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Genetic identification of native populations
of Mediterranean brown trout Salmo trutta L.
complex (Osteichthyes: Salmonidae) in central
Italy
A. R. Rossi, G. Petrosino, V. Milana, M. Martinoli, A. Rakaj & L. Tancioni
To cite this article: A. R. Rossi, G. Petrosino, V. Milana, M. Martinoli, A. Rakaj & L. Tancioni
(2019) Genetic identification of native populations of Mediterranean brown trout Salmo�trutta L.
complex (Osteichthyes: Salmonidae) in central Italy, The European Zoological Journal, 86:1,
424-431, DOI: 10.1080/24750263.2019.1686077
To link to this article: https://doi.org/10.1080/24750263.2019.1686077
© 2019 The Author(s). Published by Informa
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Genetic identication of native populations of Mediterranean brown
trout Salmo trutta L. complex (Osteichthyes: Salmonidae) in central
Italy
A. R. ROSSI
1
, G. PETROSINO
1
, V. MILANA
1
, M. MARTINOLI
2
, A. RAKAJ
2
,&
L. TANCIONI
2
*
1
Department of Biology and Biotechnology C. Darwin, University of Rome La Sapienza, Rome, Italy, and
2
Experimental
Ecology and Aquaculture Laboratory, Department of Biology, University of Rome Tor Vergata, Rome, Italy
(Received 30 July 2019; accepted 23 October 2019)
Abstract
Italian native populations of Mediterranean brown trout belong to the Salmo trutta complex. This species complex includes
many mitochondrial lineages and phenotypes that have caused taxonomic controversies over time. The spatial distribution and
the genetic diversity of these shes are threatened by habitat destruction, global warming and, mainly, by the introduction of
domestic trout of Atlantic origin. Indeed allochthonous trouts were massively restocked in Italian rivers for a century and they
admixed with native populations. In order to identify residual native populations of Mediterranean brown trout, a genetic
analysis of specimens collected within Latium region, on the Tyrrhenian slope of central Italy, was undertaken. To this
purpose, 210 trout specimens were collected from six different rivers and analyzed for the identication of their nuclear (LDH-
C1* RFLP) and mitochondrial (Control Region sequences) genotypes. Genetic characterization with these molecular markers
allowed a quantitative estimate of allochthonous genotypes, which are present in all brown trout populations of the six sites,
even if not equally distributed across the sampling area. At least three populations, inhabiting diverse lotic ecosystems
(mountain, hilly and coastal streams respectively), are characterized by a high percentage of native nuclear allele *100 at
locus LDH-C1* and typical Mediterranean haplotypes (of AD and ME lineages), which can be considered as different
management units (MUs). This nding highlighted the aquatic ecosystems of the Latium Region as an important hotspot
of salmonid biodiversity within the Italian peninsula, with important implications from a conservation perspective.
Keywords: Biodiversity conservation, salmonids, hybridization, restocking, threatened sh
Introduction
Salmonids include freshwater and anadromous shes
widespread in the Northern hemisphere (Nelson et al.
2016). In the Mediterranean area, the Italian peninsula
represents a hotspot of salmonid biodiversity, being
inhabited by a huge number of native taxa. Among
these, a number of shes of the genus Salmo, commonly
known as trout, are included in the national and inter-
national red list (Bianco et al. 2013)andpartlyin
annexe II of the European Union Habitat Directive
(Nonnis Marzano et al. 2016). Their present distribu-
tion is the result of the combination of historical natural
colonization (Bianco 1990), and recent anthropogenic
activities.Indeedthehistoryoftroutsofthe
Mediterranean area was geographically shaped by the
different impact of paleoclimatic events (i.e. glaciations)
on the isolation of ancestral populations into refuges
and subsequent secondary contact (Bernatchez 2001;
Cortey et al. 2004,2009). Since the last century, the
original distribution of these shes has been altered by
the massive restocking with domestic brown trout
mainly of Atlantic origin, that was translocated for
recreational shery and mixed with natural populations
(Splendiani et al. 2016b; Meraner & Gandol2018).
The coupling of these domestic brown trout with the
native populations has caused introgressive hybridiza-
tion and reduced the genetic integrity of native trout so
that in some geographic areas original populations have
been almost completely admixed or replaced by alien
trout (Splendiani et al. 2016b,2019). This context,
*Correspondence: L. Tancioni, Experimental Ecology and Aquaculture Laboratory, Department of Biology, University of Rome Tor Vergata, Via della
Ricerca Scientica 1, Rome 00133, Italy. Tel: +39 0672595975. Fax: +39 0672595965. Email: tancioni@uniroma2.it
The European Zoological Journal, 2019, 424431
Vol. 86, No. 1, https://doi.org/10.1080/24750263.2019.1686077
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of
the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
associated with the phenotypic plasticity and ecological
adaptation, has contributed to a confusing nomencla-
ture picture and to taxonomic ination(see Tougard
et al. 2018) of Italian native trout, with many morphae
described as species (or subspecies) whose validity is
questionable. To date, it is still very difcult to delineate
boundaries and unequivocal correspondences between
morphological species and evolutionary lineages identi-
ed by molecular markers; in order to overcome this
difculty the use of Evolutionary Signicative Units
(ESUs) has been suggested (Zanetti et al. 2013).
However the problem is not completely solved, as
Habitat Directive requires species names as labels of
protection actions.
The main point of congruence about Italian trouts is
that they are part of the Salmo trutta complex dened on
a molecular basis (Sanz 2018), and include: trout that
inhabit the Apennines and the western Alps (AD line-
age) that likely corresponds to S. ghigii Pomini, 1940;
trout present in south eastern Sicily with AT native
haplotypes, that should keep the name S. cettii
Ranesque, 1810, and whose distribution elsewhere in
Italy still needs to be dened; trout from the Tyrrhenian
area of the Italian Peninsula, Sardinia and north Sicily,
presently without a valid name, and characterized
by AD and ME haplotypes; S. breni (Zerunian &
Gandol1990)theendemictroutofthesmallkarstic
lake of Posta Fibreno (a split of the AD lineage);
S. marmoratus Cuvier, 1829 (MA lineage) present in
Italian and Slovenian rivers draining into the northern
Adriatic; S. carpio Linnaeus, 1758 the endemic trout of
the Garda Lake (AD and MA haplotypes) (Gratton
et al. 2014). In addition to these, allochthonous brown
trout corresponding to Salmo trutta derived from north
European farmed stocks (mainly AT lineage), are pre-
sent, and frequently hybridized with local native popu-
lations (Bernatchez 2001; Nonnis Marzano et al. 2003;
Splendiani et al. 2006,2016b,2019; Gratton et al.
2014; Fabiani et al. 2018).
In the last decade a 30% decline of native trout
populations, and a high rate of hybridization/intro-
gression with domestic trout, have been detected in
central Italy (Caputo Barucchi et al. 2015). The only
populations partially genetically preserved are those
isolated by natural or articial barriers that prevent or
reduce alien sh upstream movements; by contrast
the presence of protected areas seems to be irrelevant
in genetic conservation (Splendiani et al. 2019).
This work aims to identify and assess the genetic
composition of residual native populations of
Mediterranean brown trout from different sampling
sites within Latium region. Data could provide use-
ful information for conservation strategies, i.e. for
the identication of management actions on those
populations with higher genetic integrity, and for use
in a parallel habitat modelling analysis (Martinoli
et al. 2019). To this purpose, two molecular markers
that proved to be diagnostic of lineages (Cortey &
García-Marín 2002) and geographic origin
(McMeel et al. 2001) of brown trout were applied.
The mitochondrial marker (Control Region, CR)
was used for lineage identication; the nuclear mar-
ker (Lactate dehydrogenase C1, LDH-C1*), to dis-
criminate specimens of European hatchery origin
from those of Mediterranean native origin, and to
identify their hybrids.
Material and methods
A total of 210 specimens were collected from 6
different rivers/locations (Table I) using electrosh-
ing procedures. In order to minimize the risk of
underestimating the local population genetic varia-
bility, associated with the sampling of individuals
belonging to the same family group (Hansen et al.
1997), each sampling area was extended for more
than 20 times the wetted width of the bed of the
streams, taking also into account the eventual pre-
sence of small tributaries.
Specimens were anaesthetized with a 0.035% MS
222 (Tricaine Methanesulfonate) solution. A small
portion of the adipose n was removed and xed in
90% ethanol for genetic analysis. The procedures
used for sh sampling were carried out in agreement
with relevant legislation (CEN EN 14011/2003 -
Water quality - Sampling of sh with electricity),
avoided animal sacrice and permitted the live
release of sampled specimens after data collection.
Fish sampling was authorized by the Direzione
Regionale Agricoltura, Promozione della Filiera
e della Cultura del Cibo, Caccia e Pesca of the
Regione Lazio (Prot. n. 526425).
Total genomic DNA was extracted from n clips
according to Aljanabi and Martinez (1997). For
haplotype identication, a 544 base pairs (bp) frag-
ment of the mitochondrial CR was amplied and
sequenced in all the 210 specimens (Table I).
Amplications were performed using primers and
protocols reported by Cortey and García-Marín
(2002). Amplicons were puried and sequenced by
using an external service (www.microsynth.ch).
Sequences were deposited in the GenBank database
(https://www.ncbi.nlm.nih.gov/genbank/, accession
number MN223698- MN223719) and blasted for
similarity searching.
Sequences were aligned usingthesoftwareClustal
X (Thompson et al. 1997). Seventy-four CR sequences
(Supplementary material, Table SI) representative of
Mediterranean brown trout 425
Table I. Genetic data of the Mediterranean brown trout populations in the six sampling sites reported from North to South.
CR LDH-C1
*
River/
stream Code Longitude Latitude
Altitude
a.s.l. Province N. H AT Hd π90/90 90/100 100/100
Molinaro MOL 13°17ʹ44.484042°37ʹ58.5480892 RI 26 4 2 (24) 0.286±0.112 0.00161±0.00082 13 11 2
Ratto RAT 13°08ʹ40.704042°30ʹ29.6280764 RI 25 5 3 (23) 0.300±0.118 0.00201±0.00097 15 9 1
Simbrivio SIM 13°13ʹ42.024041°55ʹ32.7000798 RM 51 6 1 (2) 0.408±0.083 0.00187±0.00062 6 14 31
Fibreno FIB 13°38ʹ04.200041°41ʹ35.8080290 FR 41 8 1 (2) 0.605±0.080 0.00307±0.00077 0 8 33
Rapido RAP 13°50ʹ15.576041°28ʹ17.760032 FR 13 6 1 (1) 0.641±0.150 0.00401±0.00159 6 5 2
Santa Croce SCR 13°42ʹ57.708041°17ʹ13.416020 LT 54 3 1 (1) 0.073±0.049 0.00075±0.00055 0 16 38
total 210 22 6 (53) 0.815±0.012 0.00713±0.00018 40 63 107
Province indicate management authority (RI = Rieti, RM = Rome, FR = Frosinone, LT = Latina) and N number of specimens. CR indicates data obtained from mitochondrial control
region: total number of haplotypes (H), number of Atlantic haplotypes (AT) and in parenthesis number of individual showing them, haplotype diversity (Hd) and nucleotide diversity (π).
LDH-C1* columns report alleles combination at this nuclear locus
426 A. R. Rossi et al.
lineages AD, ME, MA and AT available in GenBank
were included in the alignment and in subsequent hap-
lotype network reconstruction. DnaSP 5.10 (Librado &
Rozas 2009) was used for the identication of the
number of haplotypes and haplogroups and to calculate
haplotype variability (Hd) and nucleotide variability (π)
(Nei & Tajima 1981). To investigate genealogical rela-
tionships among mitochondrial CR haplotypes,
a parsimony network was constructed using TCS 1.21
(Clement et al. 2000).
For the identication of allochthonous nuclear
genotypes, the LDH-C1* region was amplied and
digested using primers and protocols reported by
McMeel et al. (2001). Restriction fragments were
checked to verify the presence of the allele *90,xed
or very frequent in north Atlantic populations and
European hatchery stocks, or of the allele *100 typi-
cal of native Mediterranean populations of the
Salmo trutta complex.
Results
CR sequences
The analysis of the 210 CR sequences allowed the
identication of 22 haplotypes: six belong to the AT,
two to the ME and 14 to the AD lineage. Nine of
these haplotypes have already been described
(Supplementary material, Table SI): among these
the most common haplotypes of the ME lineage
(Me25) and of the AD lineage (Ad1) (Cortey et al.
2004).
The haplotype network shows that most of the
haplotypes observed in this study belong to the AD
or ME native lineages, and only a minority fall
within the AT lineage (Figure 1). However, the
proportion of native/allochthonous haplotypes is
not homogeneously distributed among the different
sampling locations: the percentage of allochthonous
haplotypes ranges from 1.85% in SCR to 92.3%
in MOL.
LDH-C1* RFLP
RFLP analysis of the LDH-C1* fragments allowed
the identication of native (*100/100), allochtho-
nous (*90/90) and hybrid (*90/100) genotypes. The
number of native genotypes is very close to the sum
of the allochthonous and hybrid ones. Again, there is
not a homogeneous distribution of the genotypes
among sampling locations.
The comparison of nuclear and mitochondrial
data provided a complete pattern, showing all the
Figure 1. Haplotype network based on CR sequences. Lineages are indicated: AD, Adriatic (AD-PFB haplotypes typical of Fibreno); MA,
Marmoratus; ME, Mediterranean; AT, Atlantic. Circle dimension is proportional to the number of individuals showing that haplotype,
except for haplotypes recovered from GeneBank.
Mediterranean brown trout 427
possible combination of pure and allochthonous
genotypes (Figure 2). In some of the sites, the per-
centage of pure genotypes (allele *100 and AD or
ME haplotypes) reaches 78% (FIB) while in others
no pure genotype is present (MOL). Overall, indi-
viduals showing allochthonous genotypes (allele *90
and AT haplotypes) represent 13.3% of the total;
however, this percentage is much higher in some of
the sites, reaching 56% in RAT. In addition to
these, traces of hybridization between domestic indi-
viduals and native ones are present in all the sites.
Allele and haplotype frequencies are reported in
Table II.
Discussion
Results here obtained conrm the deleterious effect
of massive introduction of domestic brown trout in
Italian rivers already observed in other Italian geo-
graphic regions (Nonnis Marzano et al. 2003;
Caputo et al. 2004; Splendiani et al. 2016b,2019)
or Latium (Fabiani et al. 2018), with a few exception
that characterize some insular areas (Zaccara et al.
2015; Berrebi et al. 2019). The most frequent AT
haplotype (At18) corresponds to that originally iden-
tied in samples from Norway, Denmark and Spain
(Cortey & García-Marín 2002) and already reported
in a hatchery stock of Atlantic origin from an
Ichthyogenic centre of central Italy (Gratton et al.
2014). These data demonstrate that in spite of law
prohibition, stocking practices with domestic trout
still continue, spreading among allochthonous geno-
types (Splendiani et al. 2019): AT haplotypes and
allele *90 are present in all the sites examined in this
study, but with a different frequency among them.
Atlantic brown trout in some of the sites have
replaced almost completely native Mediterranean
ones or at least admixed with them. When frequen-
cies of allele*90 and AT haplotype are compared, it is
clear that there is no direct correspondence between
the two sets of data, as conrmed by Spearmans
Figure 2. Map of the sampling sites, abbreviated as in Table I. Circles represent (a) genotypes observed at locus LDH-C1* and (b) CR
haplotype lineages. The size of the circles is proportional to the sample size.
Table II. Frequencies of nuclear alleles and haplotype lineages of
Mediterranean brown trout populations in the six sampling sites.
See Table I for site code.
Site N. LDH-C1*90
LDH-
C1*100 AT AD
AD-
PFB ME
MOL 26 71.15 28.85 92.31 7,69 - -
RAT 25 78.0 22.0 92 8 - -
SIM 51 25.49 74.51 3.92 92.16 1.96 1.96
FIB 41 9.76 90.24 4.88 12.2 82.92 -
RAP 13 65.38 34.62 7.69 84.62 - 7.69
SCR 54 14.81 85.19 1.85 - - 98.15
428 A. R. Rossi et al.
correlation analysis (Rs = +0.7714, P = 0.2). The two
most admixed sites are MOL and RAT, with 0 and 1
pure individual respectively, and with a high percen-
tage of AT haplotypes. These two sites are part of two
different river basins: MOL (892 meters above sea
level, a.s.l.) belongs to the Tronto basin, that drains
into the Adriatic Sea, while RAT (764 m a.s.l) is
a tributary of Velino river that drains into Nera-
Tiber River and then into the Tyrrhenian Sea. In
spite of this, the two sites are geographically close
and are managed by the same province (Rieti), the
Local Authority that controls sh introductions.
Therefore it is likely that both populations were fre-
quently affected by massive restocking activities.
An intermediate situation is observed in RAP
(32 m a.s.l.) that belongs to the Garigliano-Liri
basin: this site shows a very admixed trout popula-
tion with few pure individuals, but the majority
of AD haplotypes.
The best-preserved sites, showing a higher percen-
tage of native individuals, are FIB, SIM, and SCR.
These sites show both a high percentage of allele *100
and typical Mediterranean haplotypes. Unfortunately,
our sequences are shorter than those (1013 bp) that
allowed the denition of reference haplotypes by
Cortey et al. (2004). For this reason our Me25, mainly
characterized by SCR Mediterranean brown trout
population, corresponds to 6 different haplotypes
(Supplementary material, Table SI), including the
one (MEcs1) that is considered basic of this lineages.
This haplotype was distributed in Corsica, northern
and central Italy in late nineteen century, before com-
mon stocking with Atlantic strains (Splendiani et al.
2017) and was already present in prehistoric times in
southern Italy (Splendiani et al. 2016a). The same
problem of sequence length is present for AD lineages;
certainly the most common haplotype of this
lineage AD-cs1, originally spread across Italian penin-
sula and Sardinia (Splendiani et al. 2016a,2017)is
not present in our sampling area. Our results match
previous data. Further, Mediterranean brown trout
population from Fibreno shows typical genetic fea-
tures and forms a distinct gene pool from lacustrine
S. breni, although limited hybrid zones are known.
The very low level of introgression from Atlantic strain
detected in FIB, that is located within a Regional
Protected area, was interpreted as the result of con-
servation measures adopted more than 40 years ago,
i.e. cessation of restocking this basin (Gratton et al.
2013). The most common haplotype present in this
site (Ad-Pfb16) corresponds to that observed in the 48
specimens examined by Gratton et al. (2013).
Other AD haplotypes (Ad36, Ad21 and Ad1) corre-
spond to haplotypes already observed in different sites
(ADcs3, ADcs17, and Adcs11 respectively), by
Cortey et al. (2004), or by other authors (Ad1, see
below). The origin of these haplotypes can be
explained by considering two different hypotheses.
The rst one assumes their presence is the result of
natural dispersion. This is quite likely for Ad1, which
was identied around the Adriatic area (e.g., Cortey
et al. 2004;Sušnik et al. 2007), in Macedonia (Marić
et al. 2017) and in central Italy (Fabiani et al. 2018;
Berrebi et al. 2019). As regard to ADcs3 and ADcs17,
they were originally identied in Spain, so their pre-
sence is more difcult to explain unless considering
successive waves of colonization. The second hypoth-
esis explains their presence as a consequence of past
introduction, congruent with the absence of pure
Atlantic individuals, but with their remnants as
nuclear hybrid genomes. Concerning SIM and SCR
sites, they are geographically close to locations ZLS
and CDA, respectively, analyzed by Fabiani et al.
(2018), the results obtained being similar in both
cases. Indeed SIM is characterized by AD haplotypes,
the most common of which is, again Ad1; in addition
to this, two further new AD haplotypes (Ad2 and
Ad12) are present, plus the most common haplotypes
of lineages AD-PFB, ME and AT. Finally, SCR shows
almost exclusively ME haplotypes: these are absent in
other Latium sites here examined, but were observed
in other geographic areas (Cortey et al. 2004).
Regarding its features, SCR represents a population
with a high percentage of native individuals; these
characteristics are shared with CDA (Fabiani et al.
2018), a site geographically very close (about
0.51 Km) within the same basin. These two sites
are managed by the same province (Latina) and it is
likely that both were preserved from the massive intro-
duction of allochthonous trout, although some traces
of these activities are still present. Indeed besides
a new ME haplotype (Me57), a new AT haplotype
(At56) is also present, possibly being a remnant of past
introductions (according to the presence of allele *90).
Thus it is also possible that the particular environmen-
tal condition of these sites has determined a negative
selection of non native brown trouts: CDA and SCR
are characterized by cold springs (about 14°C) in low-
land and coastal zones (about 20 meters above sea
level), very close (< 10 Km) to the stream outlet into
the Tyrrhenian Sea.
Overall different Management Units, i.e. conser-
vation units identied on the base of population
genetic characters, can be recognized across the
sampling area; they include SIM, FIB and SCR,
the three sites with the highest percentage of native
Mediterranean trout, and with a different abun-
dance of typical AD, AD-PBF and ME haplotypes.
Mediterranean brown trout 429
This differential composition and distribution of
haplotypes may be associated with the different eco-
logical conditions (e.g. altitude and water tempera-
ture) of lotic ecosystems inhabited by these
populations. Such a high diversity outlines the aqua-
tic ecosystems of the Latium Region as a unique
hotspot of salmonid biodiversity within the Italian
peninsula, with important implications from a con-
servation perspective.
Further analyses are necessary to obtain a clear
picture of the genetic distribution of native brown
trout populations in the Latium Region and to gain
basic data for concrete conservation actions. Such
analyses should include newpopulations from
other geographic sites and which can be identied
on the basis of habitat modeling results (Martinoli
et al. 2019).
Acknowledgements
We wish to thank P.T. Colombari of ARSIAL and
G. Moccia of Latium Region for valuable support
during sampling campaigns. We are grateful to an
anonymous reviewer for providing valuable com-
ments. This work was nancially supported by
ARSIAL(to L.T. and A.R. R.).
Disclosure statement
No potential conict of interest was reported by the
authors.
Supplementary material
Supplemental data for this article can be accessed
here.
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