To extract the DNA from organisms, a direct lysis approach, utilizing WLB was employed (per 100 µl: 10 µl of 10xTAqBuffer without MgCl2 (Evrogen), 10 µl of Proteinase K (10 mg/ml), 10 µl of Chelex aqueous solution (5% w/v) in 96-well plates (dried at 60 °C), addition of 50 µl of WLB, incubation at 56 °C for 1 hour, and at 98 °C for 10 minutes). PCR was carried out with HS-ScreenMix (Evrogen) and specific primers (Table 2). Mitochondrial loci targeted included cytochrome c oxidase subunit I (COI), cytochrome B (cytB), 16S ribosomal RNA (16S), and the small (18S) rRNA subunit of nuclear DNA. The genetic diversity of specimens morphologically identified as A. tonsa was evaluated by amplifying CO1, 16S, Cytb, and the nuclear marker 18S (Table 2).

Sequencing was conducted using an ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit, with subsequent analysis of the reaction products performed on an Applied Bio systems 3500 Genetic Analyzer automatic sequencer.

Sequences were handled and aligned using the Condone Code Aligner software (Codon Code Corporation, Massachusetts). The resulting sequences underwent multiple alignments with the Clustal W program (Wang and Jiang 1994). Phylogenetic relationships were reconstructed via the Neighbor-Joining approach (Saitou and Nei 1987). Evolutionary distances were calculated employing the Maximum Composite Likelihood technique (Varin et al. 2011), expressed as the count of base substitutions per site. All evolutionary analyses were performed in MEGA 11 (Tamura et al. 2021).

The morphological examination confirmed the presence of two distinct species within the genus Acartia in the studied regions: A. tonsa and A. clausi. Detailed morphometric measurements revealed size differences between the two taxa, with A. clausi being generally larger across both sexes (Table 3). Our measurements showed that A. clausi females reached a mean total length of 1.20 mm (±0.12), while A. tonsa females were notably smaller, averaging 1.02 mm (±0.09). A similar trend was observed in males, where A. clausi (mean 1.09 mm ± 0.11) exceeded A. tonsa (mean 0.98 mm ± 0.08) in size. These findings align with the diagnostic descriptions provided by Gubanova (2000) for the Black Sea populations, where A. tonsa is typically characterized by a more compact body form compared to its native congener. Beyond total length, the species were definitively separated by the fine structure of the fifth swimming legs (P5). In all examined A. tonsa females, the P5 exopod was long and slender with distinct lateral serrations, matching the “invasive morphotype” described in previous studies of the Caspian and Baltic basins (Gubanova 2000; Caudill and Bucklin 2004; Shiganova et al. 2004). In contrast, A. clausi specimens exhibited the characteristic shorter, thicker terminal spines. Species identification for males was also confirmed through the structure of the fifth swimming legs (P5). In A. tonsa, the P5 is characterized by a highly asymmetrical right leg with a large, smooth, and curved inner projection on the second exopod, paired with a hook-shaped terminal segment. In contrast, A. clausi males exhibited a shorter, differently shaped inner projection and more robust distal spines. These distinct features, alongside the smaller mean size of A. tonsa males, ensured segregation between the two species.

The stability of these morphological traits across the sampled sites (Black, Caspian, and Baltic Seas) provided the first line of evidence that the specimens selected for genetic analysis were taxonomically distinct. These morphometric results served as the baseline for the subsequent molecular validation using CO1, 16S, Cytb, and 18S markers.

Thus, after summing up of our data and comparing them with the published information (Belmonte et al. 1994; Garmew et al. 1994; Marcus and Wilcox 2007), we may conclude that Acartia tonsa Dana 1849 possesses a translucent body with females typically measuring between 0.8 and 1.5 mm in length, while males range from approximately 0.8 to 1.3 mm. This species features a comparatively short abdomen and exhibits a larger body width compared to its sympatric congeners. A. tonsa females are only slightly larger than males, in contrast to A. clausi, whose females are substantially bigger. Key distinguishing characteristics include notably long first antennae, which reach at least half of the body length, bigamous (branched) second antennae, and a distinct joint between the fifth and sixth body segments (Fig. 1). A. tonsa females may be differentiated from Acartia bifilosa females by the morphology of the exopod on the fifth pair of legs: A. tonsa, has elongated legs with the several spinules positioned about halfway to the tip, whereas Acartia bifilosa displays a swollen base and fine spinules along the distal half (Redeke 1934).

An examination was conducted on 459 samples for the CO1 fragment, 55 samples for 16S, and 8 samples for cytB, sourced from the GenBank database and our own material. The sampling sites for the materials utilized are illustrated in Fig. 2 and are numbered for subsequent reference. The Cyt B gene tree is the only instance where our samples exhibit variations, indicating a polymorphism of 0.7%.

The phylogenetic trees for the mitochondrial gene fragments CO1, 16S, and cytB, produced by our analysis, are shown in Fig. 3. These trees will be examined in more details in the subsequent sections.

Despite the limited data available for A. tonsa in GenBank, two distinct populations can be identified, residing in European and Northeastern American coastal waters. To prevent any potential misidentification with other species within the Acartia genus, we employed for our study a more stable genetic marker – a fragment of 18S RNA. In all samples identical sequences were found, confirming that only one species i.e., A. tonsa, was examined (Fig. 4).

The 18S Nuclear Gene study utilized nine nucleotide sequences. For each sequence pair, all ambiguous positions were eliminated (using the pairwise deletion method). The resulting dataset comprised 656 positions. Examination of the nuclear fragment reveals that specimens from the North Atlantic (regions 4 and 9 in Fig. 2) and our own samples (highlighted in red in Fig. 4) belong to a single species. Subsequently, a CO1 tree analysis was conducted (Fig. 5). Our samples from the studied seas (indicated in red in Fig. 5) are derived from the North Atlantic (regions 4 and 9 in Fig. 2).

Fig. 5 depicts the common evolutionary origins of copepods inhabiting the Black Sea, Baltic Sea, Caspian Sea, North Sea, and North Atlantic. Samples were collected in the western part of the Atlantic Ocean (regions 3 and 4), at the coast of Brazil (region 6), in the Gulf of Mexico (region 15), in the North Pacific (regions 1 and 2), and in the South Pacific (region 14). These specific populations have remained confined to their initial geographic ranges (refer to Figs 5, 6). In each of these regions, except for the Pacific Ocean, the genus Acartia splits into two or three separate clades, signifying the presence of distinct genetic lineages.

Additionally, a haplotype network diagram (Fig. 6) was constructed from the analysis of 458 samples. These were grouped into 159 distinct haplotypes, with each circle in Fig. 6 representing a unique haplotype. The number of dashes between circles indicates the count of substitutions, separating the haplotypes. Circle size reflects the relative number of individuals per haplotype. Colors correspond to the geographic regions, listed in the legend, whose numbering aligns with Fig. 2. The orange, dark green and blue colors denote samples, originating from the Pacific Ocean, which appear genetically quite distinct from the others. Samples from the American coast (region 4) display considerable diversity. The Black Sea samples, including our own (light blue), form a separate cluster, suggesting they arrived there quite a long time ago and have accumulated mutations since then. A similar pattern is observed in the Caspian Sea (light green). Individuals from the Baltic Sea (green) are identical to various North Atlantic haplotype variants (region 4, red).

Our genetic investigation of the nuclear gene verified that analyzed specimens belong to the same species. The native habitat of A. tonsa is the Atlantic coast of North America, from where it expanded into the North and Baltic seas. After its initial establishment in the Black and Caspian seas, no further introductions have occurred, leading to a complete lack of genetic interchange.

Acartia tonsa is a cryptogenic organism with a global presence in the subtropical and temperate coastal zones. Some studies also suggest it may have persisted as a glacial relict in the northeastern coastal waters of North America (McAlice 1981).

A. tonsa was initially identified around 1900 in the brackish waters along the eastern coast of North America. Today, its range in the Atlantic spans from the shores of Canada to the coast of Argentina. In the Pacific, it has been recorded on the eastern side: in San Francisco Bay by 1924, and further south at La Jolla and San Diego prior to 1905, as well as along the coasts of Chile (Redeke 1934) and Peru (Garmew et al. 1994).

The specimens used for the original description of A. tonsa were collected at Port Jackson in New South Wales, Australia (Redeke 1934). However, whether Australia represents its native range is uncertain. Instead, its discovery in a port area could indicate an introduction facilitated by human activity. Thus, Acartia tonsa Dana, 1848 may be considered as an abundant neritic species, which commonly inhabits the coastal waters of the Western Atlantic, Indian and Pacific Oceans (Raymont 1983).

Within non-native habitats, warm-water Acartia tonsa expands northward (in the Northern Hemisphere) due to the climate warming. It was initially discovered in 1916 in the brackish coastal waters of the Netherlands. Nevertheless, its initial identification was inaccurate. Subsequent analysis, however, verified the species as A. tonsa. It was also proven that the species was definitively absent from this location in 1912 or prior years (Redeke 1934).

The initial documentation in Danish coastal waters occurred in the Ringkøbing Fjord in 1921 (Jespersen 1933). Today, it ranks among the most prevalent copepod species in Danish coastal areas (Sørensen et al. 2007; Tiselius et al. 2008).

In the Baltic Sea, A. tonsa was first documented in 1924 within the Gulf of Riga. It was subsequently located in the Gulf of Gdansk (Poland) in 1925, in the Gulf of Finland in 1934, and by 1939 it had extended to the waters near Helsinki (Segerstråle 1957).

From the 1990s to the present, A. tonsa has been plentiful in multiple estuaries along the Bay of Biscay (David et al. 2007; Aravena et al. 2009) and also off the coast of Portugal (Leandro et al. 2006).

In Germany, A. tonsa was initially identified in the Weser River estuary in 1930 (Redeke 1934; Gollasch and Nehring 2006). The first documented observation in Sweden dates to 1934. Today, it is present along Sweden’s western coastline and in certain coastal areas of the Baltic Sea. Its presence in the Gulf of Bothnia remains unconfirmed (Främmande 2006).

Within both the Mediterranean and Black seas, A. tonsa was identified more recently. Historically, three Acartia species were known in the Black Sea: A. clausi Giesbrecht, 1889, A. latisetosa (Krichagin, 1873), and A. italica Steuer, 1910 (Guidebook for marine fauna of the Black Sea, 1969). The first report of A. tonsa in the Black Sea occurred in 1994 (Belmonte et al. 1994), based on plankton samples collected near Karadag on the southeastern Crimean coast in September 1990. Subsequently, significant populations of A. tonsa have been observed in the southern Black Sea (Kovalev et al. 1998) and within Sevastopol Bay. The first record of A. tonsa near the Bulgarian coast was in 2000 (Kamburska 2004). It appeared with a maximum share of 62% of the overall abundance at coastal habitats in spring-summer period (Ivanova et al. 2017).

Consequently, the precise timing of A. tonsa’s emergence in the Black Sea remained uncertain. As earlier research had not differentiated between A. tonsa and A. clausi, Gubanova (2000) re-examined samples collected in 1976. She identified A. tonsa in these samples, demonstrating its presence in Sevastopol Bay as early as 1976 (Gubanova 2000). During summer, its abundance even exceeded that of A. clausi. The prior confusion between A. tonsa and A. clausi before 1994 stemmed from their close similarity in size and morphology.

A. tonsa was not documented in the Mediterranean Sea until 1985, suggesting its introduction to the Black Sea likely occurred via ballast water, rather than through natural spread from the Mediterranean (Gubanova 2000). This interpretation is further reinforced by the population density of A. tonsa in the Sea of Marmara being 25 times lower than in the Black Sea (Hubareva et al. 2008).

The species Acartia tonsa was introduced to the Caspian Sea from the Black Sea, most likely transported in ballast water following the opening of the Volga-Don Canal. It was first officially recorded in the central Caspian in 1982 and in the northern region in 1983. However, it is probable that the species was present earlier but misidentified as Acartia clausi Giesbrecht, 1889 (Kurasheva and Abdulaeva 1984). Correct identification as A. tonsa did not occur until 2001 (Shiganova et al. 2001). Within the Copepoda, A. tonsa has emerged as the most significant invasive species in the Caspian Sea. After becoming established around 1981, it progressively displaced native copepod populations (Shiganova et al. 2004). A. tonsa became the dominant species, forming the primary food source for kilka (Zabralieva et al. 2006). Its role became especially critical following the invasion of Mnemiopsis leidyi, which heavily consumed the native zooplankton.

Populations of A. tonsa can reach densities of up to 3100 ind.m^-3 in the Southern Caspian, where it has supplanted the native Eurytemora grimmi in the Middle and Southern basins. It has also replaced another abundant species, Calanipeda aquaedulcis, in coastal zones (Shiganova et al. 2004). Prior to the invasion of A. tonsa (1971–1981), concentrations of C. aquaedulcis ranged from 201 to 610 ind.m^-3 in the western Caspian and from 238 to 1013 ind.m^-3 in the eastern part. After the establishment of A. tonsa in 1981, the abundance of C. aquaedulcis declined markedly. During 1982–1993, its density varied from 15 to 844 ind.m^-3 in the western Middle Caspian and from 58 to 344 ind.m^-3 in the eastern Middle Caspian. In the Southern Caspian, C. aquaedulcis abundance fell sharply, from 563 to 0.3 ind.m^-3 in the west and from 968 to 68 ind.m^-3 in the east (Shiganova et al. 2023).

Our genetic investigation of the nuclear gene proved that analyzed specimens from the various basins belong to the same species. The native habitat of A. tonsa is the Atlantic coast of North America, from where it expanded into the North, Baltic, Black and Caspian seas. An important point that followed from our results is that after initial establishment of A. tonsa in the new habitats, no further introductions have occurred, leading to a complete lack of genetic interchange.

The first evidence of deeply diverging genetic lineages in A. tonsa was presented by Caudill and Bucklin (2004), who showed four distinct lineages discovered at the American coast of Atlantic Ocean and the Gulf of Mexico using mitochondrial 16S rRNA (mt16S) sequences. Chen and Hare (2008, 2011) identified three lineages (F, X, and S) of A. tonsa inhabiting the Chesapeake Bay and other Atlantic estuaries, based on sequences of the mitochondrial cytochrome oxidase subunit I (mtCOI) gene and ITS. The study of Figueroa with co-authors (2020) also confirms above-mentioned findings.

To conclude, this study, combining the thorough morphological examination of A. tonsa with one of its first detailed genetic analysis and geographical aspects of distribution, provides a new comprehensive overview of this ecologically-crucial species.