**Population Genetic Diversity of Two Marine Gobies (Gobiiformes: Gobiidae) from the North-Eastern Atlantic and the Mediterranean Sea**

#### **Katarína Cekovsk ˇ á <sup>1</sup> , Radek Šanda 2, Kristýna Eliášová 3,4 , Marcelo Kovaˇci´c <sup>5</sup> , Stamatis Zogaris <sup>6</sup> , Anna Maria Pappalardo 7, Tereza Soukupová <sup>1</sup> and Jasna Vuki´c 1,\***


Received: 2 September 2020; Accepted: 10 October 2020; Published: 13 October 2020

**Abstract:** Gobies (Gobiiformes: Gobiidae) are the most species-rich family of fishes in general, and the most abundant fish group in the European seas. Nonetheless, our knowledge on many aspects of their biology, including the population genetic diversity, is poor. Although barriers to gene flow are less apparent in the marine environment, the ocean is not a continuous habitat, as has been shown by studies on population genetics of various marine biota. For the first time, European marine goby species which cannot be collected by common fishery techniques were studied. The population genetic structure of two epibenthic species, *Gobius geniporus* and *Gobius cruentatus*, from seven localities across their distribution ranges was assessed, using one mitochondrial (cytochrome b) and one nuclear gene (first intron of ribosomal protein gene S7). Our results showed that there is a great diversity of haplotypes of mitochondrial gene cytochrome b in both species at all localities. Global fixation indices (FST) indicated a great differentiation of populations in both studied gobies. Our results did not show a geographic subdivision to individual populations. Instead, the data correspond with the model of migration which allow divergence and recurrent migration from the ancestral population. The estimated migration routes coincide with the main currents in the studied area. This matches well the biology of the studied species, with adults exhibiting only short-distance movements and planktonic larval stages.

**Keywords:** benthic fish; molecular tools; cytochrome b; ribosomal protein gene S7; *Gobius cruentatus*; *Gobius geniporus*; Mediterranean Sea; genetic structure

#### **1. Introduction**

Despite the fact that the seas and oceans are interconnected, marine organisms can show a strong genetic differentiation [1]. Several phylogeographical studies have shown that even the ocean is a fragmented environment. A spatial genetic structure has been discovered in different marine organisms, e.g., in sea-grasses [2], sponges [3], mollusks [4], sea cucumbers [5], sea urchins [6], crustaceans [7–9] or fishes [10–12]. However, in contrast to terrestrial and freshwater ecosystems, the delimitation of individual populations in the ocean is not an easy task because barriers to gene flow are far less apparent in the marine environment [13]. Some of the driving forces of the genetic variability of marine species are oceanographic barriers (e.g., direction of currents, presence of straits, extent of different type of habitats, temperature and salinity zonation), limited dispersal capabilities of species, isolation by distance and geological history of the area. On the other hand, factors such as hydrodynamics, long duration of the larval pelagic stage, or migratory behaviour of adults are most commonly responsible for the genetic homogenization of the population [1,14]. Final scenario of genetic partitioning can thus be a consequence of interaction of more factors. Understanding these processes is crucial for marine phylogeographical investigations, for species conservation and management of marine resources [15].

The Mediterranean Sea is a small enclosed basin, connected with the Atlantic Ocean by the Strait of Gibraltar and with the Black Sea through the Bosporus. It is subdivided into several deep subbasins separated by shallow sills. The major water exchange occurs with the Atlantic Ocean, and is strongly affected by climate [16]. Cool surface water inflows from the Atlantic Ocean to the Mediterranean Sea, while warm and more saline subsurface Mediterranean water outflows to the Atlantic Ocean [16].

Although they are adjacent and were connected during most of their past, the Mediterranean Sea and the Atlantic Ocean had partly dissimilar geological histories. Driven by a combination of climatic and tectonic forces, the Messinian Salinity Crisis (MSC, 5.97 to 5.33 million years ago, Mya) was one of the major events, which impacted geological history, and consequently the biota of the Mediterranean Sea [17,18]. It is widely accepted that at the onset of the MSC, the Mediterranean Sea became disconnected from the world ocean, and, as a consequence of evaporation, suffered a great water level drawdown [18,19]. This led to a severe change of environmental conditions in the Mediterranean Sea. However, the scenarios of the fate of the Mediterranean Sea at that time greatly differ (from almost complete desiccation of the sea to the existence of the deepwater marine environment in the first phase of the MSC [17,18,20]). The second phase of the MSC was characterized by fluctuations of environmental conditions due to the repeated connection with the Paratethys [17,21]. Accordingly with the various scenarios about the form of the Mediterranean Sea during the MSC, various scenarios about the fate of the ichthyofauna in the Mediterranean Sea were proposed, ranging from the extinction [22] to survival, possibly in refugia; the latter was corroborated by the findings of fossils of marine fishes [21,23]. The MSC ended by the opening of Gibraltar and refilling of the Mediterranean Sea by Atlantic waters in an event known as the Zanclean flooding [24] and by the onset of stable marine conditions [17].

Another event with a strong impact on the diversity and distribution of the extant species in the Mediterranean Sea and the Atlantic was the Pleistocene glaciation (2.6 Mya to 11,600 before present, BP) [25]. During the most recent ice ages the growth and decay of ice masses drove the world sea-level fluctuations in the order of 10's to over 100 m on the time scales of 100's to 10,000 years, and ranging from the sea level several meters higher than present to more than 100 m below the present level [26]. The coastline of the Atlantic Ocean and the Mediterranean Sea and its size changed accordingly, huge areas, e.g., the North Adriatic Sea, were repeatedly desiccated and reflooded. During the Last Glacial Maximum (24,000–18,000 years BP), the sea level was about 125 m lower than today, with the most intensive sea level rise between 17,000–7000 years BP, after which it reached more or less the present coastline [27]. Glacial and interglacial phases resulted in the sea temperature alterations, with the reconstructed cooling amplitude in the Mediterranean during the Last Glacial Maximum reaching up to 6–7 ◦C [28], which should have had a severe impact on the diversity and distribution of the living organisms in the Mediterranean Sea. However, the Mediterranean waters remained warmer that those of the adjacent Atlantic Ocean during glacial peaks, thus many species now present in the warm temperate Atlantic likely survived the cold phases of the glacial cycles in the Mediterranean Sea,

recolonizing the Atlantic when more favourable temperatures were re-established during interglacial phases like the present one [29].

The studied area concerns the Mediterranean Sea province and the South European Atlantic Shelf ecoregion. The Mediterranean Sea is classified as a specific, well defined biogeographic province within the Temperate Northern Atlantic realm [30]. It is further divided into seven ecoregions (Adriatic Sea, Aegean Sea, Levantine Sea, Tunisian Plateau/Gulf of Sidra, Ionian Sea, Western Mediterranean, and Alboran Sea). Its neighbouring marine provinces are the Black Sea in the east and the Lusitanian province in the Atlantic Ocean in the west. The Mediterranean Sea is connected with the South European Atlantic Shelf and Saharan Upwelling ecoregions (both belonging to the Lusitanian province and having the boundary in the Gibraltar Strait area).

Circulation of water in the studied area, and especially in the Mediterranean Sea, is very complex. The main currents and gyres are depicted in Figure 1. Within the studied region, multiple biogeographical barriers have been identified, of which the Almeria-Oran front, the Strait of Sicily and the Otranto Strait (see Figure 1) are considered to be the major ones influencing genetic diversity of various marine organisms [1,13]. However, dissimilar influence of biogeographic barriers has been found even for the closely related taxa with the same biology and ecology [31–34].

**Figure 1.** Map of the studied area, with sampling sites (black dots), main oceanographic barriers (red lines) and circulation of the main currents in the Mediterranean Sea (arrows). 1—Spain, 2—Portugal, 3—France, 4—Sicily, 5—Croatia, 6—Montenegro, 7—Greece, 8—Cyprus W (west), 9—Cyprus E (east).

Gobies (Gobiiformes: Gobiidae), with currently recognized 1915 valid species, are the most species-rich family of fishes in the world [35]. It is also the most speciose family of fishes of the European seas and, in particular, of the Mediterranean Sea. The majority of gobies are benthic fishes with planktonic larval stages. Marine gobies predominantly occupy shallow shelf bottoms, with limited number of species extending to deeper shelf, and only a few of them reaching bathyal depths [36,37]. In total, there are more than 90 marine species of gobies in European seas listed in the last review [38]. However, new species are still being discovered, e.g., [36,37,39,40], and the knowledge about the distribution of many species is still quite limited e.g., [41–43]. The information about the population genetic diversity and phylogeography of European marine gobies is very poor too. Population genetic studies have been done so far in only a few species, which are easy to collect by the common fishery techniques. Nevertheless, on the basis of limited information, it is evident that for some studied goby species there is a clear genetic differentiation between distant populations [44–50], but not for

the others [51]. Within the genus *Gobius*, the population genetic structure was studied so far in the only species, *Gobius niger*, where the mitochondrial marker nicotinamide adenine dinucleotide dehydrogenase (NADH) was analysed by restriction fragment length polymorphism (RFLP) [51]. The results suggested the existence of population subdivision in this species.

In this work we studied the population genetic diversity of *Gobius geniporus* and *Gobius cruentatus* (*Gobius*-lineage, gobiine-like clade *sensu* Agorreta et al. [52]), two bottom dwelling (epibenthic) species. *Gobius cruentatus* occurs in the north-eastern Atlantic Ocean, from south-western Ireland to the coasts of Senegal, and in the Mediterranean and the Black Seas [53,54], while *G. geniporus* is a Mediterranean endemic [53]. *Gobius cruentatus* is typically detected on mixed bottom habitats dominated by stones, boulders or seagrass [55]. It can grow up to 18 cm and occurs in depths between 1.5 and 40 m [53,54]. *Gobius geniporus* prefers sandy bottoms mixed with gravel, cobbles and boulders, with at least some amount of rocky formations present scattered over sand. It reaches a size of 16 cm and the depth at which it was observed ranges from 1 to 30 m [53,56,57]. These species are restricted to shallow shelf bottoms, so their real area of occupancy is only a narrow stripe of bottom along the coastline. Being small, epibenthic, territorial and non-migratory in adulthood, these species are expected to have only a limited dispersal capability [53].

The aim of this study was to assess the genetic diversity of seven geographically distant populations of two species of European marine gobies, *G. geniporus* and *G. cruentatus*, across their distribution ranges, using mitochondrial and nuclear markers, in order to reveal a possible population subdivision and a potential existence of biogeographical barriers which would affect the connectivity of the populations.

#### **2. Materials and Methods**

#### *2.1. Samples*

It is very difficult to collect benthic marine gobies in general, unless they live on sandy or muddy bottoms. They are usually not commercially used, so it is not possible to purchase them or to obtain them from fishermen even as a bycatch. As both species investigated in this study typically occupy mixed bottom habitats it is not possible to use common fishery techniques, such as trawl nets or push-nets, to collect them, even though these two species belong to the largest ones among gobies from the north-eastern Atlantic and the Mediterranean Sea. For research purposes, these two species are being collected individually, during scuba-diving and using hand nets and anaesthetic, which is very time consuming. They do not occur in shoals and it can be difficult to spot them. A total of 74 specimens of *G. geniporus* from seven localities in the Mediterranean Sea and 41 specimens of *G. cruentatus* from two localities in the Atlantic Ocean and five localities in the Mediterranean Sea were included in this study (see Figure 1 and Table 1). Tissue samples and voucher specimens are deposited in the ichthyological collection of the National Museum, Prague, Czech Republic.


**Table 1.** Sampling sites and number of analysed specimens of *Gobius geniporus* and *G. cruentatus* for cytochrome b and S7.

#### *2.2. DNA Extraction, Amplification, Sequencing*

Total genomic DNA was extracted from the finclips using Geneaid® DNA Isolation Kit following the manufacturer's protocol. The samples were analysed for two genes, one mitochondrial, cytochrome b (cyt b), and one nuclear, first intron of the ribosomal protein gene S7 (S7). For the amplification of cyt b, the primers GluF and ThrR [58] were used. S7 was amplified with the primers S7RPEX1F and S7RPEX2R [59]. The polymerase chain reaction (PCR) was performed in 25 μL total volume containing 12.5 μL of PPP Master Mix (TopBio), 9.7 μL of Ultrapure H2O, 0.65 μL of each primer and 2 μL of DNA isolate. Amplification of cyt b followed the protocol described in Šanda et al. [60]. For S7, a specific touch-down protocol was used with the following steps: initial denaturation at 95 ◦C for 5 min, followed by 5 cycles of denaturation, annealing, and elongation: 94 ◦C for 40 s, 60 ◦C for 1 min, 72 ◦C for 2 min, followed by 35 cycles of denaturation, annealing, and elongation: 95 ◦C for 30 s, 56 ◦C for 1 min, 72 ◦C for 2 min, and the final elongation at 72 ◦C for 20 min. PCR products were purified with the use of ExoSAP-IT and sequenced at Macrogen Europe. For the sequencing of cyt b, the specific internal primers were designed: GcruF1 (5 -GGT GCA ACC GTC ATC ACT AA-3 ) and GcruR1 (5 -AGT GGG TTG GCA GGA ATG-3 ) for *G. cruentatus* and GgenF1 (5 -GTA GGC TAT GTC CTG CCC TG AG-3 ) and GgenR1 (5 -TTG GAG CCT GTC TCG TG GA-3 ) for *G. geniporus*. Nuclear gene S7 was sequenced using the amplification primers. Sequences were deposited in GenBank under accession numbers MT774412-MT774485 (cyt b) and MT893746—MT893891 (S7) for *G. geniporus* and MT684467—MT684507 (cyt b) and MT684508—MT684585 (S7) for *G. cruentatus*.

#### *2.3. Data Analyses*

Obtained cyt b and S7 sequences were checked manually in Chromas v2.6.4 and aligned in Bioedit v7.2.6.1 [61]. The appropriate model of nucleotide substitution was determined using jModelTest v2.1.9 [62], based on Akaike Information Criterion (AIC) [63]. DnaSP v6.11.01 [64] was used to assess the haplotype diversity (Hd) and nucleotide diversity (π), as well as to perform Fu and Li's F and Tajima's D neutrality tests. The results of these tests can point to possible selection or a change in population demography. To evaluate the amount of genetic variance within and between populations, analysis of molecular variance (AMOVA) was performed using ARLEQUIN v3.5 [65]. It estimates population differentiation with the use of individual haplotypes and their frequency in the studied populations. This further enables calculation of fixation indices, a global FST and pairwise FSTs. FST expresses a degree of genetic differentiation between the individual populations. The two populations from Cyprus were grouped together, as well as the two populations from the Adriatic Sea (Montenegrin and Croatian); the grouping was made based on location, proximity and the water circulation. The remaining populations represented individual groups. The statistical significance of the FST values was tested by executing 16,000 permutations. For pairwise FSTs, a Bonferroni correction was subsequently applied to correct for multiple tests. Further, genetic distances (uncorrected p-distances) between and within populations of each species were calculated in MEGA 6 [66]. The datasets of S7 were phased by the program PHASE v2.1.1 [67]. All sequences were phased with a probability of 0.9 and the final datasets with inferred phased sequences consisted of 146 sequences for *G. geniporus* and 78 for *G. cruentatus*. The phased S7 data were then used for calculating diversity measures and constructing haplotype networks. The rest of the analyses were not performed on S7 due to a very low polymorphism of S7 datasets. A detailed reconstruction of relationships of the haplotypes of populations was performed by a statistical parsimony method under a 95% connection limit [68], using PopART [69].

Isolation by distance hypothesis was tested by Mantel test [70] using R v3.5 software (package adegenet), executing 1000 permutations. Mantel test compares genetic distances estimated by pairwise FSTs with geographical distances between locations. The matrices of geographical distances were derived from the coordinates of the individual localities. Another approach was applied using ARLEQUIN v3.5 [65], where the shortest marine paths between each pair of localities, estimated from the GoogleEarth, were used in matrices of geographical distances. Statistical significance of the Mantel test was estimated by executing 1000 permutations.

We estimated migration routes between pairs of populations using Migrate-n software v4.4. [71]. Five demographic scenarios were tested for each population pair: (1) model assuming full migration between populations, (2) model assuming migration from the population A to the population B, (3) model assuming migration from B to A, (4) model allowing divergence, where A splits off from B, with migration from B to A after the split, (5) model allowing divergence, where B splits off from A, with migration from A to B after the split. For each scenario, the migration rate and population sizes were estimated; for the scenarios (4) and (5) also the time of divergence between the two populations. Pairs of populations are listed in the Table S1. Migrate-n analyses were conducted using a static heating strategy with four short chains with temperature values of 1.0, 1.5, 3.0, and 1.0 × 106 and a single long chain. 1,000,000 steps were recorded every 100 generations with 200,000 steps discarded as burn-in to ensure the convergence of the analyses. Appropriate mutation model was assessed using jModelTest [62] resulting in Hasegawa-Kishino-Yano (HKY). Priors were set as follows: Bayes-priors = THETA \* \* UNIFORMPRIOR: 0.001, 0.000 0.0100, Bayes-priors = MIG \* \* UNIFORMPRIOR: 0.000, 100000.000, 10000.000.

Past population demography of each species was inferred using the linear Bayesian skyline plot model [72], implemented in BEAST v1.8.4 [73]. It allows observing fluctuations of effective population sizes from the present, backwards in time, to the coalescence in the most recent common ancestor, and is expressed graphically. Analyses were conducted under the Bayesian coalescent method, with corresponding nucleotide substitution model for each species and using a strict molecular clock. The x-axis of the plot shows the time in mutation units per nucleotide position and y-axis scaled effective population size. Simulations ran for 100 million Markov chain Monte Carlo (MCMC) steps with sampling every 10,000th generation. Results from three independent runs were combined using LogCombiner and burn-in was set to 20 million iterations in each run. Finally, TRACER v1.7.0 [74] was used to check the parameter estimates and visualize Bayesian skyline plots.

#### **3. Results**

#### *3.1. Gobius geniporus*

In *G. geniporus*, 74 specimens were analysed (Table 1). The alignment of cyt b had a length of 1113 bp and contained 56 polymorphic sites, while there were only two variable sites in the 594 bp long alignment of 146 sequences of S7 (Table 2). A total of 45 haplotypes were found for cyt b and only three haplotypes for S7 within seven Mediterranean populations. The best-fit substitution model selected for cyt b was general time reversible with proportion of invariable sites (GTR+I). Haplotype diversity of cyt b was high, while nucleotide diversity low (Hd = 0.969; π = 0.004) and for S7 both haplotype and nucleotide diversity were extremely low (Hd = 0.054; π = 0.0001) (Table 2). Diversity measures calculated per each locality are listed in Table S2. The values of neutrality tests (Tajima's D, Fu and Li's F) for cyt b were negative and significant, indicating a recent population expansion or purifying selection (Table 2). The Bayesian skyline plot of *G. geniporus* depicts a gradual population size growth since the coalescence and its stabilization in the present (Figure 2a).

**Table 2.** Diversity measures and results of neutrality tests for *Gobius geniporus* and *G. cruentatus* based on cytochrome b and S7 sequences. N—number of sequences, S—number of segregating sites, Nh—number of haplotypes, Hd—haplotype diversity, π—nucleotide diversity. Significant values (at α = 0.05) indicated by asterisk.


**Figure 2.** Bayesian skyline plot for *Gobius geniporus* (**a**) and *G. cruentatus* (**b**) based on cytochrome b sequence data. The graph illustrates fluctuation of the population size from recent to the coalescence (from left to right). Y-axis stands for scaled effective population size and x-axis for time scale in units of mutation per nucleotide position. Blue middle line shows the median estimate, 95% confidence interval is indicated in blue.

In the haplotype network of *G. geniporus* based on cyt b, there was an indication of a certain geographical pattern: there were two major haplotype groups. In one of them there was one more frequent haplotype shared between the central Mediterranean populations (Montenegrin, Croatian and Sicilian), while in the other one, there were two more frequent haplotypes, shared between Cypriot and Greek populations (Figure 3a and Figure S1a). Most haplotypes from Cypriot populations grouped together, as well as the majority of the haplotypes from the Sicilian one. Unique haplotypes prevailed in the network. Practically all sequences of S7 in *G. geniporus* were of the same haplotype (142 out of 146, Figure 3a) and due to this low polymorphism, all following analyses were performed only on the cyt b dataset. AMOVA for *G. geniporus* based on the cyt b showed that most of the genetic variance is distributed within populations (76%). FST index indicated a high level of genetic differentiation (FST = 0.237, *p* < 0.01). Pairwise FSTs showed in most cases a pronounced or high level of genetic differentiation between the pairs of populations, but several values were low (Table 3); however, only a half of values were significant. Statistically significant values indicating high or pronounced differentiation were for most comparisons for Sicilian and both Cypriot populations. Mean p-distances between the populations were low and ranged between 0.2 and 0.6% (Table 3). Mean p-distances within populations were of a similar range (0.1–0.5%), while the maximum intraspecific p-distance for *G. geniporus* was 1.08%.

**Table 3.** Mean genetic distances between *Gobius geniporus* populations for cytochrome b (uncorrected p-distances, in %, above the diagonal), intrapopulation distances (on diagonal), and pairwise FSTs (below diagonal). Significant values of FSTs (at α = 0.05/number of pairs) indicated by asterisk.


**Figure 3.** Haplotype networks of *Gobius geniporus* (**a**) and *G. cruentatus* (**b**) based on cytochrome b and S7 sequences. Size of circle is proportional to haplotype frequency. The number of mutational steps between closest haplotypes is indicated by hatch marks. Missing intermediate haplotypes are shown as small white circles.

Mantel test was significant using both approaches (adegenet: observation 0.299, expectation 10<sup>−</sup>5, *p*-value < 0.001; ARLEQUIN: correlation coefficient 0.69, *p*-value < 0.001), indicating a possible pattern of isolation by distance (see Figure 4a). Among the modelled migration scenarios, for each pair of populations, the model which allows divergence and the recurrent immigration from the ancestral population after the split was the one with the highest probability. The divergence directions and the migration routes are schematically depicted in the Figure 5a, while the estimates of immigration rates, divergence times and population sizes are listed in the Table S3. The system of migration routes is rather circular, anticlockwise, with a large circle between Sicily, western Cyprus, eastern Cyprus, Greece, Montenegro, Croatia and Sicily, and two smaller ones: Sicily, Greece, Montenegro, Croatia, Sicily, and Sicily, Montenegro, Croatia, Sicily. All the routes, with the exception of the one between Sicily and Greece eastwards, can be well explained by the prevailing currents (see Figure 5a). The highest rate of migration among the modelled pairs of populations was estimated between the western and eastern Cyprus, correspondingly with their proximity and the prevailing eastward current.

**Figure 4.** Histograms of the Mantel test assessing the relationship between genetic and geographic distance for *Gobius geniporus* (**a**) and *G. cruentatus* (**b**).

**Figure 5.** Migration routes of *Gobius geniporus* (**a**) and *G. cruentatus* (**b**) estimated by Migrate-n. Grey tips of arrows indicate less probable directions.

#### *3.2. Gobius cruentatus*

Mitochondrial marker cyt b was analysed in 41 specimens of *G. cruentatus*, and nuclear marker S7 in 39 specimens (Table 1). The alignment length of cyt b was 1117 bp and contained 47 polymorphic sites, while that of S7 had a length of 555 bp and contained only three segregating sites (Table 2). There were 32 haplotypes of cyt b and only four of S7. The best-fit substitution model selected for cyt b was general time reversible with proportion of invariable sites (GTR + I). An overall haplotype diversity of cyt b was high, while nucleotide diversity low (Hd = 0.985; π = 0.006), which can indicate a recent population expansion. This was also suggested by negative values of neutrality tests (see Table 2). On the other hand, for nuclear gene S7, values of haplotype and nucleotide diversity were markedly lower (Hd = 0.212; π = 0.0004). Diversity measures calculated per each locality are listed in Table S2. The Bayesian skyline plot showed a constant population size in the past, followed by a gradual population expansion and a stable population in the present (Figure 2b).

In *G. cruentatus*, cyt b haplotype network reconstruction did not reveal any well-defined spatial structure (Figure 3b). The network consisted mostly of unique haplotypes, very diverse for each locality (Figure S1b). Three haplotypes were shared between two or three distant populations (Spain/Sicily, Sicily/Croatia/Cyprus, Cyprus/Croatia). The network displayed two highly variable haplogroups separated by six mutational steps. Interestingly, there was a particular geographic pattern: while haplotypes from the central Mediterranean populations (Croatia, Sicily and Montenegro) were in both haplogroups, the haplotypes from the westernmost populations (Portugal, Spain and France) were placed only in one haplogroup, and haplotypes from the easternmost population, Cyprus, occurred only in the other haplogroup. On the contrary, there was nearly no polymorphism in S7. The network was formed by one dominant haplotype which included 69 alleles (out of 78) and was shared among all populations, one less frequent shared haplotype, which included only 7 alleles, and two unique ones. All haplotypes were very similar (Figure 3b). The remaining analyses were performed only on the cyt b dataset due to the low polymorphism in S7.

Similarly to the results of haplotype network, AMOVA performed on the cyt b showed that most of the genetic variance is distributed within the populations, with a ratio of approximately 1:4 between the variability among and within populations. Computed FST index indicated a high level of genetic differentiation (FST = 0.216, *p* < 0.01). Some values of pairwise FSTs performed on *G. cruentatus* indicated a pronounced differentiation (the highest values were between the easternmost population, Cyprus, and three westernmost populations—Spain, Portugal and France), but the majority were low or moderate; however, none of the values was significant (Table 4). The mean p-distances between the populations ranged between 0.3 and 1%, and the range of intrapopulation p-distances was similar (0.2–1.1%) (Table 4). The highest interpopulation divergences were between the westernmost and easternmost populations (0.9–1%, see Table 4). The highest overall intraspecific p-distance for *G. cruentatus* was 1.52%.


**Table 4.** Mean genetic distances between *Gobius cruentatus* populations for cytochrome b (uncorrected p-distances, in %, above diagonal), intrapopulation distances (on diagonal), and pairwise FSTs (below diagonal). None of the values of FSTs was significant at α = 0.05/number of pairs.

The result of the Mantel test was significant using both approaches (adegenet: observation 0.085, expectation 0.002, *p*-value 0.03; ARLEQUIN: correlation coefficient 0.81, *p*-value < 0.01), indicating a

possible pattern of isolation by distance (see Figure 4b). According to our results, the most probable scenario of migration of *G. cruentatus* between the studied localities was also the one allowing divergence and the migration from the ancestral population after the split. The divergence and migration routes are schematically depicted in the Figure 5b, while the estimates of immigration rates, divergence times and population sizes are listed in the Table S3. The results were not unequivocal, as can be seen from the Figure 5b, in several cases models proposing the opposite direction of divergence and migration between two populations had almost the same probability. This might be due to the low number of samples.

#### **4. Discussion**

Many previous phylogeographic studies have shown the existence of the geographical structure in populations of zoobiota within the north-eastern Atlantic and Mediterranean (i.e., Northern European Seas, Lusitanian and Mediterranean Sea provinces of Temperate Northern Atlantic realm *sensu* Spalding et al. [30]), e.g., [7,8,10–12]. In this term, however, the European marine gobies, despite being the most speciose and abundant fish family in this area, have been little studied so far.

Our results on two epibenthic goby species (*G. geniporus* and *G. cruentatus*) showed that the most plausible model which can explain the genetic structure of populations of both species is a model of divergence and recurrent migration from the ancestral population after the split. In the case of *G. geniporus*, the direction of divergence and the migration routes match well the prevailing currents between the studied localities (Figure 5a). The main feature of the migration route is anticlockwise circulation from Sicily towards east and then turning westwardly back to Sicily, and making a smaller circle from Sicily to the Adriatic Sea, following the Montenegrin and Croatian coast and subsequently the Italian coast, and back to Sicily. More dense sampling would be useful to confirm these findings, as also smaller gyres can substantially influence the genetic structure of epibenthic fishes [75]. The directions of divergence and migration between the pairs of populations of *G. cruentatus* were ambiguous. The observed pattern may be an outcome of a low number of individuals used to infer the migration routes in this species. Alternatively, it might be a consequence of higher complexity of water circulation in the species range, and/or biology of this species (see later discussion on hyperbenthic juveniles).

The lifestyle of the two studied species matches the model of divergence and recurrent migration. Being epibenthic and territorial, *G. geniporus* and *G. cruentatus* most probably exhibit only short-distance movements in adulthood, which allow the divergence between populations. Their main dispersal route is thus via a transport of planktonic larval stages, which can be dispersed by currents. The distance which a larva can reach depends mainly on the hydrodynamics and on the duration and behaviour of the larval stage, but the dispersal of planktonic larvae is much more complex and still not well understood [76]. The high multiscale variability of topography, temperature and salinity in the Mediterranean Sea generates free and boundary currents, bifurcating jets, meander and ring vortices, permanent or temporary cyclonic and anticyclonic gyres and eddies [77]. Recently, computer simulations that integrate a high number of biological and marine physical information have been successfully used in several works focused on the role of marine currents on the dispersion and genetic structure of marine organisms [75,78–80].

The influence of currents on genetic structure of the populations of epibenthic marine fish species was found for *Tripterygion tripteronotum* [75], where the population structure matched well the gyres in the Adriatic Sea, and also for other marine organisms [78–82].

Where known, the planktonic life stages in different European goby species have a variable duration, with a minimum of 13 days in *Zosterisessor ophiocephalus* to 51 days in *Gobius paganellus* [83]. However, in many Mediterranean gobies, nothing is known about their larvae, the duration of this stage, nor about their dispersion routes or distances. A similar range of planktonic larval duration (PLD) was observed in other Mediterranean fish species. In epibenthic Mediterranean littoral fish species of the genus *Tripterygion*, the PLD is estimated to be two to three weeks [84], while blennies

(Blenniidae) have a PLD between 22 and 71 days. In species of both these fish groups a population genetic subdivision was observed [75,85–87].

The dispersal capability of fish larvae can broadly differ, while it is around 120 km during 80–170 days of PLD in *Sebastes melanops* [76,88], it is only 100–500 m during 30–50 days in *Chaetodon vagabundus* [76,89]. This underlines the complexity of the dispersion process of fish larval stages.

Apart from having mobile larval stages, *G. cruentatus* has hyperbenthic juveniles, swimming in shoals within 1 m above the sea bottom. It is not known which distances this stage can cover and whether the dispersion during this stage has any influence on the gene flow. In the aquarium, this stage lasted two months [90]. It is not known whether other European gobies have hyperbenthic juveniles. Also other biological traits, such as reproduction strategy (European benthic gobies are iteroparous [53]) and timing of spawning, can influence the genetic structure of populations [75].

Our results showed that there was a high diversity of haplotypes of cyt b at each sampled locality. As discussed above, no clear population subdivision was found in two studied species, as it was disturbed by the recurrent migrations between the populations. There was a certain structuring in both species, as two haplogroups are observable in the networks (Figure 3). In *G*. *geniporus*, in the most frequent haplotypes of each haplogroup, specimens from different areas dominate: in one, the specimens from the eastern (Cyprus and Greece), while in the other, the specimens from the central Mediterranean Sea (Italy, Montenegro and Croatia). However, haplotypes of specimens from the Sicilian population are prevailing in the haplogroup with the eastern Mediterranean Sea haplotypes. In *G. cruentatus*, one haplogroup includes all specimens from the western part of the species range, from the Atlantic coast of Spain and Portugal, as well as from the western Mediterranean French coast, while the other haplogroup includes all samples from the eastern Mediterranean Sea (Cyprus). However, the central Mediterranean samples (Sicily, Montenegro and Croatia) are present in both haplogroups. Similar situation, where haplotypes from different haplogroups were found at the same geographic locality, with no clear geographical pattern, was observed also in other fish species in the Mediterranean Sea [91,92]. It was attributed to the secondary contact between the isolated populations which diverged in allopatry and came to a contact again after the removal of the migration barrier [91]. Additionally, our migration scheme for *G. cruentatus* shows convergence of the routes from the eastern and western Mediterranean Sea and the Adriatic Sea near Sicily, corresponding to the situation in the haplotype network.

Most of the research on population genetic structure of marine gobies from Europe have been conducted on epibenthic species of the genus *Pomatoschistus* (gobionelline-like gobies [52]), usually inhabiting lagoons and shallow coastal waters with fine substrates. Population genetic differentiation was observed in all four studied *Pomatoschistus* species [44–50,93]. Population genetic diversity of species from the gobiine-like gobies [52] has been studied in only two European marine species [51], epibenthic goby *G. niger*, living on the muddy substrates, and *Aphia minuta*, a pelagic shoal species. Giovannotti et al. [51] found a spatial genetic structure in epibenthic *G. niger*, while no structure in the pelagic *A. minuta*.

There are several recognised biogeographic breaks in the Mediterranean Sea and the north-eastern Atlantic Ocean. Our data did not point to the existence of any biogeographic boundary preventing a gene flow between the studied populations for neither of the two species. However, the effect of a small sample size cannot be excluded. The Strait of Gibraltar, or rather the Almeria-Oran front, which is an important biogeographic barrier for some marine organisms [4,8,10,33,94], did not have any influence on the gene flow between Atlantic and western Mediterranean populations of *G. cruentatus*. Similarly, this break does not present a barrier to gene flow of the various fish species, neither pelagic, e.g., *Sardina pilchardus* (nDNA microsatellite loci) [95], *Thunnus thynnus* (mtDNA d-loop) [34], *Scomber colias* (mtDNA d-loop) [31], *Diplodus sargus* (mtDNA d-loop, nDNA S7 first intron) [96], nor benthic ones, ranging from widespread eurybathic *Lophius piscatorius* (mtDNA d-loop), able to reach depths down to 500 m [33] to *Parablennius sanguinolentus* (mtDNA d-loop, nDNA S7 first intron), which is restricted to very shallow littoral of 0–1 m depth [86,87]. Neither did the Sicily Channel influence the genetic

structure of the two studied goby species, unlike is the case of some other fish species, e.g., *Dicentrarchus labrax* (nDNA microsatellite loci) [97], *Sprattus sprattus* (mtDNA d-loop) [11], and *P. tortonesei* (mtDNA 16S, COI) [48], where the Sicily Channel presents an important breakpoint. Although many studies showed a genetic differentiation between populations of the biota of the Adriatic and the Mediterranean Seas, separated by the Otranto strait, e.g., in *P. minutus (*mtDNA d-loop, cyt b, allozymes) [44–46,50], *Platichthys flesus* (allozymes) [98], Gouania willdenowi (mtDNA COI and 9 nDNA markers) [99] and *Sparus aurata* (allozymes) [100], neither was the Otranto Strait a biogeographic barrier for *G. cruentatus* and *G. geniporus.*

#### **5. Conclusions**

Our data revealed that the population genetic structure of the two studied epibenthic goby species (*G. geniporus* and *G. cruentatus*) can be well explained by the model of migration, allowing divergence between each pair of populations, with the ongoing migration from the ancestral population. This corresponds well with the biology of these gobies, having poorly mobile adults on one hand, and planktonic larval stages, which can be dispersed by currents, on the other hand. The population genetic structure of *G. geniporus* is influenced by currents: the estimated migration routes between the studied populations follow the main current directions in the study area.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-1312/8/10/792/s1, Figure S1: List of pairs of populations modelled in Migrate-n, Table S2: Diversity measures for *Gobius geniporus* and *G. cruentatus* calculated per each locality based on cytochrome b and S7 sequences, Table S3: Posterior distribution table of Migrate-n analyses, Figure S1: Cytochrome b haplotype frequencies at each locality, *Gobius geniporus* (a), *G. cruentatus* (b).

**Author Contributions:** Conceptualization, J.V. and R.Š.; methodology, K.C., J.V., K.E., R.Š.; software, K. ˇ C., K.E., ˇ J.V.; validation, J.V., A.M.P.; formal analysis, K.C., J.V., K.E.; investigation, K. ˇ C., J.V., R.Š., T.S.; resources, J.V., R.Š., ˇ M.K., S.Z., A.M.P.; data curation, K.C.; writing—original draft preparation, K. ˇ C.; writing—review and editing, J.V., ˇ R.Š., M.K., K.E., S.Z., A.M.P., T.S.; visualization, K.C., J.V.; supervision, J.V.; project administration, J.V.; funding ˇ acquisition, J.V., R.Š., T.S., K.C. All authors have read and agreed to the published version of the manuscript. ˇ

**Funding:** This research was funded by the Grant Agency of the Charles University (GAUK), grant number 1192217. Stay of K.C. at the University of Catania was supported by Erasmus program. RŠ received support by ˇ the Ministry of Culture of the Czech Republic (DKRVO 2019–2023/6.III.b National Museum, 00023272).

**Acknowledgments:** We are very grateful to two anonymous reviewers for their constructive comments, which helped us to improve our manuscript. We wish to thank David Villegas Rios (Spain), Konstantinos Moustakas and Ioulianos Pantelides (DFMR, Cyprus) and the Dikelas Dive Center (Karystos, Greece) for the help in the field. Sampling in Greece and Cyprus was conducted under permission of the Hellenic Ministry of Environment (through collecting licence to HCMR, no. 220965/2583/22-8-2011) and the Cyprus Ministry of Agriculture, Rural Development and Environment, through the Department of Fisheries and Marine Research (no. IΠ2921IΠ/10-11-2017) respectively. Collection of a part of the material was supported by the EU FP7 project ASSEMBLE at CCMar/Centre of Marine Sciences of Algarve, Faro, Portugal, and Observatoire oceanologique de Banyuls/Mer, Laboratoire Arago, Banyuls/Mer.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Filling the Gap of Data-Limited Fish Species in the Eastern Mediterranean Sea: A Contribution by Citizen Science**

**Roxani Naasan Aga Spyridopoulou 1,\*, Joachim Langeneck 2, Dimitris Bouziotis <sup>1</sup> , Ioannis Giovos 1,2,3, Periklis Kleitou 1,3 and Stefanos Kalogirou 4,\***


Received: 8 January 2020; Accepted: 5 February 2020; Published: 10 February 2020

**Abstract:** The biodiversity of the Mediterranean Sea is rapidly changing due to anthropogenic activity and the recent increase of seawater temperature. Citizen science is escalating as an important contributor in the inventory of rare and data-limited species. In this study, we present several records of five data-limited native fish species from the eastern Mediterranean Sea: *Alectis alexandrina* (Geoffroy Saint-Hilaire, 1817), *Ranzania laevis* (Pennant, 1776), *Dalatias licha* (Bonnaterre, 1788), *Lophotus lacepede* (Giorna, 1809), and *Sudis hyalina* (Rafinesque, 1810). All of the records were collected by a participatory process involving fishers and validated by associated taxonomic experts of the citizen science programme "Is it Alien to you? Share it!!!". This study fills an important gap for the distribution of the reported species and signifies the important role of citizen participation as a tool for extending marine biodiversity knowledge and fisheries management in an area with several gaps of knowledge on targeted and non-targeted species.

**Keywords:** Alexandria pompano; Slender sunfish; Kitefin shark; Crested oarfish; Barracudina; eastern Mediterranean Sea

#### **1. Introduction**

The Mediterranean Sea is facing several unprecedented anthropic pressures (e.g. pollution, habitat destruction, and geographical reshuffling of species) [1,2]. Along with climate change, species community shifts are regularly observed, leading to the tropicalization of the Mediterranean Sea [3,4]. Projections have indicated that at least 25% of the Mediterranean continental shelf might experience a total modification of species assemblages by the end of the 21st century [5]. Major gaps exist regarding deep species assemblages [6], particularly rare and data-limited species. This is mainly attributed to fragmented research, scarcity of observations, less fishing pressure, and practical difficulties in monitoring deeper waters.

Historically, the low economic value of non-targeted species has led to less data and a lack of vulnerability assessments. Some progress has been made in recent years to develop methods on status and risk assessments [7], such as mixed fisheries [8] and policy requirements [9,10]. Thus, the methodology has been developed, including time-series catch data [11], life history aspects [12],

and size structure [13]. Another aspect is the willingness by citizens to pay for climate adaptation and fisheries resources, as shown by Tulone et al. [14].

Citizen science is emerging as a key component for the exploration of marine biodiversity, being widely acknowledged by scientists, policy-makers, and conservationists, due to its capacity to address conservation issues that are related to rare species, climate change, and coastal systems [15–17]. Currently, there is an increasing number of citizen science efforts in the Mediterranean Sea t focused on various topics that are related to the marine environment [18–20]. This provides high potential for addressing data gaps related to least studied species. However, citizen science data should be carefully treated, since they often incorporate taxonomic uncertainties and misidentification due to photographic identification [21,22].

Here, we report several records of five data-limited fish species in the eastern Mediterranean Sea and highlight the use of citizen science as an emerging scientific tool for increasing our understanding on species distributions and contributing to fisheries management in the Mediterranean Sea. In addition, we used current published literature to highlight and give a quantitative aspect of current and historical knowledge of the studied species distribution in the eastern Mediterranean Sea (Supplementary Table S1).

#### **2. Materials and Methods**

The citizen-science programme "Is it Alien to you? Share it!!!" was initially launched in May 2016 by the Environmental Organisation iSea, with the aim of recording information on the occurrence, distribution, and expansion of marine non-native and rare species in Greece. An online easy-to-upload data repository was generated for citizens that allowed for records with pictures to be tracked. In addition, a Facebook group was created and all of the uploaded pictures to the group were automatically uploaded to the same data repository. At the time, this manuscript was written, this project's Facebook group numbers > 10,000 members, among which approximately 5000 are actively engaged. Each observer is requested to provide for each photo information on species size in total length (TL in cm.) and/or wet weight (WW; in g.). It is also requested to provide more information regarding the depth (D; in meters), the number of individuals (N), the location in latitude and longitude (LAT: LON), the date (DD: MM: YYYY), and the type of observation, as: underwater observation (UW), stranded (S) (i.e. found deceased on shore). Trained observers with silhouettes measured all of the individuals that were categorized as UW. If fishing gear/practice was provided, each observation was categorized following the FAO classification: longline (LL), bottom longline (bLL), surface longline (sLL), harpoons (HAR), and handlines and pole-lines that are hand-operated (LHP) (Figure 1, Table 1). All of the records are pre-scanned for quality and sent out for external evaluation to taxonomic experts prior to the inclusion in the final dataset of iSea. Only confirmed observations are recorded in the dataset, either through photo-identification or in-situ validation.

**Figure 1.** (**A**,**B**): *Alectis alexandrina* juvenile sighted in Protaras, Cyprus; (**B**,**C**): *Alectis alexandrina* adults caught in Zygi and Limassol port (Cyprus) respectively; (**D**): *Alectis alexandrina* adult captured in the harbour of Kos Island, Greece; (**E**): *Alectis alexandrina* juvenile sighted in Zygi, Cyprus; (**F**): *Alectis alexandrina* captured by spearfishing in Akrotiri Bay, Cyprus; (**G**,**H**): *Ranzania laevis* individual captured with surface longline in Psathoura Island, Greece.

**Table 1.** Records of five rare species in the eastern Mediterranean Sea with information on number of individuals (N), depth (m.), total length (cm.), area, coordinates (lat: lon) in decimal degrees and type of observation (underwater, UW; shore fishing, SF; spearfishing,; demersal longline, LL; Otter bottom trawl OTB).



**Table 1.** *Cont*.

All of the data used in this study has been uploaded to an electronic repository (https://wp.me/ P94Vaj-1Mm).

#### **3. Results**

#### *3.1. Alectis Alexandrina*

On 20th November 2012, two juvenile individuals (≈ 10 cm TL) were sighted and photographed near Protaras, Cyprus (Levant Sea, 35◦00 48.6" N, 34◦02 14.6" E) (Figure 1A; Table 1). On 4th February 2015, an individual with a TL of 45 cm was caught by a recreational fisherman at Zygi, Cyprus (Levant Sea, 34◦43 35.3" N, 33◦20 15.0" E), while a second individual, approximately of the same size, was caught on 30th May 2015 in the old port of Limassol (Levant Sea, 34◦40 13.8" N, 33◦02 34.8" E) (Figure 1B,C). On July 2016, a single individual with a TL of ≈ 50 cm and 1100 g in weight was caught by a recreational fisherman at the port of Kos Island (Aegean Sea; 36◦53 43.8" N, 27◦17 19.0" E; Figure 1D). On 16th March 2017, a juvenile individual (≈ 22 cm TL for 100 g) was found stranded near to Zygi village, Cyprus (Levant Sea, 34◦43 41.5" N, 33◦20 25.8" E) (Figure 1E). On 11th October 2017, an individual (≈ 60 cm TL) was captured by spearfishing at a depth of 5 m at Akrotiri Bay in Limassol (Levant Sea, 34◦33 20.2" N, 33◦00 59.8" E). During 2018 there had been two more records that were collected on 9th of October an individual of ≈ 15 cm TL by a spear fisher and another individual ≈ 6.5 cm TL was photographed by a diver; both occurrences were in Cyprus Ayia Napa, (Levant Sea, 34◦58 53.0" N, 34◦00 17.9" E) and Portaras Bay, (Levant Sea, 35◦00 30.1" N, 34◦03 48.4" E), accordingly (Table 1). Finally, there has been another stranded individual of 45 cm TL, in Zygi, Cyprus (Levant Sea, 34◦40 15.7" N, 33◦02 39.4" E), (Table 1; Figure 4).

#### *3.2. Ranzania Laevis*

On 21st December 2014, an individual of *R. laevis* was captured with a surface longline near Psathoura Island in North Aegean Sea (39◦26 46.0" N, 24◦04 46.1" E) at a depth of approximately 200 m and landed at Skopelos Island (Figure 1G, H). The specimen was 53 cm in TL. The fisherman and a local journalist contacted the local authorities, which identified the species as "Propela", the common Greek name for *Mola mola*. On 29th of July 2017 the journalist contacted iSea, in the context of the citizen science project "Is it Alien to you? Share it!!!" for sharing a log of rare species observations from Skopelos Island. Among the observations, the record of *R. laevis* was found and all of the available information was collected.

#### *3.3. Dalatias Licha*

On 5th November 2016, a single male individual of *D. licha* (≈ 150 cm TL) was caught by a demersal longline at a depth of 580 m off Amorgos Island (Aegean Sea, 36◦46 25.7" N, 26◦02 38.0" E) (Figure 2A). On 29th August 2019 an individual was caught off Levithas island at a depth of 612 m in depth by a demersal longline over muddy substrate (Aegean Sea; 36◦58 23.2" N, 26◦29 11.4" E) (Table 1; Figure 4).

**Figure 2.** (**A**): Male *Dalatias licha* caught with demersal longlines off Amorgos Island, Greece; (**B**): *L. lacepede* caught with demersal longlines between Skopelos and Euboea Islands, Greece; (**C**): *L. lacepede* caught with demersal longlines between Santorini and Anafi Islands, Greece; and, (**D**): *L. lacepede* caught off the Coast of Chalkidiki Peninsula, Greece; (**E**): *L. lacepede*, Rhodes Island, Greece.

#### *3.4. Lophotus Lacepede*

Demersal longlines set at 500 m deep bottoms between Skopelos and Euboea Islands (Aegean Sea, 36.7738◦ N, 23.5789◦ E) allowed for the capture of a large individual (≈ 160 cm TL, 10 kg WW) (Figure 2B) on 3rd March 2017. A few days later, on 9th March 2017, a slightly smaller individual (approximately 140 cm in TL and 8 kg in WW) was collected from the same area while using the same fishing gear. A third, distinctly smaller individual (≈ 60 cm in TL) (Figure 2C) was captured by demersal longlines at a depth of 540 m between Santorini and Anafi Islands (Aegean Sea; 36.3776◦ N, 25.5957◦ E). Lastly, on 6th October 2017, a fourth large individual (approximately 100 cm in TL and 6kg in WW) (Figure 2D) was captured by a longline targeting swordfish off the coast of Chalkidiki Peninsula

towards Sporades Islands (North Aegean Sea; 39◦44 10.7"N, 23◦22 56.6" E) at a depth of approximately 300 m. On September 12, 2017 an individual of ≈ 25 cm in TL was caught outside Rhodes Island, Greece (36◦27 21.6" N, 28◦13 05.5" E) (Figure 2E). On 1st of November 2018, an individual of ≈ 140 cm in TL was caught by a professional fisher with a demersal longline at a depth of 540 m (Cretan Sea, 23◦29 29.1" N, 35◦34 58.0" E) (Figure 4).

#### *3.5. Sudis Hyalina*

On 1st July of 2014, an individual was caught at 100 m in depth by demersal longline of a recreational fisher, off Crete Island (Aegean Sea, 35◦31 20.8"N, 24◦00 55.5" E). On 6th November 2016 an individual was caught by demersal longline approximately 30 miles south-east from Kastellorizo Island and at approximately 700 m in depth (Aegean Sea; 35◦51 41.4"N, 30◦06 32.0"E) (Figure 3A). The specimen was found mutilated by another fish, possibly a scabbardfish (*Lepidopus caudatus;* Euphrasen, 1788), but features of the head allowed for a uniequivocal identification. On 17th August 2017, another individual of *S. hyalina* (≈ 30 cm TL) was captured at Akrotiri, Cyprus (34◦32 46.0"N, 32◦56 56.4" E) by a recreational fisher at an approximate depth of 200 m (Figure 3B,C). During 2018, another record was added from Morfou in Cyprus; the individual was approximately 40 cm in TL and it was caught at 300 m in depth (Levantine Sea, 35◦13 43.4" N, 32◦52 01.1" E). On 19th May 2019, another individual was caught in 400 m by shore-based fishing off Crete island (Cretan Sea, 34◦54 55.1" N, 24◦54 50.6" E); on 29th August 2019, an individual was caught off Levithas island at 612 m in depth with demersal longline over muddy substrate along with a *D. licha* individual (Aegean Sea, 37◦00 02.9" N, 26◦27 32.6" E), (Table 1; Figure 4). The latest record was on 17th September 2019, where another individual of ≈ 20 cm in TL was caught by demersal longline at 100 m in depth at Saronikos Gulf (Aegean Sea, 37◦50 12.0" N, 23◦18 50.7" E).

**Figure 3.** (**A**): Head of *Sudis hyalina* collected from demersal longlines off Kastellorizo Island, Greece; (**B**) individual captured in Akrotiri Bay, Cyprus; and, (**C**): individual captured in Chalkidiki Peninsula, Greece.

**Figure 4.** Geographic density dependent bubble plot on five uncommon data-limited species (*Alectis alexandrina, Dalatias licha, Lophotus lacepede, Ranzania laevis* and *Sudis hyalina*) in the eastern Mediterranean Sea.

We found that, based on current literature (Supplementary Table S1), the reported number of individuals in this study contributed 21.6% of the reports the species in the eastern Mediterranean Sea and 27.3% to the total number of reports during the last decade. For some species, the contribution to the total number of reports in the eastern Mediterranean was higher: (a) *A. alexandrina* contributed 34.4% of the reports the species in the eastern Mediterranean Sea and 35.7% to total number of reported species in the eastern Mediterranean Sea during the last decade, (b) *R. laevis* contributed 11.1% of the reports of the species in the eastern Mediterranean Sea and 20% of the reports of the species in the eastern Mediterranean Sea during the last decade, (c) *D. licha* contributed 18.9% of the reports of the species in the eastern Mediterranean Sea and 28.6% of the reports of the species in the eastern Mediterranean Sea during the last decade, (d) *L. lacepede* contributed 46.1% of the reports of the species in the eastern Mediterranean Sea and 75% of the reports of the species in the eastern Mediterranean Sea during the last decade and (e) *S. hyalina* contributed 11.9% of the reports of the species in the eastern Mediterranean Sea and 14% of the reports of the species in the eastern Mediterranean Sea during the last decade.

#### **4. Discussion**

The present work provides additional information on five uncommon fish species for the Aegean and Levantine Sea. Rare species are often considered to be data-limited. However, spatial and temporal variations in the distribution of rare species might provide relevant signs of climate and environmental change [23,24]. Until recently, the scarce communication between researchers and citizens entailed the loss of an important part of available information on these species, while the recent increase in the use of social networks allowed for a closer communication that can result in an increase of records of rare and non-native species [20,25–28]. To date, the project "Is it Alien to you? Share it!!!" has gathered a vast amount of information regarding the distribution and establishment of species, including several first records, as well as expansion evidences [20].

*Alectis alexandrina* is a thermophilic species with a rather sporadic occurrence in the Mediterranean Sea. Fishers state that the species is increasingly caught the last years, being very common in Cyprus and rather sporadic in southern Aegean Sea, despite the few observations from the northeast Mediterranean Sea.

*Ranzania laevis* commonly known as slender sunfish (Fam. Molidae) is a pelagic-oceanic cosmopolitan species found in tropical and temperate seas and feeding on small fish, planktonic crustaceans, and jellyfish. It is one of the two Molidae species occurring in the Mediterranean Sea; however, reports of *R. laevis* from the eastern basin are scarce. Occasional records from Greece [29], Cyprus and Israel [30], Turkey [31], and Lybia [32] have been reported. Current distribution of *R. laevis* covers the whole Mediterranean Sea while quantitative information has revealed a rather sporadic occurrence and local peaks of abundance with increased zooplankton biomass [23]. The reported species is the second record from the north Aegean Sea [29].

The remaining three species reported are typical rare deep-water species in the eastern Mediterranean Sea [6]. Recent studies demonstrated the occurrence of these deep-water species, as previously overlooked in the eastern Mediterranean Sea [33,34].

*Dalatias licha*, commonly known as the kitefin shark, is one of the largest deep-sea sharks occurring in the Mediterranean Sea, being distributed at depths between 200 and 900 m, where it can be considered a top predator. It is although an uncommon species in the western and central part of the basin [35,36], while its presence in the eastern Mediterranean Sea is considered as very scarce [37,38], signifying any reports as highly valuable, also given the conservation status of the Mediterranean population of the species as vulnerable [39].

*Lophotus lacepede*, commonly known as the crested oarfish, is a large bathypelagic species that is remarkably sporadic in the whole Mediterranean Sea. Similarly, to the majority of deep-sea fish species it is more regularly observed in the western Mediterranean Sea, with only a few records in the eastern part of the basin. Currently, only four published records are known, all being from the Aegean Sea [40]. The observations that are reported in the current work highlight the deep waters between Chalkidiki Peninsula and Evoia Islands as an important area for the species.

*Sudis hyalina*, which is a rare bathypelagic cosmopolitan fish species, is considered to be regular in the western part of the Mediterranean basin, while only five documented records of the species exist from the eastern Mediterranean basin [41]. In the Aegean Sea only two records of the species have been published [29]. Consequently, the record presented in this study is the third record of the species from the Aegean Sea. Interestingly, the report from Cyprus is the first record of the species from the Cypriot territorial waters, most possibly an overlooked species.

#### **5. Conclusions**

This study reveals that the contribution of citizen-science accounts for 45.4% of the total number of the studied species published records and 58.5% of the total number of published records during the last decade in the eastern Mediterranean Sea. This signifies the important role of citizen-science efforts, both for scientific and public awareness. Nine more records of the reported species have been published since "Is it Alien to you? Share it!!!" was launched [38,42–46], whereas, in this article, we report the occurrence of 26 individuals contributing with 74.2% during the same period. Of course, it can be hard to determine whether the species density in the region has increased or if the reports were overlooked before pictures of the species were circulated on social media as something rare. Thus, post-hoc interviews with citizens and fishermen who report catches can be used to reconstruct species distributions and densities.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-1312/8/2/107/s1, Table S1. Up-to-date records of five data-limited species in the eastern Mediterranean Sea.

**Author Contributions:** Conceptualization, S.K., I.G., P.K. and R.N.A.S.; methodology, S.K., R.N.A.S.; software, R.N.A.S.; validation, S.K. and R.N.A.S.; formal analysis, S.K. and R.N.A.S.; investigation, S.K.; R.N.A.S., I.G., P.K., J.L., D.B.; resources, S.K.; R.N.A.S., I.G., P.K., J.L., D.B.; data curation, R.N.A.S., S.K.; writing—original draft preparation, S.K.; R.N.A.S., I.G., P.K., J.L., D.B.; writing—review and editing, S.K., R.N.A.S., I.G., P.K., J.L. and D.B.; visualization, S.K., R.N.A.S.; supervision, S.K.; project administration, I.G.; funding acquisition, I.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by iSea, Environmental Organisation for the preservation of the aquatic ecosystems—"Is it Alien to you? Share it!!!".

**Acknowledgments:** We are grateful to the fisherman Nikolas Katsillis (Greece) for sharing his pictures and data for three specimens of *S. hyalina, L. lacepede* and *D. licha*, Antonis Voutsas (Greece) for sharing his picture and data of *L. lacepede*, to Werner Wolf (Austria), Pabos Charalambous (Cyprus), Savvas Ioannou (Cyprus), Michalis Ilia (Cyprus) and Antonis Salachoris (Greece) for sharing data and pictures of the *A. alexandrina* specimens, Angelos Kouriefs (Cyprus) for sharing data and pictures of *S. hyalina* specimen and Georgios Poulios (Greece) for sharing data and pictures of *R. laevis* and all the other citizen scientists who contributed in gathering the information needed.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*
