The extraordinary polymorphism of major histocompatibility complex (MHC) genes is considered a paradigm of pathogen‐mediated balancing selection, although empirical evidence is still scarce. Furthermore, the relative contribution of balancing selection to shape MHC population structure and diversity, compared to that of neutral forces, as well as its interaction with other evolutionary processes such as hybridization, remains largely unclear. To investigate these issues, we analyzed adaptive (MHC‐DAB gene) and neutral (11 microsatellite loci) variation in 156 brown trout (Salmo trutta complex) from six wild populations in central Italy exposed to introgression from domestic hatchery lineages (assessed with the LDH gene). MHC diversity and structuring correlated with those at microsatellites, indicating the substantial role of neutral forces. However, individuals carrying locally rare MHC alleles/supertypes were in better body condition (a proxy of individual fitness/parasite load) regardless of the zygosity status and degree of sequence dissimilarity of MHC, hence supporting balancing selection under rare allele advantage, but not heterozygote advantage or divergent allele advantage. The association between specific MHC supertypes and body condition confirmed in part this finding. Across populations, MHC allelic richness increased with increasing admixture between native and domestic lineages, indicating introgression as a source of MHC variation. Furthermore, introgression across populations appeared more pronounced for MHC than microsatellites, possibly because initially rare MHC variants are expected to introgress more readily under rare allele advantage. Providing evidence for the complex interplay among neutral evolutionary forces, balancing selection, and human‐mediated introgression in shaping the pattern of MHC (functional) variation, our findings contribute to a deeper understanding of the evolution of MHC genes in wild populations exposed to anthropogenic disturbance.

Brown trout is considered a complex of incipient species, including several phylogenetic lineages, whose natural distribution in the Mediterranean area has been altered, since the beginning of the 1900s, by massive introductions of domestic strains of Atlantic origin to support fisheries. Introduced trout naturalize in new suitable environments and extensively hybridize with native populations. Here, we characterized putatively neutral and adaptive genetic variability and population structure of Mediterranean brown trout from six river catchments in central peninsular Italy, as revealed by both mitochondrial (Control Region) and nuclear (microsatellites, LDH-C1, major histocompatibility complex) markers. We quantified the admixture of wild populations with hatchery strains and evaluated the effects of domestic trout introductions on shaping population genetics. Our analyses indicated: (1) a composite picture of genetic variability in the area, with the presence of all native Mediterranean trout mitochondrial lineages (“Adriatic”, “Mediterranean”, “marmoratus”), various frequencies of allochthonous genotypes and different rates of introgression among sampling sites; (2) asymmetric mito-nuclear introgression; (3) increasing nuclear marker diversity with increasing levels of admixture across populations; (4) strong population structure coupled with relatively low effective population size. Data allowed the identification of five management units and we propose specific actions to support ongoing and future conservation strategies within the examined area.

Italy hosts a large number of endemic freshwater fish species due to complex geological events which promoted genetic differentiation and allopatric speciation. Among them, the South European roach Sarmarutilus rubilio inhabits various freshwater environments in three different ichthyogeographic districts. We investigated the genetic diversity of S. rubilio using two different mitochondrial markers (COI and CR), aiming to define its relationship with other similar taxa from the Balkan area and, from a phylogeographic perspective, test the effects of past hydrogeological dynamics of Italian river basins on its genetic structure and demographic history. Our analysis highlighted a marked genetic divergence between S. rubilio and all other roach species and, among Italian samples, revealed the existence of three deeply divergent geographic haplogroups, named A, B and C. Haplogroup C likely corresponds to a new putative cryptic species and is located at the northern border of the South European roach range; haplogroup B is restricted to Southern Italy; and haplogroup A is widespread across the entire range and in some sites it is in co-occurrence with C or B. Their origin is probably related to the tectonic uplifting of the Apuan Alps in the north and of the Colli Albani Volcano in the south during the Pleistocene, which promoted isolation and vicariance followed by secondary contacts.

The large number of cryptic species and extensive intraspecific phenotypic plasticity among freshwater fishes pose a challenge to the correct assessment of diversity within these taxa, which is crucial for their conservation. Recently, three different genetic lineages have been identified within the Italian endemic South European roach Sarmarutilus rubilio, a threatened species with a broad ecological niche. Using geometric morphometrics, we tested the existence of morphological differences associated with genetic common ancestry (i.e. between lineages) and/or environmental parameters. We observed streamlined body shapes in sites scarcely altered by human intervention and with fast water flow; on the contrary, we observed deeper body shapes in canals and in one reservoir with slow/still water flow. Our results suggest that morphological diversity in the South European roach is mainly a result of phenotypic plasticity in response to different environmental drivers (i.e. lotic vs. lentic hydrodynamic patterns) rather than different genetic backgrounds.

Understanding the relationship between environmental DNA (eDNA) concentration and taxa abundance is essential for the advancement of quantitative biodiversity monitoring. We experimentally manipulated biomass of three freshwater fish species of monitoring interest—the Italian‐endemic Squalius lucumonis and the exotic‐invasive Pseudorasbora parva and Lepomis gibbosus —under controlled conditions (flow‐through 310 and 1330 L tanks). Following eDNA collection (2 L water filtration) and Real‐Time PCR quantification, Generalized Additive Mixed Models (GAMMs) revealed: (i) monotonic non‐linear relationships of moderate‐to‐high magnitude (0.42 < partial‐ R ² < 0.62), with eDNA concentrations plateauing at intermediate biomasses in smaller‐sized taxa; and (ii) a significant effect of experimental replicates (tanks) in two out of three species. These findings suggest that eDNA‐based biomass quantification should not assume linearity, and emphasize the critical role of replication to account for inherent uncertainty, likely driven by inter‐ and intra‐individual variations in eDNA shedding rates.

The South European roach Sarmarutilus rubilio is a threatened freshwater fish, endemic to the Italian peninsula. Previous investigations revealed the presence of three mitochondrial haplogroups (namely HpA, HpB, and the highly divergent HpC) that originated in allopatry, despite currently coexisting at the margins of the species’ distribution. However, no information on S. rubilio contemporary genetic structure is available. In this study, we tested cross-amplification for 19 Leuciscidae-designed microsatellite loci, optimizing protocols for 12 of them that were used to analyze genetic variation, population structure, and demography in twelve S. rubilio populations representative of the species range. Our results revealed population structuring at the basin scale, which is more pronounced than differentiation revealed by mtDNA, indicating the role of local and relatively recent processes (e.g., isolation, habitat fragmentation, genetic drift, environmental selection) over ancient phylogeographic ones. Overall, we did not find evidence of compromised genetic diversity and strong bottlenecks, although in some sites a low effective population size was detected. In addition, microsatellites did not support the hypothesis of HpC as a cryptic species. These data provide practical indications to support the conservation and management of S. rubilio as required by the European Habitats Directive.