nonionella*
sp. T1 could examine its reproduction time, seasonal
dynamics, and temporal distribution to study ecological
effects at the peaks of the population.

It has been suggested that the effect of *Nonionella*
sp. T1 on the ecosystem might be a positive one.
According to Choquel et al. (2021), the significant ben-
thic denitrification performed by *Nonionella* sp. T1 could
help reduce eutrophication in locations like the Gullmar
fjord, and actually contribute to a healthier ecosystem.
However, the lack of knowledge regarding to which
degree the presence of *Nonionella* sp. T1 disrupts
native ecosystem components and services makes any
positive effects potentially insignificant. The remarkable
ability of *Nonionella* sp. T1 to denitrify might affect
native denitrifiers, including bacteria, which in turn
could alter biogeochemical cycles causing habitat
changes potentially impacting not only the native fora-
miniferal communities, but the entire benthic
ecosystem.

CONCLUSIONS

The dPCR and morphological results of this study pro-
vide evidence that the NIS *Nonionella* sp. T1 is gener-
ally absent from the open Skagerrak. Morphospecies
analysis demonstrates that it has a significant presence
in the Swedish Gullmar and Hake fjords, comprising on
average 15% of the living foraminiferal community in
the Gullmar fjord and 36% in the Hake fjord. The results
of both molecular and morphological methods indicate
that *Nonionella* sp. T1 is completely absent from the By
fjord. Overall, both datasets show a high level of agree-
ment between dPCR and morphospecies analyses.

*Nonionella* sp. T1 seems to be an opportunistic, highly adaptable species, utilizing heterotrophy, denitrification, and an efficient reproductive strategy to thrive in a variety of locations. These factors have likely allowed it to gain a competitive advantage over native foraminifera and facilitated its successful establishment on the Swedish west coast. The subsequent spread of *Nonionella* sp. T1 along Skagerrak coasts and into the Kattegat could be attributed to the ability to spread with currents by means of propagules. However, its absence in the central Skagerrak further indicates that it did not originally arrive at the Swedish west coast carried by currents, instead likely being introduced by shipping traffic.

Despite the seemingly adaptable nature of *Nonio-
nella* sp. T1, the depth and possible lack of organic car-
bon from primary production in the central Skagerrak
appear to preclude its establishment there. Similar dee-
per basins, like the Alsbäck deep in the Gullmar fjord,
might be accumulation sites for propagules that may or
may not develop into adults. These propagules would
also be detected by dPCR, necessitating care in inter-
preting results at such sites.

Fulfilling the aim of this study, dPCR has proven to be
an efficient novel method of tracking foraminifera, and the
new *Nonionella* sp. T1-specific assay can be helpful in
the further study of *Nonionella* sp. T1 distribution and
ecology, while being highly compatible with traditional
picking methods. More generally, further developments in
the use of dPCR and molecular biology techniques might
help solve the elusive problem of invasive microorgan-
isms, and shed light on these hidden aliens.

AUTHOR CONTRIBUTIONS

**Filip Morin:** Writing—original draft; methodology; formal analysis; visualisation; investigation. **Irina Polovodova Asteman, and Marina Antonina Zoe Panova:** Conceptualisation; methodology; investigation; writing—review and editing; supervision. **Magali Schweizer:** Methodology; investigation; writing—review and editing. **Marlene Wiechmann, Nicole Eliassen, and Lili Cluzel-Burgalat:** Formal analysis; investigation. **Per Sundberg:** Resources; conceptualisation; and methodology.
ACKNOWLEDGEMENTS

The authors would like to thank the crew and captain of R/V Skagerak; Martin Hassellöv, Karin Mattsson, Kjell Nordberg, Per Hall, Astrid Hylén, Sebastiaan van de Velde, and all other participants of the FACTS expedi- tion 2020 and the Swedish fjord expedition 2021 for collecting sediment samples in the field. Sophie Quinchard (LPG, University of Angers, France) is acknowledged for her technical help in molecular biol- ogy. We are thankful for financial support from the Swedish Environmental Protection Agency and Swed- ish Agency for Marine and Water Management (con- tract 802-0107-18) to Per Sundberg. Two anonymous reviewers provided positive feedbacks with constructive and thoughtful comments, which helped to improve the article. The authors received financial support from Swedish Environmental Protection Agency and Swed- ish Agency for Marine and Water Management grant no: 802-0107-18.

CONFLICT OF INTEREST STATEMENT

All authors declare that they have no conflicting interests.

DATA AVAILABILITY STATEMENT

Primer and probe sequences constituting the dPCR assay used in this project are listed in Table 1. Bathy- metric data from the GEBCO 2022 grid are available at https://doi.org/10.5285/e0f0bb80-ab44-2739-e053-6c86 abc0289c.

ORCID

Marina Antonina Zoe Panova https://orcid.org/0000-0002-4147-6473

Magali Schweizer https://orcid.org/0000-0001-5115-1616

Per Sundberg https://orcid.org/0000-0001-6726-0075

Irina Polovodova Asteman https://orcid.org/0000-0001-7300-5548

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Morin, F., Panova, M.A.Z., Schweizer, M., Wiechmann, M., Eliassen, N., Sundberg, P. et al. (2023) Hidden aliens: Application of digital PCR to track an exotic foraminifer across the Skagerrak (North Sea) correlates well with traditional morphospecies analysis. *Environmental Microbiology*, 1–17. Available from: https://doi.org/10.1111/1462-2920.16458