**1. Introduction**

In 1992, the Directive 92/43/EEC, known as the Habitat Directive (HD), was issued to promote the "conservation of natural habitats and of wild fauna and flora" by ensuring bio-diversity (HD art. 2 par. 1) in the territory of European Member States. In Annex I, the Habitat Directive lists the sites of community interest, "whose conservation requires the designation of special areas of conservation". Among these sites, some are considered of "priority interest" because they are in danger of disappearance, such as the priority habitat 1150\* "Coastal lagoons", defined as follows:

"Lagoons are expanses of shallow coastal saltwater, of varying salinity and water volume, wholly or partially separated from the sea by sand banks or shingle, or, less frequently, by rocks. Salinity may vary from brackish water to hypersalinity depending on rainfall, evaporation and through the addition of fresh seawater from storms, temporary flooding of the sea in winter or tidal exchange" [1].

These habitats are found along all European coasts and their extent in the Mediterranean Sea varies widely, from 2 ha of Chalikiopoulou Greek lagoon [2] to 57,000 ha of Italian lagoon of Venice.

In the Mediterranean biogeographic region (Table 1; Portugal excluded because it overlooks the Atlantic Ocean), there are 312 coastal lagoons included in the Natura 2000 network (the ecological network of sites to promote restoration and protection projects, in accordance with the HD's objectives, art. 3) [3] and, on average, their conservation status is unfavourable/bad (U2) [4]. The threats and pressures determining this evaluation are numerous and they can be linked both to the direct economic development of coastal areas (D03, E01, F02, J02) and to the effects of indirect activities that take place in the continental area (A02, A08; Table 2). These habitats, in fact, receive river inputs, enriched with nutrients and pollutants, leading to a substantial alteration of ecological balances and a general impairment of water quality [5]. However, the main threat is the direct destruction or reduction of habitats, due to the construction of infrastructure (e.g., ports), dredging of shipping channels and the creation of facilities for the regulation of hydrodynamism. Other threats include fishing, aquaculture, recreational development and the voluntary or accidental introduction of alien species, that has induced significant changes in environmental and ecological status [5]. From a managemen<sup>t</sup> point of view, in almost all Member States, coastal lagoons are public property, except for a few special cases where some confined portions are privately managed [2].

**Table 1.** Number of "Coastal lagoons" (habitat code 1150\*) in the Natura 2000 Network for EU Member States in the Mediterranean biogeographic region (Portugal excluded). Data were elaborated by consulting [3].


**Table 2.** Activities with highly important pressures and threats on "coastal lagoons" habitats throughout the EU. The "Pressures" and "Threats" columns show the number of Member States that have reported these activities as the ten most impacting (modified from the global report for the 2007–2012 reporting period for coastal lagoons [4]).


From morphological and hydrodynamic points of view, each lagoon has peculiar characteristics closely related to the amount of freshwater inputs, tidal fluctuations and human interventions that, very often, have modified these areas either by land reclamation or by exploitation of fish resources or for navigation. The composite mosaic of the lagoon structure, with islands, sand barriers, salt marshes, wetlands, etc., determines a high heterogeneity from which we can derive the ability to

support higher productivity, compared to adjacent seas. These characteristics, determined by the transition between continental and marine environments, may also favour the presence of endemic species of both freshwater and marine fauna [6].

However, not all organisms can tolerate these marked physio-chemical, spatial and temporal variations, so the biodiversity can be lower than that in the adjacent sea and freshwaters, because of the reduced euryvalence of most species. Among fish fauna, species can be categorized into functional guilds on the basis of the way they use transitional areas: to breed, to feed, as a nursery for juveniles, as refuge, as migratory routes between freshwaters and seas, for the entire life cycle [7–9].

The Annex II of HD includes "Animal and Plant Species of Community Interest whose conservation requires the designation of special conservation zones". Among these, more than 60 fish species are listed, including bony fishes and cyclostomes. Focusing the attention on the species recorded in the "Coastal Lagoon" habitats of the Mediterranean Sea, on the basis of data provided by Member States [3], it was possible to fill the list in Table 3 [3,10–14], further completed with the information on the Conservation status under the Habitat Directive and the red list assessments compiled by the International Union for the Conservation of Nature (IUCN) [13]. This list includes 26 species that could be grouped in functional guilds, according to the classification proposed by Potter et al. [9]: anadromous fishes (five species), freshwater fishes (17 species) and lagoon residents (four species). The present review aims at exploring the potential use of fish species of Community interest (sensu Habitat Directive art. 4, letter g), as indicators in the context of lagoon coastal habitats.

Ecological indicator species are defined as species whose presence and abundance provide information about ecosystems [15]. As the morphological alteration is one of the main threats for coastal lagoons (Table 2, [4]), the present paper describes the biology, habitat preference, distribution and conservation status of those fish species that are more strictly related to salt marshes and wetlands, in order to propose them as early warning signal of habitat loss. Bortone et al. [16] indicates that the guild of estuarine resident is the most suitable to be used as an indicator of lagoon environmental conditions. Therefore, four lagoon resident species (Table 3), that spend their entire life cycle in a transitional environment, were selected. Adapted to a naturally stressed ecosystem, these species can tolerate wide environmental variations and, hence, cannot be considered indicators of water quality. However, in the following sections, examples on how these species can be threatened by the loss or alteration of peculiar habitat structures will demonstrate their suitability as indicators. In this document, the term "ecological indicators" means the indicators that respond to environmental stressors related to changes in the structure of lagoon habitats, including morphological alterations, salinity changes, and presence of invasive alien species.

In the Habitat Directive, *Aphanius fasciatus* and *A. iberus* are referred to as belonging to the order Atheriniformes, family Cyprinodontidae, but the recent nomenclature places them in the order Cyprinodontiformes and the family is Aphaniidae [17]. Similarly, *Knipowitschia panizzae* and *Ninnigobius* (former *Pomatoschistus) canestrinii* are listed as Perciformes, family Gobiidae, while are now attributed to the order Gobiiformes, family Gonionellidae [12]. The most common species for the number of reports in the Mediterranean coastal lagoons of the Natura 2000 Network is *A. fasciatus* (109 sites), while the others are present in 20–30 sites (Table 3). None of these species are priority species, meaning a species "for the conservation of which the Community has particular responsibility in view of the proportion of their natural range" (HD art. 1 letter h) nor is present in other Annexes. Additionally, the two species belonging to the genus *Valencia* were discussed, since they are phylogenetically related to Aphanidae and have a high level of conservation priority. Moreover, *V. hispanica* and *V. letourneuxi* occur in small coastal lakes, tolerating low salinity conditions, and the former shares the endemic status with *A. iberus* along the Mediterranean Spanish coast.


**Table 3.** List of fish species in Habitat Directive's Annex II and their presence in the habitats 1150\* Coastal lagoons in the Mediterranean Sea basin. The first column included the species as listed in HD, and the second column the currently accepted nomenclature, verified by consulting [10–12]. IUCN category abbreviations as


**Table 3.** *Cont*.

### *Water* **2020**, *12*, 2059

Despite the di fferent conservation status of these six species, on the basis of the o fficial European and International documents, related to the Habitat Directive and IUCN red list, these species may represent ecological indicator species of the complex mosaic of habitats characterizing the coastal lagoons in the Circum-Mediterranean area. The first goal of the present review was to collect and present three main components of the knowledge available on these species, on the basis of the current scientific literature: (1) Biology and Distribution, including the main information on population ecology and genetics, behaviour and life history (2) Conservation status, providing useful data to obtain some insights on the estimated conservation status of these species across their geographical range of distribution (3) Management, that is, interventions and actions conducted on these species, in terms of habitat conservation and restoration, including all projects carried out until present to enhance the favourable conservation status of their population.

The second goal was to overview and comparatively analyse this information, not only to provide a state of the art on the Conservation Biology of these species, but also to identify the potential future directions of research that may promote the use of these species as ecological indicators of morphological alterations in Mediterranean transitional water systems.

### **2.** *Aphanius Fasciatus* **(Valenciennes, 1821)—Order Cyprinodontiformes**

### *2.1. Biology and Distribution*

Individuals of *A. fasciatus* are small in size (maximum total length of female approx. 8–9 cm [18]) and have a marked sexual dimorphism, with males being smaller than females, characterized by yellow to yellow/orange caudal, dorsal and anal fin coloration (in some specimens, a vertical black band can also be present on the caudal fin) and 10–12 transversal brown bands alternating with straighter, silver-white bands on a greenish brown body [18]; females have 11–17 short dark brown bands along both sides of the body and the background is grey. The life cycle can last at maximum six to seven years [18,19] but, generally, sexual maturity is achieved in the first year of age [19], favouring a large number of generations and a rapid turnover of the population [20]. Females are, generally, more abundant than males, although the sex ratio displays significant seasonal variations: males, with their brighter colours and striking courtship behaviour, could su ffer a higher mortality during the reproductive period [18,21,22]. It is omnivorous [18] and its diet varies with individuals' age and seasons: juveniles are planktonophagic while adults become benthivores [23]. Although *A. fasciatus* can be found in freshwaters, especially in the terminal sections of rivers, the most abundant populations live in coastal environments from brackish [24] to hyperhaline water bodies [20,25], both closed or subjected to tidal excursions. It is supposed that the reproduction is synchronized with the moon phases because adults exploit the rising spring tide to reach the most isolated canals within the salt marsh system [26]. During the courtship, the male, trying to exclude the competitors, pushes the female towards the optimal area for spawning, which is represented by submerged vegetation in small and isolated creeks inside salt marsh system. Males have no territorial behaviour and parental cares lack, so the cannibalism of eggs often occurs [27]. After hatching, which takes place 14 days after the spawning, when the tide height is again favourable to movements, the juveniles reach small bodies of water, isolated from open waters at low tide and, therefore, they are less at risk of predation [26]. Field observations on the life cycle of this species sugges<sup>t</sup> that it is a sedentary fish throughout all of the year, preferring isolated areas [24], well protected from predation, avoiding the open lagoon [28]. The limited home range, the large demersal eggs, the absence of larval dispersal stages [19,21] and the fragmentation of brackish habitats lead to a genetic isolation with scarce gene flow [25] and a high degree of genetic divergence among populations [24], inducing some authors to hypothesize the presence (not verified) of di fferent species within the complex *A. fasciatus* [20]. A marked genetic diversity among the populations of *A. fasciatus* has been observed in the east coast of the Adriatic Sea, where individuals of the saline in the Northern part were found to be genetically distinct from those in the south [29]. Likewise, populations from Sicilian, Sardinian and Adriatic coasts display significant osteological differentiation [30]. Bottleneck effects were documented in some Italian lagoons along the Tyrrhenian coast, where a high mortality rate due to particular events, such as dystrophic crises, significant tidal changes [31] or lack of maintenance in saltern environments [25], determined a dramatic genetic loss. However, except for these few documented cases, *A. fasciatus* populations are considered to be demographically stable, namely without recent founder events or recurrent bottlenecks [24]. In Italy, two main clusters were identified with a similar genetic structure: the first includes the Tyrrhenian populations above about latitude 41 ◦N and the second the southern Tyrrhenian and the Adriatic ones [32].

*A. fasciatus* is one of the most eurythermal and euryhaline species of the Mediterranean Sea, being able to withstand temperature changes between 4 and 40 ◦C and to reproduce at a salinity between 10 and 80 [20]. Despite this high capacity to adapt to extreme physical–chemical conditions, the genetic variation within-population is very small and is threatened by natural and anthropogenic pressures [25,32]; coupling this with the scarce gene flow among populations [24], the species survival can be locally compromised [33]. This situation was reported in the Maltese island, where only four populations were found in hydrologically isolated sites [34]. Furthermore, these populations were highly vulnerable as the abundance of adults and the percentage of juvenile survival were low [35].

It is widely distributed along the coasts of central Mediterranean basin; in the easternmost coasts it is scarcely abundant because *A. dispar* is present, even if, in some Egyptian lagoons, both species have been reported and hybrids have been observed [33]. In the westernmost areas, only *A. iberus* has always been recorded. According to the Natura 2000 network dataset, *A. fasciatus* is present along all Italian and Greek coasts, in Malta, Croatia and Slovenia and along the Corsican coasts (Table 3).
