Genetic and Phenotypic Characteristics of the Salmo trutta Complex in Italy

Abstract

Salmonid fish have become ecological and research models of study in the field of conservation genetics and genomics. Over the last decade, brown trout have received a high level of interest in research and publications. The term Salmo trutta complex is used to indicate the large number of geographic forms present in the species Salmo trutta. In Europe, the S. trutta complex consists (based on mitochondrial DNA control region analysis) of seven major evolutionary lineages: Atlantic (AT), Mediterranean (ME), Adriatic (AD), Danubian (DA), Marmoratus (MA), Duero (DU) and Tigris (TI). In several nations, the difficulty of identifying some lineages derives from their wide phenotypic and geographic plasticity and the presence of mixed lineages (due to introgressive hybridization with domestic AT populations). In Italy, the S. trutta complex populations living in the Tyrrhenian area and on the main islands (Sicily, Sardinia and Corsica) showed high genetic diversity. Currently, on the Italian Red List, the protected (near threatened) populations are the AD and ME lineages. Recent studies based on traditional (mitochondrial and nuclear markers) and NGS (next-generation sequencing) analyses have clarified some genetic differences between the populations of the Tyrrhenian region, Sicily, Sardinia and Corsica. Native populations in Sardinia belong to the AD lineage, while those living in Corsica are mainly characterized by the AD, MA and ME haplotypes. In Sicily, in the area of the Iblei mountains, an AT lineage (North African) exists. According to some authors, the term Salmo macrostigma should only be used for populations in North Africa. The use of genotyping methods based on mtDNA and nuclear markers and the latest generation sequencing techniques can improve the study of populations and evolutionary lineages in areas where there are overlaps and hybridization phenomena.

1. Phylogenetic Analysis of Brown Trout Salmo trutta

The family Salmonidae includes three subfamilies: whitefish (Coregoninae), grayling (Thymallinae) and salmonids (Salmonidae). The genus Salmo and six other genera, Brachymystax, Salmothymus, Acantholinqua, Hucho, Salvelinus and Oncorhynchus, form the subfamily Salmonidae [1].

The genus Salmo Linnaeus, 1758 is characterized by a wide distribution range and species and local ecotype subdivisions. The genus Salmo includes only two species (Salmo salar and Salmo trutta). All brown trout in Europe are referred to as the Salmo trutta complex, which includes most of the European taxa.

The ancestor of the family Salmonidae appeared early in the Cretaceous period (between 63 and 135 MYA) in freshwater [2]. Genomic duplication of Salmonidae became evident approximately 60 MYA [3,4]. The subfamily Salmonidae appeared after the Tertiary period, more precisely, during the Miocene (between 13 and 25 MYA) [5]. The separation between the ancestors S. salar (Atlantic salmon) and S. trutta occurred approximately 9-5 MYA [4], and that between S. marmoratus and S. trutta occurred approximately 4 MYA. The nucleotide divergence between S. salar and the S. trutta complex is less than 2% [6]. However, chromosomal rearrangements [6], chromosome number (S. trutta: 80 vs. S. salar: 54–58) and the degrees of residual tetrasomy [4] differ significantly between the two species.

The S. trutta complex consists of more than 60 species, including several ecotypes and evolutionary lineages. S. trutta originated from an ancestral form in the Palearctic region. Bernatchez [7] analyzed the mitochondrial DNA control region (mtDNA CR) and found that the S. trutta complex consists of five main evolutionary lines, Atlantic (AT), Mediterranean (ME), Adriatic (AD), Danubian (DA) and Marmoratus (MA), which evolved independently following allopatric fragmentation. Additional evolutionary lineages have been identified in the Duero River (Duero, DU) in Spain [8] and the Dades River (DA) [9] and Tigris River (TI) [10] in Turkey. Recently, the S. obtusirostris and S. ohridanus forms have been included in the genus Salmo as distinct species [11]. The lineage AT is distributed throughout the Atlantic basin, from Morocco to Scandinavia. The ME lineage has a native range in the Mediterranean rivers of the Iberian Peninsula, southern France, Corsica, Italy and Greece. Currently, the ME lineage is present in the middle–lower reaches of many watercourses in the Alps, from the Aosta valley to Slovenia. The ME lineage was the first to separate from the AT lineage. The AD lineage is distributed along Mediterranean rivers from the southern Iberian Peninsula to Turkey and the Apennine area in Italy and Corsica. The MA lineage originates from the rivers of the Northern Adriatic basin. The DA lineage is present in rivers flowing into the Black, Caspian, D’Aral and Persian Gulf Seas. In some areas, the AD and ME lineages show overlaps. For example, in Corsica, both forms have been reported [12], while in Sardinia, native populations are of the AD lineage. In the Iberian Peninsula, populations comprising both AD and ME and pure ME or AD populations have been reported in the Po basin [7].

The results of genetic analysis indicate that the different evolutionary lineages of the S. trutta complex were modified by glaciation processes and specific colonization histories [13]. The diversification of the S. trutta complex occurred mainly during the Pliocene and Pleistocene and was associated with the climate cooling that affected the Northern Hemisphere [14]. The oldest fossil of European trout was found in the Caucasus region and dates back to the Upper Pliocene [15]. During glacial periods, European fauna were confined to ice-free refuges in Spain, Italy and the Balkans and spread northwards during the interglacial warm periods. Populations living in these territories are the result of complex evolutionary processes involving older evolutionary lineages and local adaptations. The occupation of the territory by glaciers was a limiting factor for the distribution of the species even though brown trout can survive in near-zero temperatures. In addition, individuals could move across seas and oceans when the sea water was cool enough. It is probable, therefore, that glacial periods may have caused the isolation of some populations and, at the same time, favored the spread of others who had free access to the sea. In southern Europe, during interglacial phases, warming favored the spread and fragmentation of populations living in single catchment areas (Figure 1). Based on the current southern limit of the anadromous populations of S. trutta, which approximately corresponds with the northern Atlantic coast of the Iberian Peninsula, we can deduce that S. trutta could form stable anadromous populations until the surface temperature of the seas reached approximately 16–8 °C [14]. During glacial periods, most of the Mediterranean Sea was suitable for salmonid life, providing the possibility of colonizing all accessible watercourses (Figure 2).

During interglacial periods, in the different sectors of the Mediterranean basin, summer temperatures were too high to allow salmonids to live (always exceeding 20 °C) (Figure 2).

The spatial genetic differentiation of the S. trutta complex is confirmed by the biogeographic hypothesis. The evolutionary lineages in the Mediterranean area (ME–AD–MA) originated from a common ancestor [7,17]. Within anadromous migratory populations, only a portion of progeny return to the sea, while some individuals remain as residents and grow to sexual maturity in freshwater. During the Pleistocene, the chemical and physical conditions of the Mediterranean Sea were not always favorable to the survival of anadromous populations, causing alternating situations characterized by phases of isolation and redistribution within the entire Mediterranean basin [14]. The three Mediterranean lineages separated from an anadromous ancestral population approximately 150,000 years ago [15]. This date corresponds with the date of the glacial maximum of the penultimate glaciation (Riss glaciation). Refuge areas have been identified to the west in the major catchment areas of the Rhone and Ebro rivers and to the east in the Anatolian Peninsula, which was scarcely affected by glaciation [7]. The earliest separation was probably followed by a subsequent fragmentation within the Mediterranean basin, leading to the divergence of the ME, MA and AD lineages. Compared to the AT line, ME retains more ancestral features in its genetic sequence [18]. The presence of these lineages confirms the hypothesis that the Mediterranean area was a refuge during glaciations. The richness and diversity found in Southern Europe is probably the result of the persistence of refugia and the accumulation of variations during glaciations, while the rapid postglacial colonization was the result of the less complex evolutionary lineages in northern Europe.

2. Mitochondrial (mtDNA) and Nuclear DNA Analysis

Genetic markers based on mtDNA analysis are simple to use and inexpensive. Mitochondria contain most of the genes that code for cellular energy production and electron transfer (the NADH dehydrogenase subunit, the cytochrome oxidase subunit, ATPases 6 and 8, cytochrome b, rRNAr, RNA, 12S and 16S) [19,20,21]. Compared to nuclear DNA, mtDNA markers show a higher rate of nucleotide substitution (by 5 to 10 times), no (or little) recombination, maternal inheritance and neutral (or nearly neutral) evolution [7]. The choice of the sequence used for genetic analysis depends on the phylogenetic hypothesis to be tested—the D-loop is used in the case of rapid evolution; cytochrome b is used in the case of moderate evolution; and 12S or 16S rRNA and cytochrome oxidase I are used in the case of slow evolution. mtDNA is used to study the direction of hybridization and the incidence of introgression [22,23]. Note that in the case of hybridization, the use of mitochondrial markers alone is not sufficient and may generate erroneous inferences. For this reason, other analyses (e.g., based on nuclear genes) are also considered in phylogenetic and phylogeographic studies [24]. Recently, Ravinet et al. [25] suggested a Bayesian coalescence-based method for mitochondrial DNA analysis.

Traditionally, the nuclear genes most often used in phylogenetic analyses are histone (H3) and ribosomal (18S and 28S) genes. At the population level, introns or noncoding regions are usually used, as their evolutionary rate is higher, and they are not affected by selection [26]. To resolve the simplest phylogeny cases, ITS (internal transcribed spacer) regions offer sufficient variability, although in some cases, paralogy effects and recombination events can complicate analyses [18].

Electrophoretic analyses of proteins and the LDH-C1 allozyme (lactate dehydrogenase-C1 locus) were used in early studies of brown trout [12]. Genetic differences between the AT and ME evolutionary lineages were confirmed using mtDNA and restriction fragment length polymorphism (RFLP) analyses. This method facilitated the distinction between the AT and ME lineages by amplifying the mitochondrial 16S rDNA and control region (D-loop) genes using the restriction enzyme RsaI and amplifying the nuclear LDH-C1* gene using the restriction enzyme BslI [27]. The LDH-C1* 90 allele is specific to the AT line. Several authors have used mtDNA markers for genetic analysis of brown trout [14,28,29]. The control region sequence is the marker most widely used to identify main evolutionary lineages [14]. This gene is characterized by the absence of introns and recombination. S. marmoratus is characterized by the mtDNA MA and LDH-C1*(120) alleles [30,31]. Meraner et al. [32], using the complete mtDNA control region in S. trutta, identified the MA, AT and DA lineages. In a recent study, ancient DNA was used for phylogeographic analysis of the S. trutta complex in Italy [31], highlighting that the AD and ME evolutionary lines are also present in museum-preserved samples.

2.1. Microsatellite Analysis

Microsatellites are also known as simple sequence repeats (SSRs) or short tandem repeats (STRs). Microsatellites are distributed throughout the genome, with a greater presence in noncoding regions. Microsatellites are mainly neutral and unaffected by selection [33]. Regions flanking repeated sequences tend to be highly conserved. The advantages of using microsatellites are their high reproducibility and high resolution. The most common repeats are di-, tri- and tetra-nucleotides, and the maximum length is approximately 200 bp. Microsatellite loci with a dinucleotide motif are the most widely used due to their higher density (on average, one dinucleotide per 30–50 kb) [34]. Microsatellite markers are very useful in analyses of population structures and gene flow.

The main limitations of the use of SSRs derive from possible errors during PCR amplification [35] and the occurrence of homoplasy products (equal types and numbers of different alleles) [36]. These phenomena lead to underestimations of the true level of divergence between two populations. Genotyping errors can also occur due to the preferential amplification of small alleles (while larger alleles are not amplified) [37] or replication slippage, which can produce additional elements (in the form of allelic prepeaks) [38]. Most of these problems can be solved by using markers from different sources, e.g., nuclear and mitochondrial genes. Microsatellites can be easily identified in new species by de novo techniques [39]. Numerous authors have used microsatellite markers for genetic analysis of brown trout [32,40,41].

2.2. Combined Analysis of Different Genetic Markers

Some authors have used different combinations of mitochondrial and nuclear genetic markers in genetic analyses of the S. trutta complex [32]. Berrebi et al. [41] used a set of 6–12 microsatellites in combination with 16S rDNA (or D-loop) and LDH-C1* markers. Spendiani et al. [42] used two genes, mtDNA (control region) and LDH-C1*, in their first experiment, and three genes, mtDNA (control region), 11 microsatellites and LDH-C1* in their second work [43]. Fabiani et al. [44] used an mtDNA gene and microsatellite markers, while Chiesa et al. [45] used the mtDNA 16SrDNA gene and microsatellite markers in a study of S. marmoratus.

3. Next Generation Sequencing (NGS)

Since the 2000s, the development of single nucleotide polymorphism (SNP) analysis has brought ecological genetics into a new era. The development of new sequencing techniques has allowed for the simultaneous analyses of thousands of markers [46]. The first sequencing technology was proposed by Sanger [47] and produced a sequence for each fragment (>700 bp) with a very low error (<0.01%). This technique has been used to sequence various fish, and it is still used in some laboratories to sequence single genes or microsatellites. New second- and third-generation sequencing techniques (such as single molecule real-time (SMRT) sequencing) have improved genomic assembly by the parallel sequencing of millions of fragments [48] and have been used for Atlantic salmon [4], rainbow trout (Oncorhynchus mykiss) [49] and brown trout [50]. Currently, several high-density genomic maps are available for brown trout [6], Atlantic salmon [51] and Artic char [52]. Several research projects are underway to study the genomes of Atlantic salmon and rainbow trout (including cGRASP, Consortium for the Genomic Research of all Salmonids, and ICSASG, International Collaboration for the Study of the Sequence of the Genome) [53], perform comparative genome analyses of similar species and conduct genome resequencing and de novo transcriptome analyses of Atlantic salmon, rainbow trout, brown trout and Arctic charr [54,55]. These initiatives have enabled the creation of an expressed sequence tag (EST) database and the development and commercialization of a number of microarray SNPs for rainbow trout, Atlantic salmon and other salmonid species. The creation of the PhyloFish database (which published the transcriptomes of 15 fish species) provided another important contribution to the development of the sector [56].

The availability of reference genomes for the main salmonid species is essential for analyzing their complex traits [48]. The discovery and annotation of genomic variants allow the production of new microarray SNPs and, accordingly, the implementation of specific genomic programs. Recently, the availability of microarray SNPs for salmonids has allowed the simultaneous analysis of thousands of SNPs at low cost. In the aquaculture sector, recent studies have shown that the genomic selection method can be used to improve several production traits and disease resistance in rainbow trout and Atlantic salmon [57,58]. Population genomic data also provide valuable information for studying aspects of the eco-evolutionary history of different salmonid species, such as population structures, local adaptations and genetic diversity between populations. In several fish species, genome-wide distributed sets of SNP markers have been successfully used for phylogenetic analyses. The use of SNPs has also contributed to the development of new conservation strategies [59]. Hand et al. [60] used 10,267 SNPs in the USA to study the introgression of O. mykiss into wild populations of Oncorhynchus clarkii lewisi and to identify chromosomal regions affected by selection. To date, these methods have rarely been used for S. trutta, and currently, only a limited number of SNP markers for the species have been developed. Recently, for the first time, a commercial SNP microarray derived from a 57K SNP chip originally developed for rainbow trout was used in a S. trutta complex selection program [61]. The identification of approximately 900 polymorphic sites facilitated studying the genetic structure of wild Mediterranean brown trout populations in Italy and to begin the selection of native broodstocks. Zhang et al. [62] used a 57K DNA chip developed for rainbow trout in different salmonid species, and Drywa et al. [63] used a SNP array developed for Atlantic salmon to analyze a population of lake brown trout (S. trutta trutta).

In fish phylogenetic and phylogeographic studies, the most popular NGS methods are restriction site-associated DNA sequencing (RAD-seq), transcriptome sequencing (RNA-seq), targeted amplicon sequencing (TAS) and hybrid enrichment using probes. In salmonid population genetic studies, the RAD-seq technique has been widely used [64]. RAD-seq technologies facilitate overcoming some limitations of traditional methods by increasing the number of genes analyzed [6]. The main applications of the RAD-seq method are the identification and annotation of novel SNPs and the genotyping of thousands of SNPs in population genetic studies. The RAD-seq method proposed by Baird [65] showed some limitations, and therefore, subsequent modifications were proposed (ddRAD [66] and 2b-RAD [67]). In Baird’s method [65], sequences between known restriction sites and random sites are obtained, while with the ddRAD method, sequences between two known restriction sites are obtained. The results of RAD-seq analysis can be analyzed using several software programs, such as STACKs [68], IQ-TREE [69], UNEAK (TASSEL) [70], PyRAD [71], dDOCENT [72] and AftrRAD [57,73]. The most popular software is the STACKs program. The results obtained in the first analytical step using this software are then transferred to the GENEPOP or STRUCTURE programs for further analysis. The ddRAD method was used by Letwein et al. [6] to obtain a dense association map for S. trutta by crossing parents of the AT and ME lineages. Lecaudey et al. [74] used a RAD-seq method to study the different genera of the subfamily Salmonidae. Leitwein et al. [75] analyzed the introgression of the AT lineage into local ME populations in southern France using the ddRAD technique, and Magris et al. [76] used the same method for different populations of the S. trutta complex in Italy (northern Italy, the Tyrrenian area, Sardinia and Corsica). Vendrami et al. [77] and Lemopoulos et al. [33] reported improved genetic analyses using RAD-seq methods compared to those using microsatellite markers, particularly in resolving population structures. Saint-Pé et al. [78] used a RAD-seq method to identify 12,204 SNPs throughout the genome of S. trutta and a low-density SNP array (192 SNPs).

Transcriptome sequencing (RNA-seq) is used to analyze the sequences of coding regions, which usually account for approximately 1.5% of the total genome. Carrauthers et al. [54] obtained 35,736 protein-coding transcripts for the AT lineage.

4. Description of the Broun Trout Salmo trutta Complex in Europe

The S. trutta complex, with all its variations, is present throughout Europe. The northern limit of its range extends from Iceland to Russia (territories north of the Volga) and the northern part of Scandinavia. The southern limit of its territory is the Atlas Mountains (between Algeria and Morocco) and includes both Italian islands of Sicily and Sardinia. From west to east, brown trout are present from territories along the Atlantic European coast to the Caspian Sea and Aral Sea. The anadromous marine form is present in the rivers of the Cheshkaya Gulf, Baltic Sea, North Sea, Irish Sea, English Channel, Black Sea, Caspian Sea and Aral Sea. Sea trout are absent from the Mediterranean area. Lake trout occur in lakes in the Alps, Scandinavia, Great Britain and northern central Europe [79]. The distribution within this range varies according to the following specific characteristics:

• a water temperature within a very narrow range (averaging less than 20 °C in the summer);
• fast currents;
• good water quality with pH values close to neutral;
• easily accessible spawning areas (clean bottoms with coarse gravel and pebbles).

Examples of mixed evolutionary lineages [7] have been reported in several European countries [41].

5. Description of the Brown Trout Salmo trutta Complex in Italy

In its wide distribution range, the S. trutta complex shows extreme phenotypic diversity and considerable variation in biological characteristics. Italy, due to its latitude, central position in the Mediterranean area and abundant mountains, is a very important hotspot of the S. trutta complex. In Italy, the S. trutta complex is widely distributed in all suitable waters. From north to south, country ranges from the rivers, streams and lakes of the Alps, which are characterized by seasonality, to the streams of the southern Apennines, which are characterized by summer heat waves at the survival limits of salmonids. In Italy, available habitats range from the cold springs of the great rivers of the central Apennines to the streams that flow through the middle of the Mediterranean scrub on the main Italian islands.

In recent decades, knowledge of the state and distribution of native populations of the S. trutta complex in Italy has received a considerable boost, especially for conservation purposes. This attention has led to the publication of numerous scientific papers that have revealed a certain degree of introgression of domestic populations of northern European origin (AT lineage) into native strains [80,81]. Clearly, this characteristic makes the systematic classification of the S. trutta complex even more complicated.

In recent decades, several taxonomic classification methods have been proposed. Bianco in 1995 [82] identified three species, S. trutta, S. marmoratus and S. carpio, considering S. marmoratus and S. carpio to be the only Italian endemic species. More recently, Bianco [83] identified six species, S. fibreni, S. carpio, S. cettii, S. marmoratus, S. cenerinus and S. rhodanensis, the latter of which is endemic to the Roja River on the Italian–French border. In 2004, Zerunian [84] identified two species (S. fibreni and S. carpio) and a superspecies consisting of three semispecies: S. trutta semi species, S. trutta macrostigma, and S. trutta marmoratus. In this case, the endemic species of the Garda and Fibreno lakes were identified as species, and the other forms present in Italy were combined into a single superspecies (S. trutta). It is worth noting that this approach was based on the hypothesis existing at the time, which was that the native trout of central–northern Italy were similar to the trout in northern Europe and that in the Tyrrhenian area, some populations similar to S. macrostigma occurred, as suggested by Vinciguerra in the early 1900s [84]. Later, according to the first studies based on molecular markers, it became clear that the brown trout of Italy were phylogenetically separated from northern European trout [14]. During this period, Mediterranean Alpine and northern–central Apennine trout were referred to as “Mediterranean brown trout”, distinct from S. macrostigma [42].

The taxonomic classification of the S. trutta complex in Italy was revised by Kottelat and Freyhof in 2007 [85]. According to their study, the term S. macrostigma should be used only for the populations of Morocco, and the correct name for the “Tyrrhenian and Apennine trout” was S. cettii Rafinesque, Schmaltz 1810, which was originally used by the Franco-German zoologist to describe trout living in the rivers of eastern Sicily. Additionally, these authors proposed the name S. cenerinus Chiereghini, 1847 for the populations of the S. trutta complex living along the Adriatic side of the Apennines (up to the Vomano River in Abruzzo) and in the high portions of Alpine streams in the Po basin. Using several mitochondrial markers (the mtDNA control region) and nuclear markers (11 microsatellites and eight intronic genes), Gratton et al. [86] identified two main native evolutionary lineages: a “peninsular lineage”, comprising samples of S. cenerinus, S. cettii and S. fibreni, and the lineage of S. carpio and S. marmoratus. S. marmoratus is the only native brown trout present in the middle and lower river courses of the Po River and the basins of the upper Adriatic area [87]. According to Tortonese [88], the MA lineage represents a subspecies within the S. trutta complex. S. carpio was also considered a subspecies by Tortonese [88].

In 2014, the Italian Association of Fresh Water Ichthyologists (AIIAD) [89] proposed a classification system based on evolutionarily significant units (ESU, populations that are partially genetically differentiated as a result of significant evolutionary separation). According to this classification, the five evolutionary lineages present in Italy (S. trutta complex) are as follows: S. marmoratus, distributed in the Po valley, Croatia and Slovenia; S. cettii (former name S. macrostigma), distributed in Sardinia and Sicily, Lakes Posta Fibreno and Ninfa and some Tyrrhenian waterways; S. ghigii, distributed in the Apennine and Tyrrhenian basins; S. Fibreni, distributed in the Posta and Fibreno Lakes; and S. carpio, distributed in Lake Garda. Lake trout are not considered a separate entity but an ecotype of S. trutta and S. marmoratus. In the literature, Tyrrhenian and Adriatic trout are often associated with the ME and AD mitochondrial haplotypes. In fact, this classification is inaccurate because the ME haplotype is present in some Adriatic populations, and the AD haplotype is common in some populations living in central Italy and on the main islands [86].

Native S. trutta are present in the Western Alps (20%), the Apennines (2.8%), Sardinia (50%) and Sicily (33.3%) [42]. Watercourses with non-native brown trout populations are common in the Eastern Alps (94.3%), Central Alps (100%) and Western Alps (60.0%) and less common in the Apennines (59.2%) and Sardinia (50.0%). For S. marmoratus, all populations in the Central Alps are introduced, while original populations include 75% of those in the Western Alps and 100% of those in the Eastern Alps [43]. Although the mechanisms behind the variation in the rate of introduction remain poorly documented, geological and climatic conditions seem important in the distribution of the S. trutta complex. Native trout tend to be absent or rare in rivers with modified flow regimes. Other threats to the S. trutta complex include poaching, habitat fragmentation, pollution, water diversion and competition with invasive non-native species.

The IUCN Red List of Threatened Species (Version 2016) considers S. cettii to be almost threatened, and the populations themselves are listed (as S. macrostigma) in Annex II to the Habitats Directive (92/43/EEC). For these populations, the main threats are sport fishing and restocking with non-native fish. The marbled trout was included on the IUCN Red List as critically endangered [90]. S. carpio and S. fibreni are considered to be in a critical state due to illegal fishing, the introduction of non-native species (e.g., Coregonus sp.), pollution and habitat degradation.

6. Description of the Morphological Characteristics of the Mediterranean Brown Trout of Italy

All S. trutta complex populations living on the Italian mainland and islands (Sicilia, Sardegna and Corsica), except the Sicilian populations in the Iblei mountains, are included in this group [91]. Certain morphological features can help identify the native forms, even though most of these characteristics are not universal and vary relative to the environment [92].

In general, the Mediterranean brown trout is described by the following characteristics:

• parr spots (dark, rounded spots on the flanks typical of juvenile salmonids) also present in adults;
• an evident preoperative dark spot;
• fine black and red spots with no or a slight white halo.

These general characteristics are valid for many of the populations of the central and northern Apennines and the Alpine area [91]. These features have recently been updated to include additional frequent characteristics:

• a dark or bluish preopercular spot, with other retromandibular and suborbital spots of the same color in some populations;
• parr spots inconsistently present in adults (parr spots are always present in numbers greater than nine and have a narrow oval shape compared to the round shape of trout of northern European origin), with multiple rows of parr spots often occurring;
• black and red spotting present in varying proportions, with some individuals displaying only red or black spotting;
• primary spotting often consisting of irregularly shaped patches and never including perfectly oval spots with white halos;
• populations displaying four vertical dark bands alternating with three light bands, which may be more or less evident in different populations;
• extensive spraying is absent in the dorsal zone, except in some rare hyperpigmented individuals who also have spots on the upper and lower jaws;
• from two to four dark ocular spots frequently symmetrically arranged around the pupil;
• complete absence of the black margin, with white bands occurring on the first rays of the anal and dorsal fins (in adults), although this reference characteristic is only valid for peninsular populations; and
• the fatty fin frequently occurring without points, with the exception of a few peninsular populations and many island populations.

Mediterranean brown trout may have two “extreme” eco-phenotypes: one phenotype with black dots and another with predominantly red dots. These two eco-phenotypes show adaptations to different environmental conditions. The phenotype with black dots is usually present in areas with medium–low slopes that are rich in macrophytes. The phenotype with red dots lives in areas with steeper slopes and/or waters that are always very cold and oxygenated. This general scheme is simplified and does not take into account all environmental variables and possible Mediterranean trout habitats [85,93,94].

7. Native Mediterranean Brown Trout in the Alps

The presence of Mediterranean trout in Alpine areas has long been debated. Some authors have reported that the brown trout is an indigenous species of the Alps. Zerunian [84] reported that the brown trout originated in Alpine areas and the northern part of the Apennines, stating, however, that most current populations are totally or partially composed of material of alien origin (AT). Forneris et al. [95] reported that the ME lineage cannot be considered native to the left tributaries of the Po River or the tributary waterways of the Adriatic Sea. The only exceptions are some watercourses in the western Alpine area. It should be noted that in the southwestern Alpine area (Liguria and Piedmont), the presence of ME and AD lineage has been reported by several authors, and recently, Splendiani et al. [96] (using the mtDNA control region and LDH-C1* genes) confirmed the native origin in this territory. According to these authors, in the southwestern section of the Alps, during the last glaciation, a unidirectional corridor of dispersion was active from the Adriatic side to the French side. For this reason, the French origin hypothesis for the Mediterranean trout populations in the western sector of the Alps must be definitively rejected. Gibertoni et al. [97] pointed out that the Po basin (west of the Garda Lake) can be considered a potential region of spreading by native Mediterranean trout. This hypothesis was recently reinforced by studies conducted by Splendiani et al. [31], who analyzed museum DNA samples collected from the western Alpine sector, and the results indicated the presence of haplotypes characteristic of Mediterranean brown trout. For example, an ME haplotype was found in a museum sample (dated 1876) from Lake Moncenisio (Dora Riparia basin), and AD haplotypes were isolated in samples from Lake Maggiore (dated 1879) and Lake Garda (dated 1877). Giuffra (1994) reported the presence of the ME lineage in the Dora Riparia basin (MtDNA: cytochrome b and D-loop), and this presence was also confirmed by reports from Cuvier and Valenciennes in approximately the mid-19th century [28]. Genetic studies have been conducted on the S. trutta complex in Switzerland using microsatellites and AFLP (Amplified Fragment Length Polymorphism) markers. In these studies, eight populations in various river basins, including Torrente Poschiavino (Adda basin), Allaine (Rhône basin), the Rhine basin and the Ticino basin, were analyzed. Although the aim of the research was to highlight possible neutral and adaptive genetic divergences, the authors found a clear separation between populations in the Allaine, Poschiavino and Rhine Rivers. The authors reported that although alien S. trutta have been introduced on a large scale, it was still possible to distinguish three distinct genetic groups, which attests to the residual original native genetic pool. In particular, the authors describe that AD and MA genetic variants were observed in the Poschiavino Stream [98].

8. Brown Trout of Sicily

In 1810, in a catalogue of Sicilian fish, Rafinesque included the new species S. cetti, dedicating it to the zoologist Francesco Cetti, who published the book “Amphibians and Fish of Sardinia” in 1778. Rafinesque described a generic trout with mixed black and red spotting present in the waterways of the Noto Valley and the Demon Valley. In 1896, Vinciguerra identified the Algerian S. macrostigma trout described by Dumeril in 1858, which has rounded black spots that are larger than normal and almost no red spots, in the Ragusa area. The similarity between the two fish is evident upon comparing the specimen collected in Sicily with the original description. Indeed, oval-shaped spots with clear white halos, a coarse morphology in large and well-contracted patches and the presence of black margins on the anal and dorsal fins are distinctive features of Sicilian trout compared to the ME lineage [99].

Trout in the Iblei Mountains of Sicily have a small number of points (on average, 21) and a lower number of spots compared to the ME lineage. The shape of their parrs is also different, presenting as more rounded [99]. These morphological differences have also been confirmed by molecular genetic analyses [100]. The first studies focused on the analysis of mitochondrial DNA, also identifying the AT haplotype in North Africa and the Atlantic regions of the Iberian Peninsula. The presence of the same haplotype in Palaeolithic fossils found in the caves of Praia a Mare in Calabria [43] suggests that colonization was not limited to Sicily. No other populations belonging to the AT lineage have been reported in other areas of southern Italy. Therefore, the Sicilian populations of the Iblei Mountains are the only AT S. trutta complex of South Atlantic origin [30]. Tougard et al. [30] analyzed the complete mtDNA sequences and the cytochrome b gene of four museum samples (attributed to S. macrostigma) and concluded that they belonged to the AT lineage. In the same study, samples from Corsica and Sardinia were attributed to the AD, ME and AT lines [41]. Zaccara et al. [94] (based on mtDNA and LDH-C1*) and Sabatini et al. [101] reported similar results for S. trutta complex populations in Sardinia. The results of a recent study definitively clarified the genetic differences between the Sicilian, Sardinian and peninsular populations [102]. The term S. cettii originally used by Rafinesque (1810) to identify the trout in southeastern Sicily should reasonably be used only for S. trutta populations in the Iblei Mountains.

9. Brown Trout of Sardinia and Corsica

Brown trout from Corsica and Sardinia, along with several other Mediterranean trout, are often referred to as S. macrostigma [103], which is a species native to Algeria. The name S. macrostigma refers to parr signs preserved in adults (Duméril, 1858). Recently, Berrebi et al. [41,104] analyzed morphological and genetic characteristics of populations in Sardinia and Corsica. According to the study, S. trutta populations living in Sardinia show larger head sizes than those of the AT lineage. Conversely, other characters, such as body spotting, black and white edges of fins, body depth and number of epurals in the caudal skeleton, are polymorphic. The morphological data available for populations living in Corsica are limited to the number of pyloric caves and color descriptions among populations [105]. Autochthonous S. trutta populations in Sardinia (without introgression) are characterized by the AD haplotype and the LDH-C1 100/100 allele [101], while hybrid populations in Sardinia and Corsica are characterized by the AD, MA and ME haplotypes [41,93].

10. Conclusions

Genetic studies carried out over the last decade have shown considerable genetic heterogeneity within the S. trutta complex in Europe and Italy. S. trutta evolutionary lineages living in Italy, particularly in the Tyrrenian area and on the main islands (Sicily, Sardinia and Corsica), have complex genetic structures that are still not fully known in some areas. Results of numerous research have provided clear evidence of the introgression of AT lineages into other populations, especially in central Italy. The combined use of traditional genotyping techniques (based on mtDNA and nuclear genetic analyses) and latest-generation sequencing techniques (e.g., the SNP microarray and ddRAD methods) facilitates more accurate identification of native evolutionary lineages and the measurement of their levels of hybridization with alien lineages. In particular, RAD-seq techniques can be used to identify different lineages and the level of inbreeding within populations. In-depth knowledge of the genetic characteristics of S. trutta complex populations present in Italy is important for defining the objectives and priorities of conservation activities and for improving native fish resource restocking and management methods.