Comparative genetics of scyphozoan species reveals the geological history and contemporary processes of the Mediterranean Sea

Abstract Jellyfish are useful genetic indicators for aquatic ecosystems as they have limited mobility and are highly exposed to the water column. By using comparative genomics and the molecular clock (timetree) of Rhizostoma pulmo, we revealed a divergence point between the East and West Mediterranean Sea (MS) populations that occurred 4.59 million years ago (mya). It is suggested that the two distinct ecological environments we know today were formed at this time. We propose that before this divergence, the highly mixed Atlantic and Mediterranean waters led to the wide dispersal of different species including R. pulmo. At 4.59 mya, the Western and Eastern MS were formed, indicating the possibility of a dramatic environmental event. For the first time, we find that for the jellyfish we examined, the division of the MS in east and west is not at the Straits of Sicily as generally thought, but significantly to the east. Using genomics of the Aurelia species, we examined contemporary anthropogenic impacts with a focus on migration of scyphozoa across the Suez Canal (Lessepsian migration). Aurelia sp. is among the few scyphozoa we find in both the MS and the Red Sea, but our DNA analysis revealed that the Red Sea Aurelia sp. did not migrate or mix with MS species. Phyllorhiza punctata results showed that this species was only recently introduced to the MS as a result of anthropogenic transportation activity, such as ballast water discharge, and revealed a migration vector from Australia to the MS. Our findings demonstrate that jellyfish genomes can be used as a phylogeographic molecular tool to trace past events across large temporal scales and reveal invasive species introduction due to human activity.

exposed to the physio-chemical conditions in the marine environment. In addition, their restricted movement and lack of fast swimming or active dispersal abilities force them to adapt to environmental fluctuations. Jellyfish have an ancient evolutionary lineage, with the oldest scyphozoans dated between 635 and 577 million years ago (mya) in the Neoproterozoic, the last era of the Precambrian (Van Iten et al., 2014). They successfully inhabit all oceans at all depths and in different niches. In this work, we aim to shed light on the puzzle of the Mediterranean Sea (MS) history. By examining the DNA of three jellyfish representative of Mediterranean macro-jellyfish, some of the contemporary processes that are occurring in the MS may be better understood.

| Oceanography
The Mediterranean Sea (MS) is a unique, semi-enclosed marine system, which is naturally connected to the Atlantic Ocean via the Strait of Gibraltar. This waterway is 12.9 km wide and 286 m deep. A shallow sill at a depth of 350 m in the Strait of Sicily divides the MS into two basins, eastern and western. The Balearic, Ligurian, and Tyrrhenian subbasins constitute the western side of the Mediterranean, and the Ionian, Adriatic, Aegean, and Levantine the eastern. Seawater circulation in the MS is complex and divided into three major meridional and zonal vertical circulation belts. The shallow belt, 0-500 m (Nagy et al., 2019), is the most relevant to the jellyfish in this study. The Atlantic water mass enters through the Strait of Gibraltar and flows generally eastwards via the Strait of Sicily. However, part of the water deviates toward Corsica and exits the MS again, never passing the Strait of Sicily eastwards (Nagy et al., 2019). Most of the water that enters from the Atlantic flows to the eastern MS and then to the Levantine Sea via the Libyan and Israeli coasts. When reaching the Levantine Sea, the water becomes warmer, saltier, denser, and sinks to form the Levantine Intermediate Waters (LIW). The western basin is approximately 3,400 m deep and the eastern basin 4,200 m (Cavaleri et al., 1991;Ignatiades et al., 2009;Malanotte-Rizzoli et al., 1997;Millot, 1999;Patarnello et al., 2007;Vidussi et al., 2001). The climate is relatively cold in the northern part, with an increasing temperature gradient toward the south (Cavaleri et al., 1991). Therefore, the MS is an ideal model region as it contains many different ecosystems within a relatively small area.

| Geology
During the Jurassic and Cretaceous period (208-63 mya) in the Mesozoic era, the Tethys Sea separated Europe from Africa. The exceptional conditions of the MS genesis produced a remarkable diversity in marine life and made it a suitable model of the world's oceans in terms of faunal shifts, invasive species introduction, and spread in marine ecosystems (Hsü et al., 1977;Lejeusne et al., 2010). The eastern MS is a tectonically complex region that has been evolving over a long period of time and is located in the midst of the progressive Afro-Eurasian collision (Eppelbaum & Katz, 2011). It formed its present shape during the late Miocene (23. 03-5.33 mya). Today, the eastern MS is characterized by an arid climate and is considered the most oligotrophic basin in the MS and one of the least productive seas in the world (Azov, 1991).
Around 7.24 mya, a complex combination of tectonic and other processes gradually led to a dramatic geological event that began 5.96 (± 0.02) mya, known as the "Miocene Desiccation of the Mediterranean" or "Messinian Salinity Crisis" (MSC) (Hsü et al., 1973;Krijgsman et al., 1999). This created the Earth's most recent saline giant (an extensive salt deposit, produced by the evaporation of a hypersaline sea). The MSC began with a gradual restriction of seawater exchange between the Atlantic Ocean into the MS. Apparently, a full isolation between those two bodies of water was established between 5.59 and 5.33 mya. The MSC ended at 5.33 mya as the Strait of Gibraltar opened again and new water from the Atlantic Ocean refilled the MS (Grothe et al., 2020;Hsü et al., 1973;Krijgsman et al., 1999;Roveri et al., 2014;Simon et al., 2017). During the MSC, the MS water level reached a minimum of 800-1200 m below the Atlantic sea level (Mas et al., 2018). The low sea level led to high ambient temperatures that accelerated the desiccation process. The exact amount of sea-level change during this extreme event is still under debate; it is possible that the sea level fell much lower than the suggested 1,200 m (Hsü et al., 1973;Krijgsman et al., 1999;Sternai et al., 2017). This model was supported by the erosion of the sea bed and canyons cut into the slightly older marine sediment (Hsü et al., 1973). Apparently, the MS transformed from a small ocean into a large evaporated pool and then into a brackish water lake-all within 700,000 years (Roveri et al., 2014). Tectonic processes and sea-level changes triggered the restriction and isolation of the MS from the Atlantic Ocean and the Red Sea, which is pivotal to understanding the oceanographic history of the MS. The MSC had a dramatic effect on Mediterranean fauna. Thus, this remarkable event should be expected to leave some DNA fingerprints on the living creatures of the Mediterranean.
Around 5.33 mya, the MS experienced a new revival, starting with flooding of the desiccated MS through the Strait of Gibraltar.
This was the end of the Messinian Salinity Crisis and has been described as the largest known flooding in Earth's history (Garcia-Castellanos et al., 2009). It is known as the "Zanclean flood" since it started at the beginning of the Zanclean age. A gigantic waterfall characterized this flood, eroding its way down to the desiccated Mediterranean salty lakes (Clauzon, 1973;Garcia-Castellanos et al., 2009;Popescu et al., 2017). As the MS refilled with water from the Atlantic, the flood may have begun with a smaller discharge that continued for several thousand years. However, Garcia-Castellanos et al. (2009) suggested that 90% of the water was transferred over a few months, up to a 2-year time period. Regardless of the time frame, the result of the Zanclean flood was that the Atlantic Sea reclaimed the MS with its fauna and flora. In addition to reaching sea level, this event fostered a new water composition within the MS basin, as the highly salty Mediterranean water blended with fresher Atlantic water, and introduced new Atlantic species to the local, more isolated Mediterranean biota. The removal of the land barrier and the incoming flood introduced Atlantic species, but whether the Atlantic and Mediterranean populations mingled remains controversial (Patarnello et al.,2007).
The modern MS marine environment is suffering from major anthropogenic impacts that disturb the ecosystem, such as transportation, climate change, overfishing, degradation of water quality, pollution, and heavy tourism; it makes up 33% of the world's tourism industry (Curr et al., 2000;Jackson et al., 2001). The eastern MS is the world's most affected anthropogenically and zoogeographically marine environment (Golani, 1998), and it is under significant invasion from alien species (Streftaris & Zenetos, 2006). This started with the opening of a waterway, the Suez Canal, between the Indo-Pacific and the MS in 1869 (Por, 1971). The connection between the two seas caused many marine species to migrate in both directions, either by active or passive transport, or via human transportation.
This migration, known as "Lessepsian migration" or "Erythrean invasion," mostly occurred and is still occurring along a migration vector from the Red Sea to the MS and presents a heavy impact on the marine ecosystem (Golani, 1998;Mizrahi et al., 2015). The Suez Canal was widened in 2015 to increase transportation capabilities, and the results from this construction are yet unknown. In the MS, the Suez Canal is likely the main route for introducing nonindigenous species; as much as 53% from all the species in the MS are considered Lessepsian migrants (Galil et al., 2014(Galil et al., , 2015. The Aswan High Dam, constructed in 1964, also placed a heavy ecological toll on the marine ecosystem (Golani, 1998). The major effect is the obstruction of sediments (60-180 million tons each year; Sharaf El Din, 1977) and nutrient transportation from the Nile River to the MS as a result (Biswas & Tortajada, 2012;Shalash, 1982).
The marine environment seems to have no visible geographical (allopatric condition) border, as is generally the case in terrestrial environments regarding speciation (Hickerson et al., 2010), but the marine fauna exhibits adaptation and speciation to different ecological or microecological conditions. The oceanographic history of the marine habitat offers changing parameters for the genetic structure of marine species (Neethling et al., 2008;Stopar et al., 2010). Phylogeography is viewing molecular evolution as spatial distribution of genetic lineages (Avise et al., 1987) and is a highly integrative approach used to investigate the relationship between the history of the Earth, ecology, and biotic diversification (Arbogast, 2001;Pelc et al., 2009;Via, 2009). The dispersal ability of a marine species is related to its phylogeographic capabilities; species with planktonic dispersion abilities possibly reflect contemporary oceanographic changes, while species with restricted dispersal abilities are more likely to reflect historical processes (Galindo et al., 2006;Pelc et al., 2009;Ramšak et al., 2012). Thus, the phylogeographic data we retrieve from marine jellyfish can reveal the geological history of the sea and oceanographic barriers (both visible and invisible), such as currents, temperature, salinity, predation pressure, nutrients. Phylogeographic data also depend on the species' dispersal ability, and their tolerance to overcome those barriers and become cosmopolitan (Dawson, 2005;Kuo & Avise, 2005;Ramšak et al., 2012).

| DNA markers
DNA markers are an important and valuable tool for evaluating taxonomic biodiversity and ecological studies (Hebert, Cywinska, et al., 2003;Hebert, Ratnasingham, et al., 2003). DNA barcoding is rapidly becoming the main approach for identification of species by using genetic markers mostly from mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA). mtDNA is used for a wide range of genetic applications, such as global bioidentification systems, taxonomy, population genetics, and phylogeographical analyses (Avise et al., 1987;Hebert, Cywinska, et al., 2003;Hebert, Ratnasingham, et al., 2003). For example, cytochrome c oxidase subunit I (COI) is a mtDNA gene, used widely as a barcode for species identification and discrimination (Radulovici et al., 2010;Sun et al., 2012). mtDNA is mostly maternally inherited DNA in the zygote, and a lack of heterologous recombination in mtDNA leads to a high probability of its formation from strictly maternal transmission (Hebert, Cywinska, et al., 2003;Hebert, Ratnasingham, et al., 2003;Radulovici et al., 2010;Shearer et al., 2002;Wolff & Gemmell, 2008). Heterologous recombination is a mechanism that can create a distinct class of genomic rearrangements (León-Ortiz et al., 2018). The ribosomal biogenesis in eukaryotes is taking place in the cytoplasm and in the nucleolus.
The rRNA gene, also known as ribosomal DNA (rDNA), is a tandem repetitive cluster and is the most abundant gene in the eukaryotic genome. Since it is required for the ribosome biogenesis, rDNA is critical for the cell's functions and is highly conserved from bacteria to humans (Dentinger et al., 2011;Kobayashi, 2008;Takehiko Kobayashi, 2011;Lam et al., 2007;Schoch et al., 2012). By combining multiple genes, as the nuclear 18S rDNA and 28S rDNA genes, it is possible to attain longer nucleotide data in order to reconstruct the evolutionary impact on a species (Li et al., 2012).
In this work, we used the most appropriate genetic tool for our research question (locating the small changes in base pairs of the genetic code for a species) and recent innovations in molecular technologies (Komoroske et al., 2017). Most marine jellyfish genetic studies to date have used mtDNA. Nevertheless, our work is dependent on worldwide data available for comparison with our collected data. This data limitation led us ultimately to using the more classic genes like COI, 18S and 28S. To create a reliable evolutionary clock tree (timetree) and determine the molecular clock for our examined scyphozoa, we found the most appropriate gene for sequence alignment and a proper outgroup taxon. We calibrated the specific taxon divergence time, which is the basis for calculating nucleotide rates of change and for forming a timetree (Hedges et al., 2006). The DNA mutation rate is unique for each taxon; here, we tracked the most recent common ancestor to establish our nearest taxon nucleotide rate of change. With this database, we calculated the divergence time for our specific jellyfish and calibrated the molecular clock. Scyphozoa have a life cycle with a pelagic predatory stage involving complex senses and traits . Although there are many fossils of medusoid-like animals from the late Neoproterozoic and Cambrian period, most of them lack distinctive evidence of the softpart anatomy .
Recent methods utilizing the geological and genomic records enable us to estimate divergence times based on the limited number of fossil records (Hedges & Kumar, 2003;Park et al., 2012;Peterson et al., 2007) and made it possible to calibrate the phylogenetic evolutionary timetree nodes for our examined scyphozoa Nowak et al., 2013;Ronquist et al., 2012). Usually, the classic molecular clock refers to the evolutionary rate as constant (Zuckerkandl & Pauling, 1962). However, there is much variability in mutation rates between taxonomic groups, and these are affected by population size, generation length, and other factors. To minimize our mtDNA mutation rate error, we adopted the local clock based on a publication from Yoder and Yang (2000).
Jellyfish possess some unique traits that make them suitable as genetic biological indicators for aquatic environments. Their body is constructed from gelatinous soft tissue with a water content of 95% (Hsieh et al., 2001;Tucker, 2010), and they do not have any solid skeleton or shell. Thus, they are highly exposed to the physics and chemistry of the water column. These gelatinous zooplankton are large, exhibit slow swimming speeds, and lack highly complex behavior (Hamner & Dawson, 2009;Larson, 1992). They are highly dependent on environmental conditions and on their flexibility to adapt. They cannot escape, swim, or migrate to a new environment like fish or other creatures with more complex behaviors.
Jellyfish are among the oldest creatures to inhabit the Earth and are among the first multicellular creatures that left their sessile state and began swimming and wandering in the oceans (Fedonkin & Waggoner, 1997). Fossil evidence indicates that the cnidarians originated in the early Cambrian, and some of today's jellyfish species resemble their ancestors' body shape Han et al., 2010).
We selected three representative scyphozoa from among the Mediterranean jellyfish species that represent different means, vectors, strategies of migration, and phylogeographic patterns (Mizrahi, 2014;Mizrahi et al., 2015) in order to achieve a more detailed picture of the processes occurring in the MS. These three jellyfish species are meroplanktonic and are spending the greater parts of their lives in the benthic region. Their reproduction is characterized by the alternation between sexual and asexual states.
R. pulmo is native to the northern Atlantic, was first documented in 1875, and has been frequently observed since, except for the period between the years 1930 and 1960 (Kogovšek et al., 2010). In the Levant Basin, R. pulmo ( Figure 1) is known as the "Common Jellyfish" and was first reported along the Israeli coast by Bodenheimer (1935).
Israeli R. pulmo specimens have been collected and recorded continually since 1990 (Galil et al., 1990;Mizrahi, 2007). One of the oldest known images of a jellyfish from the region is a mosaic from the 5th century A.D., in Jerusalem. The image resembles R. pulmo or Rhopilema nomadica (Figure 2), the species that are most abundant in the region today. From the Lebanese Mediterranean waters, there are reports since 1971 (Galil et al., 1990) with records of R. pulmo proliferating during 1986 (Turan & Ozturk, 2011). In northern Cyprus, the R. pulmo species reports have stated that abundance is rare and it has been recorded mostly during the months March to August (Turan & Ozturk, 2011). Historically, R. pulmo blooms in the northern Adriatic Sea were first recorded from 1883 and cited by Avian and Rottini Sandrini (1994), and have been very common in this region (Kogovšek et al., 2010). There is evidence that R. pulmo blooms in European seas were confined to semi-enclosed bays that received distinctly more fresh water and nutrients from rivers. This may be the reason for the more frequent observations of R. pulmo along the western coast of the North Adriatic, where influence from fresh water is more pronounced (Kogovšek et al., 2010;Lilley et al., 2009) From the North Atlantic Ocean, we have data of R. pulmo abundance (Costello, 2001), but some data from the South Atlantic as well (Horton et al., 2018). Furthermore, a lone report exists from Pakistani waters (Muhammed & Sultana, 2008). Wavelet analysis showed that the periodicity of occurrences has shortened in recent decades and the recurrence of blooms has increased (Kogovšek et al., 2010). We can consider R. pulmo as the most representative scyphozoa in the MS, as it is well documented throughout the Mediterranean and adjacent seas. In a published phylogeographic study on R. pulmo from 2012 (Ramšak et al., 2012), the authors concluded that "no genetic structure was detected in R. pulmo from the MS," but the extensive presence of R. pulmo jellyfish throughout the Mediterranean requires us to find out if that is really the case.

| Aurelia sp
Aurelia aurita (Linnaeus, 1758) is known as the moon jellyfish and is the most-studied group within gelatinous zooplankton. It is probably the most displayed Scyphozoa in aquaria. Although this genus is well studied, its taxonomic status remains unclear due to its high morphological variability (Schroth et al., 2002). The number of Aurelia species is controversial among scientists and was changed frequently over the years. Part of this uncertainty emanates from traditional taxonomic methods (Häussermann et al., 2009). Though Mayer (1910) suggested there are 12 species belonging to the genus Aurelia, Häussermann et al. (2009) stated that by the end of the 20th century only two species were recognized. Kramp (1961) describes seven species and in a later work, Kramp (1968) describes as much as 20 Aurelia species.
However, DNA analyses revealed 16 phylogenetic species (Schroth et al., 2002), and today, the number of 13 known species of Aurelia is commonly accepted, although this number is not final. This confusion only emphasizes the need for molecular tools to assist classification. Dawson (2003) stated "Molecular data therefore provide an important opportunity to evaluate independently the utility of morphological data in systematic studies." Dawson and Martin (2001) pointed out that, despite morphological complications concerning the Aurelia genus, there were molecular analyses employed that identified the same divisions as did morphological analysis.
Aurelia sp. is considered a cosmopolitan species with a worldwide distribution in neritic waters between 70°N and 55°S (Dawson & Martin, 2001). They can be found in different coastal and continental shelf marine environments and have been reported in Japan, North America, the Black Sea, northwestern Europe (Lucas, 2001), and in semi-enclosed bays and inlets (Purcell et al., 2000). In the Israeli eastern MS, Aurelia aurita has been observed for a long time, but recently their abundance has decreased. The first record of Aurelia aurita was in 1935 by Bodenheimer (1935). In Israel, samples were collected and reported at Beit Yanai in 1972, 1983, and 1984(Galil et al., 1990. As for our work, finding specimens of Aurelia au- can serve as a good model to demonstrate the power of organismal, ecological, and molecular data, as suggested Schroth et al. (2002).
Phylogeographic analyses were conducted worldwide on Aurelia sp. by using DNA markers and revealed cryptic species, demonstrating that Aurelia sp. is distinctly separated from other Aurelia species and F I G U R E 2 Jellyfish depicted in an ancient mosaic from the 5th century, Jerusalem, which potentially indicates that jellyfish were known in ancient times in this area. The depicted jellyfish is either Rhizostoma pulmo or R. nomadica. Source: Dr Beverly Goodman, University of Haifa F I G U R E 3 An eastern Mediterranean Aurelia sp. specimen in Haifa Bay, Israel, and its horseshoe-shaped gonads exhibits comparative or parallel phylogeographic patterns (Dawson & Jacobs, 2001;Ramšak et al., 2012). Ramšak et al. (2012) concluded that the Aurelia sp. is successfully distributed in the MS.

| Phyllorhiza punctata
Phyllorhiza punctata (von Lendenfeld, 1884; Figure 4) was first recorded outside of the Indo-Pacific Ocean only after the mid-20th century and was likely introduced to the MS by means of vessel transportation (Galil et al., 2009). The first record and reported observation in the MS was from the Israeli shoreline in 1965 at Beit Yanai (Galil et al., 1990). Since then, it has been reported in different Mediterranean locations (Abed-Navandi & Kikinger, 2007;Boero et al., 2009;Galil et al., ,1990Galil et al., , , 2009Gülşahin & Tarkan, 2012).
Phyllorhiza punctata and Rhopilema nomadica ( Figure 5) are two jellyfish species that are considered Lessepsian migrants and new immigrants to the Mediterranean (Galil et al., 1990). P. punctata (Figure 4) is indigenous to the tropical Western Pacific and is mainly distributed in Australian, Philippine, and Japanese waters (Graham et al., 2003;Mariottini & Pane, 2010). In the years 2005 and 2006, impressive numbers of P. punctata were reported after many years, wherein it was very rare or not sighted at all in the Levant (Mizrahi, 2014). In the 1950s, P. punctata was recorded in southern Brazil and was misidentified and considered a new species until the misconception was detected. With the help of genetic tools, we aim to determine whether the presence of P. punctata in the MS is new and caused by human interference and to understand its migration vector. The abundance of P. punctata is fluctuating, like most jellyfish populations. In 2013, it was highly abundant in the eastern MS (Mizrahi, 2014).

| MATERIAL S AND ME THODS
DNA markers from three thriving Mediterranean macro-jellyfish species were used for aligning the sequences. This genetic data analysis is based on our own collection of samples and on supplementary existing data from GenBank (Geer et al., 2009).

| DNA amplification and sequencing
Samples from the collected jellyfish were immediately processed in situ (Figures 1 and 3). Sample cuts from the oral arms or gonads were excised and, for most of them, DNA extraction was initiated in situ.
Total genomic DNA was extracted using the Wizard® SV Genomic DNA Purification System kit (Promega). Other samples were preserved in situ in 95% ethanol for further processing in the laboratory. Throughout the work, care was taken to use sterilized tools and containers, and gloves were worn. The genetic classification was done by aligning DNA sequences to global and local data to achieve the most accurate species identification for the sampled specimens (Bayha et al., 2010;Pett et al., 2011). We used "universal" DNA primers (Folmer et al., 1994) as well as our own primers that were developed by our laboratory (Table 1). DNA concentration was measured using NanoDrop, absorption at 260 nm. DNA preparation was done using the Promega kit for polymerase chain reaction (PCR) and sequencing amplification. After a final dilution to 2 ng/µl, DNA quality was assessed by running samples on 1% agarose gels. All amplifications were carried out in a T100™ Thermal Cycler (BIO-RAD) using GoTaq® Green Master Mix (Promega). PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega).
Sanger sequencing of the specimens was performed by "HyLabs" Israel. DNA sequences were partly submitted (including corresponding primers) as in Table 1 to the European Nucleotide Archive (ENA).

| Evolution tree reconstruction
Sequences were manually trimmed, edited, and aligned using BioEdit version 7.2.5 (Hall, 1999). Evolutionary trees (Figures 5, 7 and 8) were created using MEGA X (Kumar et al., 2018). To test what is the best fit for evolution models we used MEGA X "Find Best DNA/Protein Models Maximum Likelihood (ML)" (Kumar et al., 2018). Evolutionary history was inferred by using the maximum likelihood method based on the general time-reversible model (Nei & Kumar, 2000), using neighbor-joining analysis (Mega X). The initial tree for the heuristic search was automatically obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances and then was estimated using the maximum composite likelihood approach, and then the topology (with the superior log likelihood value) was selected. Specific parameters for each analysis are listed below.

| Rhizostoma pulmo (Figure 5)
The evolutionary tree ( Figure 5 in Haifa Bay and the Israeli Red Sea (Eilat) Aurelia sp. There was a total of 658 bp of partial COI mtDNA marker used in the final dataset. The evolutionary history was constructed using the maximum likelihood method based on the general time-reversible model (Nei & Kumar, 2000). The tree with the highest log likelihood (−5091.54) is shown. A discrete Gamma distribution was used to model F I G U R E 4 Phyllorhiza punctata off the coast of Acre, Israel (L). In Australia, source: Adam Greenberg (R) F I G U R E 5 Evolutionary tree calculated by using 508 bp of partial COI mtDNA markers. The full black circle marks the Levantine Rhizostoma pulmo samples, the empty rhombus represents the Levantine Aurelia sp., and the empty circle represents the Levantine Rhopilema nomadica evolutionary rate differences among sites (five categories (+G, parameter = 4.2722)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 59.89% sites). The tree is drawn to scale, with branch lengths measured for the number of substitutions per site. The analysis involved 43 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The number of bootstrap replications is 1,000.

| Phyllorhiza punctata (Figure 8)
This evolutionary tree shows the worldwide dispersal of Phyllorhiza punctata, built on 18S and 28S rDNA markers. The evolutionary tree of P. punctata (Figure 8) is composed of two combined rDNA markers, 18S and 28S. As a result of a lack of global genetic information, and in order to achieve longer alignment sequences, we used the technique of connecting two different segments of genes that overlap and then manually trimmed them. There was a total of 2,844 bp positions of 18S and 28S rDNA markers, (or nucleotide pairing) in the final dataset. The evolutionary history was constructed by using the maximum likelihood method based on the model of Kimura 2-parameter (Kimura, 1980). The tree with the highest log likelihood (−5197.16) is shown. The analysis involved seven nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding evolutionary analyses were conducted MEGA X (Kumar et al., 2018). The number of bootstrap replications numbered 1,000.
Sequences were manually trimmed, edited, and aligned using BioEdit version 7.2.5 (Hall, 1999). The evolutionary tree and molecular clocking were conducted using MEGA X (Kumar et al., 2018). To find the best fit for the popular evolution models, we used the MEGA X "Find Best DNA/Protein Models Maximum Likelihood (ML)" (Kumar et al., 2018). We computed ( Figure 6) an evolutionary clock tree (also known as an evolutionary timetree) and determined the molecular clock to understand the Mediterranean oceanographic processes and historical biogeography with the help of DNA markers. To achieve this objective, we needed to estimate rate and time of divergence of the jellyfish species (Peterson & Butterfield, 2005;Thorne & Kishino, 2002). Our model is based on an estimated relative node for the divergence time from the most recent common ancestor (MRCA) in our taxonomic scyphozoan groups (Thorne & Kishino, 2002).

| Rate of change calibration
In order to calibrate the divergence time and rate of change, we used existing published data describing the history of three recent, common ancestral scyphozoa (Geer et al., 2009;Park et al., 2012).
We employed two divergence points to achieve one calibration constraint time rate. The first divergence time node we used was a point between two members of Scyphozoan class Chrysaora quinquecirrha, (accession ID: HQ694730) and Aurelia sp. (accession ID: NC_008446 and HQ694729) whose divergence occurred around 440 mya, apparently at the beginning of the Silurian period. The second divergence point was based on two species of Aurelia sp. (accession ID: NC_008446 and HQ694729), whose divergence occurred around 120 mya, in the Cretaceous Period (Park et al., 2012). This allows us to calibrate the rate of mtDNA changes, and both minimum and maximum divergence time enabled us to calibrate the DNA evolutionary clock and determine the mtDNA rate of change for the examined jellyfish.

| Rhizostoma pulmo DNA reveals existing East and West MS populations
To assess the phylogeography of Rhizostoma pulmo, we analyzed the available published data of R. pulmo (Geer et al., 2009)

| Rhizostoma pulmo timetree reveals the divergence time of East and West MS populations
The timetree (clock tree) for the Mediterranean R. pulmo was created by using the DNA marker of mtDNA cytochrome c oxidase subunit I (COI). The graph (tree) shows a clear picture of the divergence of R.
pulmo into different populations ( Figure 6) and in agreement with the phylogenetic analysis ( Figure 5). The most distinct point in the timetree is the divergence that occurred around 4.59 mya and gives us a picture of the separation between the eastern and western R. pulmo populations at this time point. In this tree (Figure 6), the emergence of subgroups that are happening at a much later time can be seen, and this change mostly points to a process that likely occurred and is still ongoing in modern times ( Figure 6).

F I G U R E 6 Evolutionary timetree of
Rhizostoma pulmo. The timetree shows the divergence time in a scale of mya. Notice the divergence time of 4.59 million years ago (mya), which separates Israeli R. pulmo samples from the rest. The "Messinian Salinity Crisis" (MSC, dashed gray) and the Zanclean flood (dashed light blue) times estimates are marked. Color represents R. pulmo sampling locations: red-Israel; purple-Italy; blue-western MS; yellow-Tunisia; green-Slovenia. Rhizostoma luteum (gray) was used as an outgroup

| Evolutionary tree of Aurelia sp. shows unique population and separation between Red Sea and MS populations
Since Aurelia sp. is among the few scyphozoan species found both in the MS and the Red Sea, we focused on this species to study connectivity between populations in the two seas and potential migration across the Suez Canal. We constructed an evolutionary tree of Aurelia species (Figure 7), using the COI mtDNA marker

| D ISCUSS I ON
The current outline of the Mediterranean Basin is a consequence of geological history and has a drastic influence on jellyfish biodiversity (Casal-López & Doadrio, 2018). In this work, we investigated F I G U R E 7 Evolutionary tree focusing on Aurelia sp. generated using the COI mtDNA marker. The tree exhibits the worldwide dispersal of Aurelia sp., with emphasis on the eastern MS (Haifa Bay, Israel; black rhombus) and the Red Sea (Eilat, Israel; black circle) historical geologic events and contemporary processes by taking advantage of evolutionary forces that triggered and promoted genetic changes. We are attempting to understand the correlation and the relationship, if any, among genetic alternations and environmental processes.

| Rhizostoma pulmo as a valued genetic biological indicator
R. pulmo was described in a recent study as a jellyfish species without any trace of genetic structure across the MS (Ramšak et al., 2012).
Our DNA results of R. pulmo (Figures 5 and 6) reflect the opposite and present this jellyfish as an important phylogeographic species.
These novel DNA results were made possible by trace evidence ob-

| Rhizostoma pulmo reveals a new boundary in the Mediterranean Sea
The results of Figure

Rhizostoma pulmo DNA timeline
In Figure  period of a few months to 2 years (Garcia-Castellanos et al., 2009) and created a new, mixed water composition. The removal of the land barrier and the incoming flood introduced Atlantic species (Patarnello et al., 2007) and also mixed and dispersed those species that survived the long salty period. A new physically and chemically uniform water body was created from the salty MS and allowed R. pulmo to establish itself all over the MS. Our R. pulmo ancestors may have successfully and effectively made a habitat in this new water body. Examining the R. pulmo timetree (Figure 6), it took as much as 740,000 years (from 5.33 to 4.59 mya, Figure 6) for the revived MS to stabilize and to produce some environmental  (Woodruff, 2003) that indicates that sea levels were 100 m above the present sea level during the early Pliocene (5.5-4.5 mya). Such a big decline in the sea level ending at the time we see genetic divergence at 4.59 mya could be a result of an environmental change and indicative of a long dry period (Bohlen et al., 2020;Woodruff, 2003).
This subject should still be studied more extensively, but one or more of these possible events were dramatic enough to leave its evolutionary stamp on jellyfish DNA. These changes occurred in the early Pliocene, which was a crucial time for Holarctic carnivoran faunal assemblages (Werdelin & Lewis, 2020). In Africa, one of the first human ancestors, the hominin Ardipithecus, appearedthe last common ancestor of humans and living chimpanzees and bonobos (White et al., 2009).
By that estimated time of divergence, 4.59 mya (Figure 6), it seems the eastern and western part of the Mediterranean established their unique marine ecosystems and developed different ecological habitats such as we know today. We suggest that the uniqueness and harsh aquatic ecosystems of the eastern MS developed oligotrophic conditions (Turley et al., 2000) leading to stronger local ecological forces that promoted speciation, genetic pressures and induced higher evolutionary selection than in the other Mediterranean areas. R. pulmo from the central and western 5 and 6) also exhibited some DNA changes. This may also show that oceanographic processes occur in these areas, which requires further research.

| Is the eastern MS Aurelia sp. an isolated local population?
The results from the mtDNA COI marker of the Aurelia sp. introduced in Figure 7, present, for the first time in the eastern MS, the Aurelia sp. (marked with a black rhombus) as an exclusive branch within the evolutionary tree. These results (Figure 7) resemble the data from R.
pulmo (Figures 5 and 6) and emphasize the uniqueness of the local eastern MS species and support the hypothesis that Aurelia sp. exhibits concordant phylogeographic patterns (Ramšak et al., 2012) as we see in the Aurelia sp. from the East MS as an isolated local jellyfish population. The comparison of the Red Sea, Eilat Aurelia sp.
(marked with a full circle, Figure 7) data with the eastern MS Aurelia sp. from Haifa Bay (marked with a black rhombus, Figure 7), shows clearly that they are distanced from each other. The Eilat Aurelia sp. seems to be closely related to the Aurelia sp. species from the Pacific Ocean, Papua New Guinea, and the eastern MS Aurelia sp.
from Haifa Bay is located closer to the Mediterranean species, from Croatia and Slovenia (Figure 7). This picture eliminates the idea that our examined Aurelia sp. arrived via Lessepsian migration or that there was any crossbreeding between populations. The Aurelia sp. is very common and blooms heavily in the Red Sea of Eilat.

| Phyllorhiza punctata vector of migration
Phyllorhiza punctata sheds light on contemporary processes in the MS through human activity. The evolutionary tree was constructed by combining two rDNA markers, 18S and 28S. Having made this connection of the two genes, the result proved itself and allowed us to have long sequences as a total of 2,844 bp (nucleotide pairing) that enabled good observation and a clear result.
The results suggest that the eastern MS P. punctata (marked with a black rhombus, Figure 8) from the Israeli shoreline is genetically closer to the Australian P. punctata than to those from Mexico ( Figure 8). We consider the Phyllorhiza sp. to be indigenous to the Southwestern Pacific (Graham et al., 2003). Based on the results, P. punctata was recently introduced to the MS as a result of anthropogenic impact on the environment, including introduction via ballast water (Ivanov et al., 2000;Richardson et al., 2009).

| CON CLUS ION
Our results inferred that each of the chosen jellyfish has its own characteristic dispersal strategy, although they are quite similar to each other in size and shape. It is common to find them together in swarms, but they exhibit different survival strategies (Dańko et al., 2020, Patrick et al., 2021 that enable them to experience ecological changes differently. The benefit of investigating these similar jellyfish species is that they are each telling a different part of the story of the sea. They shed light on different parts of the geologic history, as we presented in the timetree of the Rhizostoma population by marking the divergence time at 4.59 mya. The contemporary processes and anthropogenic impact are presented mostly by Aurelia sp. and P. punctata.
This work demonstrates that the DNA of today's living creatures is of great value to environmental history. We discovered that the characteristics and traits of each species, such as the unique ability and flexibility to fit in and survive in changing environments, specialization in migration, reproduction and feeding strategies, and where the journey of their species' ancestors took them, affect its DNA and provides us with information on the environment in the past and the present. Various genes and different species show us a different resolution for the time period we are investigating. Here, we used the most common DNA markers because they are widely researched, and we were able to acquire extensive worldwide data that helped us accomplish our task. Our findings demonstrated that jellyfish genomes can be used as a phylogeographic molecular tool to trace past events across large temporal scales and revealed that the introduction of invasive species was a result of human activity.

ACK N OWLED G M ENTS
We would like to express our deepest appreciation to Mizrahi Jenny for her assistance with this work, taking part in both the field work as well as contributing to writing this article.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
This genetic data analysis is based on our own collection of samples and on supplementary existing data from GenBank (Geer et al., 2009