The name of the trout: considerations on the taxonomic status of the Salmo trutta L., 1758 complex (Osteichthyes: Salmonidae) in Italy

Abstract The systematic status of the Italian trout in the Salmo trutta L., 1758 complex (including marble, Mediterranean and lacustrine trout), has long been - and is still today – subject of controversies among ichthyologists. The specific name and the taxonomic rank changed several times in the last years, and the natural occurrence of this salmonid fish in some Italian areas was debated due to spread of alien strains. The main difficulty with the taxonomy of the Italian trout stems from the impossibility of disentangling it “on paper” or, even worse, trying to face this systematic issue considering only a very limited (local/national scale) part of the brown trout range. The taxonomy of the Italian trout population is inextricably linked to the necessity of clarifying first phylogeny and phylogeography in an overall Mediterranean context. The opportunity of a non “self-referential” taxonomy is even more fundamental for a vulnerable salmonid like the Italian brown trout, for which there is a very conflicting management problem related to sport fishing and, at the same time, the urgent need for effective conservation measures. It is however necessary to emphasize that conservation is independent from taxonomy but must start from the level of the local population. In fact, management units need stability and they cannot, therefore, coincide with entities – the Linnean species – requiring continuous taxonomic revisions. Modern molecular methods are the best tools for defining these units of management and conservation in an evolutionary perspective.

An historical overview on taxonomy of the Italian trout (including Mediterranean, marble and lacustrine trout) The salmonid fish genus Salmo Linnaeus, 1758 harbours in Italy different phenotypes living in different freshwater habitats, like i) mountain streams along south-western Alps, Apennines and main islands (the Mediterranean trout, characterized by medium-small size and more or less numerous dark and/or reddish spots on flanks superimposed on vertical Parr-marks), ii) upper and lower reaches of the Po river basin (the marble trout, bigger in size and with irregular brown lines forming a marbled pattern) and iii) the lacustrine trout (the "carpione") from Garda (up to 500 mm in standard length with uniform, silver coloration and few dark spots) and Fibreno (small size, with large reddish or dark brown ocellated spots on flanks, superimposed on Parr-marks) lakes ( Figure 1). These forms are collectively attributed to the trout of the Salmo trutta Linnaeus, 1758 complex (see Caputo et al. 2009;Splendiani et al. 2019), to which a systematic review was recently devoted, with reference to Mediterranean (Europe and North Africa) river basins (Lobón-Cerviá et al. 2018).
A paragraph in the work of Lobón-Cerviá et al. (2018) focused on the systematic status of the Italian brown trout, long sinceand still todaysubject of controversies among ichthyologists. In fact, the specific name and the taxonomic rank attributed to populations living along the Alpine and Apennine chains and in the major islands (Corsica, Sardinia and Sicily) changed several times in the last years (see Kottelat 1997;Kottelat & Freyhof 2007;Bianco & Delmastro 2011;Zerunian 2013;Bianco 2014) and the natural occurrence of this salmonid fish in some Italian areas was also questioned (see Gandolfi et al. 1991). The controversial autochthony of brown trout represents the clear consequence of a long-lasting history of introduction of domestic-strains of Atlantic provenance belonging to the nominal species Salmo trutta L., 1758, starting between the second half of the nineteenth century and the early twentieth century (e.g., Bettoni 1895; Figure 2) and still persisting despite European and Italian laws contrary to the spread of alien stocks (see UZI 2018). Indeed, already in the early 50s, Sommani (1951) did not include the populations of central Italy in his zoogeographic revision, because, in his opinion, they were too compromised by the spread of Atlantic hatchery trout. In this work, the author consider the Mediterranean trout of Italy belonging to the nominal form, Salmo trutta, with the exception of Sicilian, Corsica, Sardinian and south Latium populations, attributed to Salmo macrostigma (Dúmeril 1858), due to the typical phenotype characterized by large and sparse dark spots on the body sides (see Duchi 2018) (Figure 1(c)). The occurrence in Sardinia of a trout species originally described by Dúmeril for Algeria was reported at first by Boulenger (1901), and the "macrostigma trout" was later recognized as typical of the circum-Mediterranean countries (e.g., Tortonese 1954). The Adriatic trout described by Pomini (1941) as Salmo ghigii on specimens from a tributary (Sagittario River) belonging to the Aterno-Pescara river basin (centraleastern Italy) was successively considered by Sommani a synonym of Salmo trutta. In this case, Sommani argued that the morphological variability observed at intra-basin level was often greater than that subsisting between different basins, so it was not possible to distinguish clearly two taxa of trout comparing the Tyrrhenian and Adriatic-Jonian slopes of the Italian Peninsula.
The zoogeographic picture described by Sommani (1951) was substantially taken up by Tortonese (1970) which, however, did not recognize as valid the status of species for S. macrostigma because in Sardinia, as already pointed out by Pomini (1940), there was a certain chromatic variability that would not allow to clearly distinguish the macrostigma trout from the typical Salmo trutta "not even at the level of subspecies" (Figures 1(d) and 3). In more recent years, Gandolfi and Zerunian (1987), Gandolfi et al. (1991) and Zerunian (2004) attribute the status of semi-species to macrostigma and consider it potentially distributed along the Tyrrhenian side of the Italian peninsula and in its major islands. In addition, Zerunian and Gandolfi (1990) raise a lacustrine population living in a small karstic lake in southern Latium (Fibreno Lake) to the rank of species, Salmo fibreni, distinct from the macrostigma trout. The taxonomic scenario for the Apennine and insular Italian trout was profoundly revised by Kottelat (1997) and Kottelat and Freyhof (2007), according to which Salmo macrostigma should be restricted to Maghreb populations, so the correct name for insular and Apennine Tyrrhenian Mediterranean trout would be Salmo cettii Rafinesque Schmaltz 1810, originally used by the Franco-German zoologist to describe trout living in rivers of eastern Sicily ("fiumi del val demone e val di Noto", Rafinesque Schmaltz 1810, p. 55). On the other hand, the authors recognize specific rank also for the Mediterranean trout populations living along the Adriatic side of Apennine (up to the Vomano River, in Abruzzi region) and in the upper reaches of the Alpine streams belonging to the Po plain basin, for which they propose the name Salmo cenerinus Chiereghini, 1847. However, Bianco and Delmastro (2011) and Bianco (2014) considered cenerinus as a junior synonym for Salmo marmoratus Cuvier, 1829, so suggested to use the name Salmo farioides Karaman, 1938 originally established for the Krka River and several others Dalmatian rivers. To corroborate the same specific status of the trout of the two sides of the Adriatic Sea, Bianco (2014) argued that "these basins include numerous primary or primary-like representatives of the Padany-Venetian district … Also, for palaeogeographic history, dispersal events occurred during the last Würmian glaciation, when the extended Po basin reached the meso-Adriatic ditch in the central Adriatic Sea, joining rivers of the two Adriatic slopes".
As for northern Italy, the historical picture is completed considering two other taxa of the genus Salmo, namely the marble trout, S. marmoratus (Figure 1(b)) and the lacustrine form, S. carpio L., 1758 (Figure 1(a)). The first one was long time synonymized with Salmo trutta (e.g., Festa 1892), but it was considered as a valid taxon since the 1930s of the twentieth century and the only native trout present in the medium and lower river courses in the Po plan and upper Adriatic basins (Gridelli 1935;Pomini 1937;Sommani 1960). According to Tortonese (1970) "marmoratus" would represent instead a subspecies within Salmo trutta, allowing for the possibility of fertile crossings with the nominal form. Also S. carpio, endemic of Lake Garda, was considered a subspecies by Tortonese (1970) "for the close affinities with Salmo trutta with which is interbreeding".
The most recent overall review of Lobón-Cerviá et al. (2018), only based on distributional and bibliographic data, attribute to Salmo cettii all the Mediterranea trout populations living in peninsular and insular Italy, in addition to recognizing a full species status to the marble trout (S. marmoratus) and the two lacustrine endemics (S. carpio and S. fibreni).

Italian trout biodiversity enters the molecular systematics era
The taxonomy of Italian populations of the brown trout was significantly influenced by the "molecular revolution", started in the early 90s, with the pioneering works of Patarnello et al. (1994) and Giuffra et al. (1994Giuffra et al. ( , 1996. The first one was based on direct sequencing of segments of the cytochrome b and 16S rRNA mitochondrial genes on Italian samples of marmoratus, macrostigma, carpio, fibreni and trutta samples from Italy and north Europe (Ireland). This work failed at evidencing significant differences among the compared taxa, with a possible exception, due to subtle differences, between trutta and marmoratus. The problem with this study was probably related with both the low sample size and the molecular markers used, with poor resolution power among trout genomes. In fact, a more exhaustive picture was obtained in the almost contemporary work of Giuffra et al. (1994) due to a combined sequence/RFLP analysis on coding (ATPase subunit VI and Cytochrome b) and non-coding (control region or D-loop) regions of mitochondrial DNA. This approach was already used in the seminal work of Bernatchez et al. (1992; see also Bernatchez 2001) to distinguish five European phylogenetic lineages, namely Atlantic (AT), Adriatic (AD), Danubian (DA), Marmoratus (MA) and Mediterranean (ME) ( Figure 4). Three of them are widespread in native Italian trout populations, namely MA, mostly linked to lowland marble trout, AD and ME found in mountain Mediterranean trout (Table I). In fact, Giuffra et al. (1994) describe that all marmoratus populations were monophyletic in origin and represented a distinct evolutionary lineage among the north Italian trout populations examined and demonstrated the wide spread of exotic AT strain in Po plan drainages. However, the origin of the phenotypically distinct Salmo carpio was not associated with any phylogenetically distinct branching but included four mtDNA lineages (AD, ME, MA and AT, the latter, however, considered absent by Gratton et al. 2014;Meraner & Gandolfi 2018), thus suggesting a recent post-glacial origin by mixing of allopatrically evolved genetic strains. In a subsequent work, Giuffra et al. (1996), based on allozyme loci and mtDNA approach, deepened the question of the endemic Po plain Salmo. They confirmed the possible hybrid origin of S. carpio, resulted in rather recent post-glacial times by introgressive hybridization between Mediterranean and marble trout. Concerning these latter forms, the authors suggested a process of incipient parapatric speciation, driven by pre-zygotic barriers and adaptation to different habitats (lower and upper parts of the rivers, respectively). According to Giuffra et al. (1996), these delicate evolutionary processes, still ongoing, would be at risk due to massive stocking with fish-farm trout originating from the Atlantic drainages that have already introgressed many wild trout populations and could act as 'genetic bridges', favouring gene flow between the two species. More recently, contributions of Splendiani et al. (2006), Splendiani et al. (2007) underlined the role of possible ancient natural contacts in shaping the current genetic makeup of brown trout in central Italy, with unique marmoratus-like genotypes harboured in Mediterranean trout-like phenotypes along Adriatic Apennine streams. This would be a consequence of expansion to the south of the Po plain in the Glacial maxima, favouring paleo-introgression phenomena between marble trout and Apennine Mediterranean trout. The same would apply for the occurrence of MA haplotypes in native trout from Greece (Apostolidis et al. 1997), Dalmatia (Bernatchez 2001) and Albania (Snoj et al. 2009) that would represent the southernmost offshoots of marble trout in consequence of Pleistocene glaciations. What are more difficult to explain are the MA haplotypes found in Corsica (Lerceteau-Köhler et al. 2013), but in this case it is possible to invoke the role of the Apennines as a semipermeable barrier permitting crossing of MA and other Adriatic lineages on the Tyrrhenian side of Italy, including Corsica (Bianco 1990(Bianco , 1994. However, a different interpretation by Meraner and Gandolfi (2018) proposed that MA lineage was already established before the divergence among other brown trout major mitochondrial lineages (AT, AD and ME) occurred. This lineage would have evolved in an ancestral Mediterranean brown trout and successively became fixed, probably as a consequence of genetic drift, in northern Adriatic marble trout populations.
The paper of Gratton et al. (2014) was the first attempt to face taxonomy and evolution of the genus Salmo in Italy with a wider multilocus Bayesian approach including mtDNA control region, 11 microsatellite loci (non-coding nuclear DNA) and 8 nuclear genomic fragments (mostly intronic sequences). This work analyzed over 500 trout individuals belonging to   Berrebi et al. 2019;3, Fabiani et al. 2017;4, Fruciano et al. 2014;5, Giuffra et al. 1994;6, 1996;7, Gratton et al. 2014;8, Lerceteau-Köhler et al. 2013;9, Meraner & Gandolfi 2018;10, Sabatini et al. 2011;11, Schöffmann et al. 2007;12, Snoj et al. 2011;13, Splendiani et al. 2006;14, 2007;15, 2017;16, Zaccara et al. 2015 Tortonese (1970) the different Italian Salmo taxa, namely marmoratus, carpio, cenerinus, cettii and fibreni. However, also in this case, only two main evolutionary lineages seem to emerge from the study, namely Salmo marmoratus and a "peninsular" lineage. The Lake Garda endemic S. carpio would be mostly derived from an ancestral population genetically close to the current "peninsular" lineage with a very limited contribution, if any, from a marble trout ancestor. Alternatively, the presence of both AD and MA mtDNA haplotypes within genome of S. carpio could be the result of an ancestral polymorphism within 'peninsular' brown trout, thus pointing to the non-private character of MA mtDNA haplotypes for the marble trout taxon (see also Meraner & Gandolfi 2018). The presumed species occupying the two sides of the Apennines, namely S. cettii and S. fibreni in the Tyrrhenian and S. cenerinus/farioides in the Adriatic slope, would be indeed no more than two evolutionary lines separated very recently, after the LGM (last glacial maximum, ca 18,000 years ago), probably representing a single species. Unfortunately, the paper of Gratton et al. (2014) used for comparative purpose only domestic trout belonging to Atlantic drainages, without considering Balkan, Rhone basin, Iberian or Maghreb samples, where probably populations related to the Italian ones are present. Trout from the Italian major islands (Corsica, Sardinia and Sicily) are also not analyzed, and these limitations make the work of Gratton et al. (2014) rather weak for wider taxonomic purpose. However, other useful contributions have shed light on the trout biodiversity of the principal Italian islands. The native trout inhabiting Corsica, Sardinia and Sicily were classified at first as "Salmo macrostigma", for the presence of specimens with sparse and large black dots on the body sides, although Tortonese (1970) observed that "Sardinian and Corsican trout are more variable in ornamentation, also showing red and brownish spots" (Figures 1(d) and 3). Mitochondrial DNA molecular studies of different authors indicated that these insular populations harbour indeed very different genetic lineages. Corsican and Sardinian specimens are characterized by AD, MA and ME haplotypes (Sabatini et al. 2011(Sabatini et al. , 2018Lerceteau-Köhler et al. 2013;Zaccara et al. 2015;Berrebi et al. 2019), while Sicilian ones are the only Italian trout having haplotypes belonging to the southern AT or African sub-clade (Schöffmann et al. 2007;Snoj et al. 2011;Fruciano et al. 2014;Tougard et al. 2018) (Figure 4). Thus, on the base of these consistent molecular data, the name Salmo cettii is useless for designating Tyrrhenian and insular (e.g., Kottelat & Freyhof 2007) or even all Italian Mediterranean trout (see Rondinini et al. 2013;Lobón-Cerviá et al. 2018). In fact, the species "cettii" was described on specimens from rivers of eastern Sicily (see above), phylogenetically linked to Maghreb trout populations (see also Duchi 2018). Therefore, Salmo cettii should be considered as a senior synonym of Salmo macrostigma, and eventually be used for the trout belonging to the southern Atlantic clade (e.g., Cortey et al. 2009;Snoj et al. 2011), assuming North-African and Sicilian trout populations are worthy of taxonomic distinction (but see Tougard et al. 2018).
Another significant study examined the possibility to use ancient DNA (aDNA) to disentangle the phylogeography of Italian trout (Splendiani et al. 2017). In this paper, a partial sequence of D-loop was obtained from a trout collection deposited at the Zoological Museum "La Specola" of the Florence University. The trout specimens were collected by the former Director of the Museum, Professor Adolfo Targioni Tozzetti  that in the 1880s was commissioned by the Italian Government to evaluate the distribution and abundance of the Italian trout for possible exploitation just after the Italian unification (Bettoni 1895). The analysis of 17 specimens coming from different Italian localities (peninsular and the two major islands, namely Corsica and Sardinia, including Salmo marmoratus, S. cettii and S. carpio, Figure 1) indicated, very interestingly, that probably in the second half of the nineteenth century allochthonous trout belonging to the nominal form Salmo trutta L., 1758 (AT lineage from northern Atlantic drainages, sensu Bernatchez 2001) was not yet widespread within the Italian rivers. This observation is in line with the history of restocking, which massively started between the end of the nineteenth century and the beginning of the twentieth century (see Bettoni 1895; Tortonese 1970; Figure 2). Another interesting finding is that, in addition to marble trout (MA lineage), in North-Western Italy genetic lineages referred to as native Mediterranean trout (namely AD and ME) are also detected in historical samples. The autochthony of these evolutionary lineages was thus confirmed in this area of Italy, contrary to what was claimed in a publication of the Italian association of freshwater ichthyologists (AIIAD 2013; see also Forneris et al. 2011). In fact, giving credit to local rumours, this paper considers that the Mediterranean trout was introduced in Piedmont by the Queen Elena of Savoy (1873-1952)passionate angler (see Siccardi 1996) in the first half of the twentieth century. On the other hand, a clear description of native trout phenotypes was provided by Festa (1892) and Casalis (1833Casalis ( , 1852 for specimens collected in the central-western Alps well before the "restocking era". In populations from Corsica, Sardinia and Latium, formerly attributed to Salmo macrostigma (Figure 1(c)), aDNA revealed the presence of AD and ME lineages, also observed in other museum specimens from Tyrrhenian Apennine side, and currently classified as Salmo cettii (see above). Similar findings were recently obtained by Fabiani et al. (2017) for the "macrostigma" Latium population, harbouring indeed AD and ME haplotypes. As for S. carpio, a single specimen belonged to AD lineage, thus confirming that this lacustrine trout does not represent a peculiar evolutionary line, but probably no more than an ecotype of the lake Garda (Splendiani et al. 2017).
Lastly, the huge spread of AT mitochondrial lineages belonging to the nominal form Salmo trutta, due to restocking, was repeatedly emphasized as the main threat to the survival of native trout biodiversity and the consequent impossibility for taxonomic clarification (e.g., Bianco 1991Bianco , 2006Nonnis Marzano et al. 2003;Caputo et al. 2004Caputo et al. , 2009Splendiani et al. 2019). In Splendiani et al. (2013), Splendiani et al. (2016b) focused on the role of biotic and abiotic factors promoting the spreading of alien domestic trout strain in Italy, with particular reference to the geologic substrate of river basins and the ice Alpine cap during glacial maxima. The role of permeable rock (determining suitable habitats) is of strong importance to determine the resilience of native trout to introgression of alien strains, while the expansion of glacial cap on the Alps explains well the reason for the presence of only alien brown trout in the central-east Alps, in consequence of introduction in recent times of AT strains after extirpation of native trout, due to the destructive action of the Alpine ice sheet.

Concluding remarks: taxonomy and conservation implication
The overall picture that emerges from the works published so far on the taxonomy and evolution of Italian trout is unfortunately still controversial and not conclusive. For instance, the last paper in order of time which purported to clarify the taxonomy of the Italian trout (Lobón-Cerviá et al. 2018), actually did not even notice the impossibility of using the specific name Salmo cetti on a national scale, being it a senior synonym for S. macrostigma (see above). In our opinion, the main difficulty with taxonomy of Italian trout is that it is impossible to disentangle it "on paper" or facing the question only at local/national scale. The taxonomy of Italian trout population is inextricably linked to the necessity of clarifying first phylogeny and phylogeography in an overall context, with the help of powerful molecular tools available today (e.g., Next Generation Sequencing). The necessity of a non "self-referential" taxonomy is all the more fundamental for a fish like the brown trout, for which there is a tremendous conflicting interest between biological conservation and sport fishing management (e.g., UZI 2018). In fact, due to European and local law restrictions in the use of alien stocks for supportive breeding (see Council of the European Communities 1993; UZI 2018), there is a great pressure by angling associations to have domestic "autochthonous" trout to bypass these limitations. In this context, the taxonomic confusion about native Italian trout represents an ad hoc opportunity for the trade in presumed native stocks produced by trout farmers and/or fishing associations. In the next years, this latter practice will probably represent a further threat for the conservation of the native genetic diversity of brown trout populations from the Italian Peninsula. First, based on the analysis of recent stocking records (years 2008-2018), domestic Mediterranean stocks of brown trout have been used irrespective of their geographic origin. For example, the water courses of the Provinces of Como, Sondrio, Lecco and Bergamo (central Alps) are yearly stocked with the same Mediterranean trout coming from a presumed native Apennine strain (Splendiani et al. in preparation). The use of wild animals of different Italian provenances for breeding in captivity can favour translocation phenomena, as in the case illustrated by Splendiani et al. (2019), with the observation of a Tyrrhenian haplotype in a hatchery producing "native Mediterranean" trout for restocking purpose on the Adriatic slope of the Apennines. Furthermore, this presumed native trout stock was actually a mix between Atlantic and native trout. The production and spreading in nature of this kind of trout, fraudulently being passed off as native, will lead to the future impossibility to delineate the phylogeographic history of the original populations, and therefore will represent an obstacle to describing a reliable taxonomic picture of the Italian native trout. Added to this is the still huge and illegal use of Atlantic domestic trout that are poured into the Italian rivers in tons of specimens every year for stocking (Splendiani et al. in preparation). In this context, the paradoxical disinterest of the main Italian environmental associations is a real pity, as they give priority to conservation of the "most charismatic" homeothermic vertebrates (see Fenoglio et al. 2018;Tiberti 2018) regardless of the freshwater fish so severely threatened with extinction (e.g., Zerunian 2002;Rondinini et al. 2013). It is equally paradoxical that for the Italian Ministry of the Environment major angling associations are recognized as environmental associations: in a sense, entrusting the trout to the fishermen is like "having the fox guard the henhouse"! To conclude, it is increasingly clear that conservation strategies cannot be merely based on the Linnean species, as there is the risk of useless taxonomic inflation (e.g., Isaac et al. 2004). In fact, since in the case of the trout the identification of the management units can only start at the river basin or sub-basin level, giving a species name to each of these units would make the species list disproportionate (e.g., https:// www.fishbase.de/Nomenclature/). Second, management units need stability and they cannot, therefore, coincide with entitiesthe Linnean speciesrequiring continuous taxonomic revisions (Mace 2004). It seems, therefore, necessary to emphasize once again that conservation should be taxonomy independent. On the contrary, the conservation and even the management of the native trout biodiversity must start from the level of the local population, considering genetic structure even at micro-geographical scale (see Laykre 1999;Sanz 2017;Berrebi et al. 2019). The project "Life + TROTA" ("Trout populations RecOvery in central iTAly", LIFE 12 NAT/IT/ 000940), financed by the European Commission, adopted the above approach for a proper and rational conservation strategy of the Mediterranean trout in Italy (see . The Bayesian analyses performed on multi-locus genotypes (i.e., microsatellites) indicated that in the study area, a clear genetic discontinuity of native genetic diversity was still recognizable at both inter and intra-river level . Therefore, based on the native genetic make-up observed, trout were selected as source of fishes to keep in captivity to create local management units of native origin to be used for supportive breeding and/or reintroduction activities in nature. Modern molecular methods represent thus the indispensable rationale for defining these units of management and conservation in an evolutionary perspective.