Biological invasions constitute a major influence on global biodiversity. Exotic species, especially those that succeed in establishing sustainable populations in their new region, have been the subject of numerous studies (Roman and Darling 2007; Simberloff et al. 2013; Katsanevakis et al. 2014; Havel et al. 2015). One of the main questions that arise from these research studies is: are there characters that facilitate success in colonization, since the new environment differs from the original habitat. One hypothesis predicts a decrease in genetic variability of the invasive population as compared to its native population, due to genetic bottleneck, where the founder group of the colonizers consists of a small number of individuals, which represents a sub-sample of the native population. The opening of the Suez Canal in 1869 caused a massive unidirectional migration of biota from the Red Sea into the Mediterranean; this phenomenon is often termed Lessepsian migration (Por 1978; Golani 2010). This ongoing migration phenomenon includes over 120 fish species (Golani et al. 2021 and unpublished data) and provides a unique opportunity to examine the hypothesis of reduction of genetic variability in the new population in the Mediterranean.

The Yellowspotted Puffer Torquigener flavimaculosus Hardy & Randall, 1983 (Pisces: Tetraodontidae) was first recorded in the Mediterranean in 1987 from the coast of Israel (Golani 1987). Several years later, it established a large population throughout the eastern Mediterranean (Golani et al. 2021). The family of Tetraodontidae includes 192 species worldwide in 28 genera. Most species live in marine environments while a few species inhabit brackish and freshwater (Nelson et al. 2016; Fricke et al. 2023). In the Red Sea the family is represented by 13 species (Golani and Fricke 2018), of which five species were recorded in the Mediterranean as Lessepsian migrants (Golani et al. 2021). Torquigener flavimaculosus is a benthic small species up to 11 cm Total Length (with one exception of a single specimen of 18.5 cm Total Length). Its native distribution includes the Red Sea, the Arabian Gulf, East Africa and Reunion (Gadenne et al. 2021). It was first recorded in the Mediterranean in 1987 (Golani 1987) and remained rare in its new region for 15 years, until it was recorded from Türkiye (Bilecenoğlu 2003) and spread to Greece (Corsini-Foka et al. 2006) and Libya (Al-Mabruk et al. 2018). Though little is known of its biology and ecology, in the Mediterranean, it is caught in large numbers as by-catch by trawlers at 20–50 m (Edelist et al. 2013). Golani and Lerner (2007) found it in large numbers in the sandy beach of Eilat (Gulf of Aqaba, Red Sea). It feeds mainly on slow moving invertebrates, such as crustaceans, mollusks and echinoderms (Bilecenoğlu 2005; Chartosia et al. 2021). In the Gulf of Suez, the reproductive period peaks in the spring and summer months. The specimens reach sexual maturity at 8–10 cm total length (Ramadan and El- Halfawy 2019).

The objective of this study is to better understand the mode of migration by comparing the genetic variability of the original, native populations to the migrant populations. Specifically, we compare our samples from Eilat (at the northern tip of the Gulf of Aqaba, Red Sea) with the migrant population in the Eastern Mediterranean, several decades after the first record of their invasion. This enables us to assess whether there was a decrease in genetic variability in the Mediterranean, due to a bottleneck or a founder effect. In order to support our findings, we compare the genetic variability within T. flavimaculosus populations to other species of the Tetraodontidae family (Takifugu rubripes and Takifugu obscurus), studied by Katamachi et al. (2015).

The initial objective of this study was to determine the nature of Torquigener flavimaculosus colonization into the Mediterranean Sea. Studying two mitochondrial markers, the cytochrome c oxidase 1 (COI) and the control region (D-loop), we found no evidence of a bottleneck type migration. The genetic variability within the migrant population of the Mediterranean is by no means smaller than that of the source population of the Red Sea.

This finding is compatible with previous studies, e.g., Upeneus moluccensis and U. pori by Golani and Ritte (1999), Atherinomorus forskali by Bucciarelli et al. (2002), and Siganus luridus and S. rivulatus by Hassan et al. (2003). However, in two Lessepsian migrants, Fistularia commersoni and Nemipterus randalli, the genetic variability of the Mediterranean population was distinctly lower (Golani et al. 2007; Tikochinski et al. 2019). These authors explained that the difference between these species, as compared to those that did not show genetic reduction in the colonizing populations, was that they were sampled relatively shortly after their introduction in the new environment, by relatively small number of founder individuals. However, Torquigener flavimaculosus was also sampled quite soon after its introduction, but the lack of bottleneck effect may be due to their mode of introduction, by a large number of individuals in the founding group.

Golani (1993) and Golani and Lerner (2007) showed that Torquigener flavimaculosus is very abundant in its native range in shallow waters (0.5–1.5 m) on sandy beaches with calm sea. The similar condition in the Suez Canal allowed this species to establish a sustainable population within the Canal, where it was recorded already as Tetrodon poecilonotus by Gruvel and Chabanaud (1937) half a century prior to its invasion. This population gradually dispersed northward, eventually entering the Mediterranean with large number of individuals, representing much of the genetic variability of the source population.

It is interesting to note, that in the Mediterranean Torquigener flavimaculosus changed its habitat to much deeper waters (20–60 m). The shores of the Mediterranean experience much rougher seas which probably prevent establishing a population in sandy beaches in this region. In the past three decades, over 300 hauls were conducted on the shallow beaches along the Mediterranean coast of Israel with the same experimental beach seine used in the Red Sea, yet not a single specimen of Torquigener flavimaculosus was collected (Golani, unpublished).

Surprisingly, we found in our sample that both the native and the colonizing populations are divided into two distinct phylogenetic tree branches, suggesting a division of the sampled population into two clades. This branching into two subpopulations, found in both the source population and the migrant population, raises the question of a possible speciation process. Obviously, the accumulating mutations in the mtDNA are not the cause of speciation – they only serve as markers for the general mutation rate, which can vary greatly in different taxonomic levels. Therefore, we compared the D-loop variation within T. flavimaculosus to that of a few other species of the same family. It turned out that genetic variability within our branched Torquigener flavimaculosus database is quite high – the mean divergence within each of two different species of Takifugu, T. obscurus and T. rubripes, that were randomly chosen, were significantly lower than the mean divergence within T. flavimaculosus, which in turn resembles the total divergence within a group of several Takifugu species.

Based on the mitochondrial COI gene, Turan et al. (2017) described eight distinct pufferfish species in the Mediterranean waters of Türkiye. However, their description of T. flavimaculosus as a distinct species was based on merely two individuals. Recently, Bilecenoğlu and Yokeş (2022) proposed that Torquigener flavimaculosus is a junior synonym of Torquigener hypselogeneion (Bleeker, 1852). However, these authors discovered the taxonomic complexity of the genus Torquigener and concluded that it is yet to be solved. In our study, T. flavimaculosus displays a mean inter-clade COI distance of only 2%. COI is considered as the most common gene for fish barcoding and population analysis. According to Mabragaña et al. (2011), the mean COI divergence within the 125 Argentine fish species of their study is 0.23%, compared to 4.04% within genera. Bagley et al. (2019), who studied the COI divergence of 39 freshwater fish species in Brazil, report a mean distance of 1.3% within species, and 1.8% within genera. In their study of the COI divergence within and among 66 fish species from Zhoushan coastal waters in China, Wang et al. (2023) report a mean intraspecific distance of 0.16% (ranging from 0.00 to 1.86%), compared to a considerably larger mean distance of 16.36% between congeneric species. Hence, we hesitate to define the two clades in our T. flavimaculosus study as two distinct operational taxonomic units (OTUs). More research, molecular as well as morphological, should be carried out to clarify the taxonomy of this genus.

Research conceptualization by DG and YT. Sample design and methodology, investigation and data collection, data analysis and interpretation, writing, review and editing by all authors.

With submission of this article the authors have complied with the institutional and/or national policies governing the humane and ethical treatment of the experimental subjects, and that they are willing to share the original data and materials if so requested.