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Ecology and Evolution. 2018;1–14.
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www.ecolevol.org
1 | INTRODUCTION
Until recently, brown trout (Salmo trutta sensu Lato) was considered
to be a widespread western Eurasian fish species with facultative
anadromy. Its natural range stretches from West Siberia to the Atlantic
and throughout West Asia (Maitland & Linsell, 2006). Simultaneously,
the taxonomy of brown trout is controversial. In older treatises
(e.g., Sabaneev, 1875), it is separated into purely freshwater forms
Received:19May2017
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Revised:7December2017
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Accepted:2January2018
DOI:10.1002/ece3.3884
ORIGINAL RESEARCH
Phylogeography and taxonomic status of trout and salmon
from the Ponto- Caspian drainages, with inferences on
European Brown Trout evolution and taxonomy
Levan Ninua | David Tarkhnishvili | Elguja Gvazava
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2018 The Authors. Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.
Institute of Ecology, Ilia State University,
Tbilisi, Georgia
Correspondence
DavidTarkhnishvili,InstituteofEcology,Ilia
State University, Tbilisi, Georgia.
Email: david_tarkhnishvili@iliauni.edu.ge
Funding information
Ministry of Environment and Natural
ResourcesProtectionofGeorgia,Grant/Award
Number:2014/#148;IliaStateUniversity
Abstract
Current taxonomy of western Eurasian trout leaves a number of questions open; it is
not clear to what extent some species are distinct genetically and morphologically. The
purpose of this paper was to explore phylogeography and species boundaries in fresh-
water and anadromous trout from the drainages of the Black and the Caspian Seas
(Ponto- Caspian). We studied morphology and mitochondrial phylogeny, combining
samples from the western Caucasus within the potential range of five nominal species
of trout that are thought to inhabit this region, and using the sequences available from
GenBank.OurresultssuggestthatthegeneticdiversityoftroutinthePonto-Caspian
region is best explained with the fragmentation of catchments. (1) All trout species
from Ponto- Caspian belong to the same mitochondrial clade, separated from the other
trout since the Pleistocene; (2) the southeastern Black Sea area is the most likely place
ofdiversificationofthisclade,whichiscloselyrelatedtothecladesfromAnatolia;(3)
The species from the Black Sea and the Caspian Sea drainages are monophyletic; (4)
except for the basal lineage of the Ponto- Caspian clade, Salmo rizeensis, all the lineages
produce anadromous forms; (5) genetic diversification within the Ponto- Caspian clade
is related to Pleistocene glacial waves; (6) the described morphological differences
between the species are not fully diagnostic, and some earlier described differences
depend on body size; the differences between freshwater and marine forms exceed
those between the different lineages. We suggest a conservative taxonomic approach,
using the names S. rizeensis and Salmo labrax for trout from the Black Sea basin and
Salmo caspius and Salmo ciscaucasicus for the fish from the Caspian basin.
KEYWORDS
anadromous forms, brown trout, Ice Age, morphological evolution, phylogeography, Ponto-
Caspian region
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NINUA et Al.
S. t. lacustris and S. t. fario, and the anadromous S. t. trutta. Anadromy
may not be a heritable character for brown trout (Berg, 1959, 1962);
hence, these names probably do not have any taxonomic meaning.
Fishbase (www.fishbase.org), considering recent changes in taxonomy,
lists more than 20 species that formerly were qualified as geographic
populations or subspecies of S. trutta. Eight of those are poten-
tially present in the Caucasus Ecoregion (as defined in Zazanashvili,
Sanadiradze, Bukhnikashvili, Kandaurov, & Tarkhnishvili, 2004) and,
broader, in the basins of the Black and the Caspian Seas (from here
onwards—Ponto- Caspian Basin: the drainages of the Black and the
Caspian Seas formed a contiguous body of water separated from the
Mediterranean in the geological past—Popov et al., 2004). Salmo cis-
caucasicus(Dorofeeva,1967; syn. S. trutta ciscaucasicus) is native for
the northwestern drainages of Caspian Sea, including the basin of the
Terek (Tergi) River (Kottelat & Freyhof, 2007). Salmo caspius (Kessler,
1877; syn. S. trutta caspius) is native to the southern Caspian and the
rivers of the northern Iran (Turan, Kottelat, & Engin, 2009). Salmo
coruhensis (Turan et al., 2009) is an anadromous form from the south-
eastern and, possibly, eastern Black Sea basin in Turkey and Georgia.
Salmo rizeensis (Turan et al., 2009) is a riverine form from the same
area and other rivers of the southern Black Sea basin. Salmo ischchan
(Kessler, 1877) is a lacustrine form from the Lake Sevan in Armenia
(Berg, 1962). Salmo ezenami (Berg, 1962; syn. S. trutta ezenami) in-
habits Lake Kezenoi- Am in the northeastern Caucasus (Bogutskaya &
Naseka, 2002). “Black Sea Salmon” (Salmo labrax Pallas, 1814) is an
anadromous fish reproducing in the rivers draining into the Black Sea
(Kottelat & Freyhof, 2007). Lastly, European brown trout (Salmo trutta
Linneaus, 1758) is also found in some rivers of the Caspian and the
Black Sea basins (Svetovidov, 1984).
Genetic and/ormorphological and/or geographic distinctiveness
was not demonstrated sufficiently well for some of these species.
Turan et al. (2009) showed nearly fixed morphological and genetic
differences between S. coruhensis and S. rizeensis from the southeast-
ern Black Sea drainage. The individuals of S. labrax from the northern
Black Sea drainage, described in the same paper, differ morphologi-
cally from another anadromous form, nominal S. coruhensis, although
the differences are not fully diagnostic and the individuals were not
studied genetically. It is not clear whether there are fixed differences
between S. labrax and S. trutta from the rivers draining into the Black
Sea from the north and the west, although some differences are men-
tioned (Kottelat & Freyhof, 2007). Fixed differences are suggested be-
tween S. caspius and S. labrax in the number of gill rakers (Turan et al.,
2009); however, the studied individuals of S. caspius were smaller than
those of the latter, and the influence of size on morphology cannot be
excluded a priori. Moreover, the described S. caspius individuals were
not studied genetically.
One could expect that speciation in brown troutshould follow
geological patterns, that is, catchments separated later should have
more closely related trout lineages. This can be used as a null hy-
pothesis challenged by the observed taxonomic diversity, which as-
sumes the presence of more than one species in the same catchment:
S. coruhensis, S. rizeenzis, but also potentially S. labrax in the western
drainage of the Black Sea; S. ischchan and S. caspius in the Kura- Aras
catchment (Lake Sevan is connected with the Aras River through the
River Razdan); S. labrax and S. trutta in central and eastern European
rivers.
Aiming to clear up these taxonomic and evolutionary puzzles and
to infer which nominal species of brown trout are present throughout
the Caucasus Ecoregion, the authors collected samples from six river
drainages in Georgia, including four flowing into the Black Sea and
two into the Caspian, we characterized external morphology of these
fish, and analyzed their mitochondrial haplotypes, along with the hap-
lotypes of brown trout from different sea and river drainages available
from GenBank.
2 | MATERIAL AND METHODS
2.1 | Sampling
The sampling locations (and exact or approximate locations of fish
for sequences that were downloaded from Genbank) are shown
in Figure 1. The total number of samples used in genetic and mor-
phological study is shown in Table 1. Fish were caught by net-
ting, using the net with the diameter of 1.5 cm from August 2014
through November 2014. Permits were obtained from Ministry of
Environmentand NaturalResourcesProtectionof Georgia(permit#
4029,21/07/2014). Pictures ofcaughtfishwere taken formorpho-
metric study. Samples of tissue (fin clips) were stored in 95% ethanol
forsubsequentDNAanalysis.
2.2 | DNA extraction, PCR, and sequencing
DNAwasextractedfromgillormuscletissueoftroutusingtheQiagen
DNeasytissuekitaccordingtothemanufacturer’sinstructions.Amito-
chondrial cytochrome b gene fragment was amplified using the following
primers:nSsaL14437(5′-GCTAATGACGCACTAGTCG-3′)(Warheit&
Bowman,2008)andStrCBR (5′-GGGGGCGAGRACTAGGAAGAT-3′)
FIGURE1 Sampling locations, according to our data (1–4, 9–11)
andexistingpublications(seeTableS1forthelocation/publication
list). The numbers correspond to the location names listed in Table 1.
Catchments: A—Black Sea, B—Caspian Sea, C—AtlanticOcean/Baltic
Sea, D—Mediterranean Sea, E—PersianGulf/IndianOcean
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NINUA et Al.
TABLE1 The number of samples used for the morphometric and genetic analysis original samples and downloaded sequences) from
individual sea and river catchments
Drainage, Basin Species of Salmo as referred Morphology Cyt bControl region
Kura (Mtkvari) (CA) Salmo sp.a8 6 4
Terek (Tergi) (CA) Salmo sp.a8 6 7
Enguri (BS) Salmo sp.a12 5
Rioni (BS) Salmo sp.a73
Kintrishi (BS) Salmo sp.a10 6
Coruh (Chorokhi) (BS) Salmo sp.a66+3b6
Black Sea (Georgia) Salmo sp.a9 8
Danube(BS) S. trutta 10b11b
Plitvica (BS) S. trutta 1b
Aral Sea S. trutta oxianus 2b
Caspian Sea (CA) S. caspius 1b
Arpa (CA) S. caspius 1b
Rivers of southern Caspian (CA) S. caspius 2b11b
Iyidere (BS) S. coruhensis 2b
Iyidere (BS) S. rizeensis 1b
Cayeli (BS) S. rizeensis 2b
Turkey (not specified) S. trutta 11b
Soguksu Stream (ME) S. platycephalus 1b
Stilaro (ME) S. trutta 1b
Göksu River (ME) S. trutta 1b
LakeOhrid(AD) S. ohridanus 1b
Zala(AD) S. marmoratus 1b
Buna(AD) S. obtusirostris 1b
Shkumbini(AD) S. trutta 1b
Seta(AD) S. trutta 1b
Voidomatis(AD) S. trutta 1b
Leksa (AT) S. trutta 1b
Ims (AT) S. salar 1b
Dades(AT) S. trutta 1b
Tensift (AT) S. trutta 1b
OumerRbia(AT) S. trutta 3b
Moulouya (AT) S. trutta 1b
IsliLake(AO) S. trutta 1b
IfniLake(AO) S. akairos 2b
AtlanticOcean(AO) S. salar 1b
NorthSea(AO) S. trutta 1b
BalticSea(AO) S. trutta 2b
BalticSea(AO) S. salar 1b
Euphrates(IO) S. truttac2b
IndianOceanBasin(Introduced) S. trutta 5b
BS,BlackSeadrainage;CA,CaspianSeadrainage;PG,drainageofPersianGulf;ME,MediterraneanSeadrainage;AO,drainageoftheAtlanticOcean.
aSalmo labrax or S. coruhensis or S. rizeensis.
bSequences downloaded from GenBank.
cAccording to Turan et al. (2009).
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NINUA et Al.
(Turan et al., 2009). Mitochondrial control region was amplified with
primersBrtD-F20(5′-GAGATTTTAACTCCCACCCT-3′)andBrtD-R20
(5′-TAGGGTCCATCTTAACAGCT-3′)(Segherloo,Farahmand, Abdoli,
& Bernatchez, 2012). Amplification conditions were the same for both
mitochondrial fragments. Twenty microlitre PCR reactions contained
3μl of template DNA, 1U Promega Tag polymerase, 1X promega
buffer, 2.5mmol/L MgCl2, 0.1mmol/L of each dNTPs, and primer
concentrations 0.1 μmol/L.Thermalprofilewasasfollows:3-minini-
tialdenaturationat94°C,followedby35cyclesat94°Cfor40s,50°C
for 1 min, and 72°C for 2 min, and 10 min at 72°C for final exten-
sion. 5 μl from each PCR was run on 1% agarose gel to visualize the
DNAfragments.TheampliconsweresequencedonanABI3130Gene
Analyser. PCR fragments were sequenced in both directions using the
samePCRprimersandBigDyeTerminator3.1,aspermanufacturer’s
protocol. The unique sequences of the mitochondrial cytochrome b
gene and that of the mitochondrial control region from our samples
weredepositedtoGenBank(accession#MG029536–MG029553for
Cyt- b and MG214765–MG214775 for the control region).
2.3 | Sequence analysis
We analyzed an 842- bp- long region of the mitochondrial cytochrome
b (48 samples and 27 sequences downloaded from GenBank) and a
505-bp-longcontrolregion fragment (26samplesand37 sequences
downloaded from GenBank, Table 1 and Table S1). Unfortunately,
the cytochrome b and control region sequences downloaded from
GenBank were from different publications and described different
individuals and populations; hence, we did not concatenate the stud-
ied fragments while inferring phylogenies and conducted the analyses
separatelyforthesetwofragmentsofmitochondrialDNA.
We used three methods for the analysis of cytochrome b sequences:
(1) Minimum spanning network was constructed only for the trout
from the rivers drawing into the Black and the Caspian Seas (Ponto-
CaspianBasin)using NETWORK 5.0 (Bandelt, Forster,&Rohl,1999).
(2) Maximum likelihood (ML) tree was built and bootstrap support was
estimated using software MEGA 7.0.21 (Kumar, Stecher, & Tamura,
2016). In this analysis, besides the fish from the Ponto- Caspian drain-
age,the fish from the drainage oftheIndianandtheAtlantic Oceans
and Mediterranean (nominal S. trutta, S. marmoratus, S. platycephalus,
and S. akairos) were included along with Balkan species S. ohridanus
and S. obtusirostris (Crête- Lafrenière, Weir, & Bernatchez, 2012), and
Atlantic salmon (S. salar) was added to the analysis as the outgroup. For
thisanalysis,onlyonehaplotype/locationwasincludedifmorethanone
individual from the same location had the same haplotype. The best-
fit model (that with the lowest Bayesian Information Criterion, BIC, as
recommended in Nei & Kumar, 2000) was identified with MEGA 7.0.21
using ML method(3) Bayesian inference (BI) treewas built using the
same taxa included in the ML analysis, for validating the tree topology
using different approach, counting posterior probability of the branches,
andinferringsplittimesusingBEASTv.1.8.4(Drummond,Suchard,Xie,
& Rambaut, 2012). The Bayesian analysis was initiated from random
starting trees, assuming uncorrelated log- normal relaxed clock model
and a coalescent model with constant population size. Posterior distri-
butions of parameters were approximated using Markov chain Monte
Carlo with chain length set at 100,000,000 to provide sufficient sample
size for each parameter (i.e., effective sample size ≫ 100). The same
substitution model was used as in the ML analysis.
We inferred the best- fit model and conducted ML analysis for the
obtained and downloaded sequences of control region (see Table 1 for
the list of the individuals used in this analysis). These sequences did
not show much informative variability among the studied taxa, and we
did not apply BI for this dataset. The software used was MEGA 7.0.21.
We used BEAST v. 1.8.4 (the uncorrelated log- normal relaxed mo-
lecular clock model) to account for variable rates of evolution among
thelineages and to infer the 95% HPDintervals forthe node ages,
based on cytochrome b gene variation. For calibrating the tree, we
used an estimated time of split between the reference cytochrome b
sequences included in our analysis, drawn from multiple publications
(www.timetree.org; Hedges, Marin, Suleski, Paymer, & Kumar, 2015).
The divergence time between S. ohridanus and S. obtusirostris, in one
clade, and S. marmoratus, S. platycephalus, and S. trutta in the other
was set as a time of the initial split.
We also used the alternative scaling by Schenekar, Lerceteau-
Köhler, and Weiss (2014), not considered by www.timetree.org, and
based on the sequence analysis of two distant lineages (“Atlantic”
vs. “Black Sea” lineages) from Austria (Schenekar et al., 2014), which
suggests a much later split between the lineages than other authors
(Alexandrou,Swartz,Matzke,&Oakley,2013;Crête-Lafrenièreetal.,
2012;Macqueen&Johnston,2014).
2.4 | Morphometry
Fish caught in Georgia were photographed from the lateral side. The
images were used for scoring the 28 conventional distances among
FIGURE2 Landmarks (1–14) and the
distances between the landmarks using in
the morphometric analysis of brown trout
and salmon used in the analysis
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NINUA et Al.
the14landmarksshowninFigure2,usingsoftwareImageJ(Rasband,
2016). The distances were log- transformed to control for allometric
effects. Subsequently, the regression of each of the 27 distances on
the first distance (landmarks 1–8, reflecting body length of an individ-
ual) was calculated and the standardized residuals on the regression
line (size-removed body proportions) were used for final calculations as
recommendedbyThorpeandLeamy(1983),insteadoftheoriginal28
distances. Principal component analysis (PCA) was applied for extract-
ing principal components with loadings exceeding unity, and for cal-
culating the individual scores. The individual scores on PCA loadings
1–4 were used for ordination of the individuals and inferring possible
differences among the species, which revealed genetic groups, and
life forms (riverine vs. marine). All calculations were conducted using
SPSS 21.0 for Windows. River trout from Georgia was included in the
analysis along with the marine form (“Black Sea salmon”), and two in-
dividuals of North American rainbow trout (Onychorhynus mykiss), an
introduced/invasivespeciesinGeorgia’sriversandlakes.
Besides this morphometric study, we counted (1) the number of
raysindorsalfins,(2)thenumberofmainraysinpectoralfins,and(3),
thenumber ofgillrakersatthefirst gill arch(Andersson,Johansson,
Sundbom, Ryman, & Laikre, 2016; Kara, Alp, & Gürlek, 2011).
3 | RESULTS
3.1 | Minimum spanning network
Seventeen haplotypes were revealed among the Cyt- b sequences of
brown trout and salmon from the Ponto- Caspian drainages, includ-
ing those obtained from our samples and those downloaded from
Genbank(Figure3). The sequences ofS. rizeensis from the paper of
Turan et al. (2009) were the most distant from the others and had
at least five substitutions that separate them from the next closest
haplotypes. The rest of the sequences is subdivided into those from
the Black Sea (from the catchments of the Rivers Coruh (Chorokhi)
withitstributariesAjaristskali,Kintrishi,Rioni,Enguri,theDanube,the
coast of the Black Sea in Georgia) and those from the Caspian Sea
drainage (from the catchments of the Rivers Tergi (Terek), Mtkvari
(Kura) with its tributaries Ktsia and Aragvi, and coast of the Caspian
Sea in Iran) (Figure 1). There were a maximum of 10 substitutions
between the most distant haplotypes from the Black Sea basin, and
a maximum of four substitutions between the haplotypes from the
Caspian Sea basin. A single individual with the “Caspian” haplogroup
was found in drainage of Coruh, and one also from the Euphrates
(Figure3).
3.2 | Phylogeny based on cytochrome- b sequences
The optimal substitution model for the ingroups included in the
analysis was TN93+G (Kumar et al., 2016). Both ML and Bayesian
analyses suggested the presence of 22 well- supported major mi-
tochondrial clades within the analyzed sequences (Figure 4). The
topology of the tree suggests that: (1) Trout form a monophyletic
clade including “Balkan” species (S. ohridanus + S. obtusirostris). (2)
All trout sequences of cytochrome b gene, including new sequences
from this research and those available from the GenBank, exclud-
ing S. ohridanus and S. obtusirostris (which are sister species) form
a monophyletic clade. (3) All trout sequences of the cytochrome
b gene, excluding S. ohridanus and S. obtusirostris, are subdivided
into two monophyletic clades: one from the rivers flowing into the
Atlantic Ocean (including trout of European origin introduced to
India), and the other comprised of fish from the Mediterranean and
Ponto- Caspian drainages, including fish from the Euphrates River.
(4) Almost all (68 of 69) samples from the rivers and lakes of the
Ponto-Caspian drainages, and from the upper Danube in Austria
FIGURE3 Median- joining network
showing the revealed cytochrome b
haplotypes of trout and salmon from
the Ponto- Caspian basin. Size of pies
proportional to the number of fish with
a specific haplotype among studied
specimens. Numbers at the lines—the
number of substitutions separating the
haplotypes if exceeding one). The colors
showdrainagesofdifferentrivers/seas.
S. rizeensis is shown in color different from
other fish from Coruh (Chorokhi) drainage.
“Mtkvari,” “Terek,” and “Caspian Sea” belong
to the Caspian Sea drainage, “Euphrates”—
to the drainage of Persian Gulf, and all
others—to the Black Sea drainage
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NINUA et Al.
and the rivers flowing into the Caspian Sea in Iran, including Black
Sea salmon and Caspian salmon, belong to a monophyletic mito-
chondrial clade, nested into the “Mediterranean- West Asian” clade
(hereafter Ponto- Caspian clade); (5) The Ponto- Caspian clade is
subdividedintofivemonophyletichaplotypesand/orhaplogroups:
one (with two exceptions) from the Caspian Sea drainage, one from
DanubeRiver basin, and three fromtheeasternandsoutheastern
Black Sea drainage, including nominal S. rizeensis, which belongs to a
separate monophyletic clade. (6) The clades from the eastern Black
Sea drainage, except S. rizeensis clade, are not geographically dis-
tinct.Oneof these clades was found in catchments oftheRivers
Coruh (Chorokhi), Rioni, and in the Black Sea; in the catchments of
FIGURE4 Phylogenyofthebrowntrout/salmonfromthePonto-Caspianbasin,Anatolia/basinoftheIndianOcean,basinofAtlanticOcean,
and the Balkans, based on the mitochondrial cytochrome b gene sequences. The topology inferred using Bayesian analysis (BI). Left to the
nodes: bootstrap support of the respective clade inferred using maximum likelihood (ML) algorithm before the slash, posterior probability as
inferred by BI after the slash. Identical haplotypes shown separately if found at more than one location. Clades with bootstrap support below 50
and PPs below 0.5 shown as polytomies. Red branches—samples from the drainage of the Black Sea; blue branches—samples from the drainage
of the Caspian Sea; green branches—samples from the Atlantic, Northern, and Baltic Sea drainages
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NINUA et Al.
the Rivers Coruh (Chorokhi), Kintrishi, and in the Black Sea; in the
catchments of the Rivers Coruh (Chorokhi), Kintrishi, Rioni, Enguri,
and in the coastal area of the Black Sea. The trout individuals iden-
tified as S. coruhensis by Turan et al. (2009) were present in both
clades.
We conclude that anadromous life mode is typical for all trout lin-
eages from the eastern Black Sea drainage, with the exception of S. ri-
zeensis from the northeastern Turkey. The same is true for the clade
from the Caspian Sea drainage, which contains at least one salmon
captured in the Caspian Sea. This clade contains, additionally, a well-
supported subclade of fish from the basin of the River Terek (Tergi)
(nominal S. ciscaucasicus). Another important conclusion is that the
basal genetic diversification of brown trout from the Ponto- Caspian
region occurred in the northern and eastern Anatolia, where the split
between the Mediterranean and Ponto- Caspian lineages most likely
occurred.
3.3 | Inferred time of split among the clades
If the time of split between S. ohridanus and S. obtusirostris, on one
side, and S. marmoratus, S. platycephalus, and S. trutta on the other
(5.6 mya) is set as a time of the initial split, the divergence among the
clades is dated as shown in Figure 5. The split between the brown
troutfromtheAtlanticdrainageandAnatolian/Mediterranean/Ponto-
Caspianlineage occurred3.5(range 2.1–5.4) mya;thesplit between
the Mediterranean/Euphrates basin and the Ponto-Caspian lineage
occurred2.4 (1.9–3.6) mya; the split between thefive subclades of
the Ponto- Caspian brown trout—1.5 (0.9–2.4) mya.
If calibration used by Schenekar et al. (2014) is applied, these fig-
ures should be corrected. The average split time between the sub-
cladesofthePonto-Caspiancladeisinthiscaseonlyca.230kya.This
does not affect the main conclusion: The earliest genetic diversifica-
tion of brown trout from the Ponto- Caspian Basin occurred at the
southeastern coast of the Black Sea and the rivers located in this area.
3.4 | Control region
The optimal substitution model for the ingroups included in the analy-
sis was T92 (Kumar et al., 2016). Both novel and downloaded se-
quences showed little variation within the studied fragment. However,
all sequences from the drainages of Black and Caspian Seas were clus-
tered in a monophyletic clade, albeit with modest bootstrap support,
and related to trout from the Mediterranean (it was not possible to ex-
actly identify locations for the sequences from Turkey). Remarkably,
two sequences of S. trutta oxianus from the Aral Sea watershed be-
longed to the same clade. However, the sequences could not resolve
FIGURE5 Timescale for the divergence of the clades shown in Figure 2, according to the consensus calibration of www.timetree.org. The
numbersatthenodes—estimateddivergencetime(millionsofyears,mya);grayhorizontalbars—95%HPDintervals
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NINUA et Al.
phylogenetic relations of fish from the Ponto- Caspian basin, and failed
to separate Black Sea, Caspian, and Aral Sea lineages (Figure 6).
3.5 | Morphometry
Principal component analysis based on the standardized residuals of
27 size- removed body measurements extracted seven principal com-
ponents with eigenvalues exceeding one. Cumulative weight of the
components 1–4 exceeded 68% (Table 2a). Measurements showing
relative height of fish bodies had the highest loadings on the first PC.
Measurements of the head (relative length of snout) had the highest
loading on the second PC. The distances in the hind part of the body
had the highest loading on the third PC. Relative distances between
the dorsal and adipose fins had the highest loadings on the fourth PC
(Table 2b).
The first PCA axis separated river trout (including rainbow trout)
from the trout caught in the coastal area of the Black Sea (“Black Sea
Salmon”). The second axis separated the fish from the basin of Terek
(Tergi; nominal S. ciscaucasicus) from the rest of the samples (Figure 7).
The third axis did not add any valuable information to the analysis, and
the fourth axis separated rainbow trout (Onychorhynchus mykiss) from
Salmo spp (result not shown). Simultaneously, the first and the second
axes partly separated river trout from the Black and the Caspian (ex-
cluding Terek) watercourses (Figure 7).
The number of gill rakers at the first gill arch varied between 14
and 22, and did not show stable differences between the individuals
from different populations, or between Salmo and Oncorynchus indi-
viduals. The two smallest individuals (body length 10 and 12 cm) had
14rakerseach, and thelargestindividual(28cm)had22rakers;30
individualswithintermediatebodylength(13–25cm)had16–18rak-
ers, the number coinciding with those reported by Turan et al. (2009)
for S. coruhensis. Correlation between body length and the number of
rakers was significant (r2 = .81, n=32,p < .01).
Ofthe 32 studied individuals, the majority had 10 raysindorsal
fins, and three individuals, two from the Caspian, and one from the
Black Sea catchment, had nine rays. Finally, the number of rays in pec-
toral fin variedbetween 12 (12 individuals) and 13 (20 individuals),
without respect to body size, genetics, or river catchment.
There are more or less stable differences in color pattern between
the marine and freshwater forms of trout (Figure 8). The background
FIGURE6 Maximum likelihood tree
based on the control region sequences of
Salmo. Bootstrap support shown above
the nodes. Identical haplotypes shown
separately if found at more than one
location. Clades with bootstrap support
below 49 shown as polytomies. Red
branches—samples from the drainage of
the Black Sea; blue branches—samples
from the drainage of the Caspian Sea;
green branches—samples from the drainage
of the Aral Sea
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9
NINUA et Al.
was silver in marine form but yellowish in the freshwater trout. The
individuals from Terek (Tergi) River were gray rather than yellowish.
Red spots on the lateral side are paler in marine forms.
In conclusion, the studied qualitative characters did not help much
to distinguish between individuals of different genetic or geographic
groups, in contrast with body shape dimensions.
TABLE2 The outcome of principal component analysis based on 28 size- removed measurements of fish body (only trout from Georgia
included). (a) Eigenvalues and % of variance for the PC exceeding one; (b) loadings of individual distances (see Figure 2 for details) on the first
seven PC axes
(a)
Component
Initial Eigenvalues
Total % of Variance Cumulative %
1 9.652 35.750 35.750
24.379 16.219 51.969
32.471 9.152 61.122
4 1.941 7.187 68.309
5 1.845 6.833 75.142
6 1.417 5.247 80.389
7 1.190 4.406 84.795
(b)
Distances
Component
1 2 3 4 5 6 7
1–2 −0.109 0.893 −0.076 −0.050 0.143 0.186 0.027
1–3 0.150 0.746 0.227 −0.033 −0.204 −0.043 −0.254
3–4 0.511 −0.614 −0.098 0.197 0.464 0.023 −0.104
4–5 0.159 0.427 0.156 −0.086 −0.452 −0.577 0.004
5–6 −0.142 −0.006 0.392 −0.793 0.251 0.019 0.185
6–7 0.606 −0.312 −0.368 −0.021 −0.475 0.175 −0.139
7–8 −0.711 0.190 0.100 0.450 0.178 −0.055 0.242
8–9 −0.602 0.294 0.243 0.267 0.127 −0.063 0.293
9–10 0.280 0.272 −0.586 −0.554 −0.143 0.201 0.205
10–11 0.418 −0.067 0.481 0.294 −0.420 −0.259 0.088
11–12 −0.025 −0.320 0.390 0.259 −0.316 0.611 −0.023
12–13 0.626 −0.069 −0.496 0.086 0.290 −0.386 0.067
13–1 0.075 0.691 0.149 −0.139 0.202 −0.036 −0.547
3–13 0.741 0.442 0.138 0.170 −0.009 −0.036 −0.123
1–8 0.759 −0.234 −0.202 −0.028 0.428 −0.275 −0.026
3–12 0.835 −0.060 −0.075 0.361 0.155 0.021 0.018
4–13 0.923 0.150 0.158 0.079 0.077 −0.070 0.077
4–12 0.669 −0.125 0.506 −0.250 −0.052 −0.178 0.032
4–11 0.894 0.120 0.115 0.238 −0.040 0.108 0.058
5–12 0.764 −0.263 0.380 −0.165 0.217 0.120 0.087
5–11 0.523 −0.445 0.534 −0.292 0.064 0.124 0.150
5–10 0.712 0.500 0.134 0.122 0.046 0.024 0.287
6–11 0.775 0.345 −0.090 0.206 0.096 0.221 0.191
6–10 0.770 0.000 −0.296 −0.004 −0.247 0.231 −0.235
6–9 0.493 0.174 −0.340 −0.132 −0.381 −0.047 0.523
7–10 0.835 0.218 0.095 −0.162 0.080 0.080 −0.184
7–9 −0.071 0.751 0.002 0.040 0.337 0.333 0.154
10
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NINUA et Al.
4 | DISCUSSION
Brown trout from the Black and the Caspian Sea drainages comprises
a monophyletic evolutionary lineage (matrilineal clade) distinct from
brown trout from other parts of West Eurasia, including those from
thedrainagesofAtlantic,Mediterranean,andtheIndian Ocean.This
lineage separated from other clades native to the rivers of Anatolia.
Most of the subclades within this clade produce both riverine and
anadromous forms, or may spontaneously switch between these two
life modes. There are some differences in body proportions between
different lineages of brown trout, although morphology should be
used very carefully while differentiating among the nominal species:
Ouranalysessuggestthatbodyproportionsandnumberofgillrakers,
commonly used in taxonomy, may be associated with highly variable
body size of the fish.
4.1 | Diversification of brown trout and
geological past
Basal diversification within Salmo, separating Atlantic salmon (S. salar)
from the brown trout lineage occurred, according to different authors,
from9.6to15.4mya(Alexandrouetal.,2013;Campbell,López,Sado,
&Miya,2013;Crête-Lafrenièreetal.,2012;Maetal.,2015;Shedko,
Miroshnichenko, & Nemkova, 2012). The split between the widely
distributed European S. trutta and recently described species S. ohri-
danus and S. obtusirostrisoccurred3to9Mya,inthelateMioceneor
Pliocene(Crête-Lafrenièreetal.,2012;Macqueen&Johnston,2014;
seehttp://www.timetree.org/;Hedgesetal.,2015forreview).Ifthese
dates are accepted, the Ponto- Caspian lineage has been separated
from the populations of the Anatolian rivers draining into the Persian
Gulf and Mediterranean Sea ca. 2.4 mya, shortly after early Pleistocene
FIGURE7 Individual scores of
brown trout (river and marine forms) and
naturalized rainbow trout from Georgian
rivers and costal area of the Black Sea
on the first and the second principal
component axes based on 27 size- removed
body measurements (see Figure 2 for
details)
FIGURE8 River form (upper image;
Kintrishi catchment) and marine form
(lower image; Black Sea coastal area
near mouth of Kintrishi) of Salmo labrax
from Black Sea drainage in Georgia; both
individuals have identical mitochondrial
haplotype
|
11
NINUA et Al.
glaciationsbegan(SubcommissiononQuaternaryStratigraphy,2017).
Consequently, all trout populations from the Black, Caspian, and Aral
Sea drainages, from Austria to Iran and Kazakhstan, descend from this
lineage dispersed during the Pleistocene, probably in Pleistocene in-
terglacials, from the rivers of the northeastern Anatolia.
Schenekar et al. (2014) suggested that the divergence time be-
tween brown trout from the Atlantic and Black Sea basins in Austria
occurred 320,000–745,000years ago, based on the 1%–2% per
MY divergence rate for Thymalus spp. (Koskinen, Haugen, Primmer,
Schlotterer, & Weiss, 2002; Weiss, Persat, Eppe, Schlötterer, & Ublein,
2002). If the scale used by these authors is applied, the separation of
the Ponto- Caspian lineage should have happened only ca. 200 kya,
hence in late Pleistocene. However, this dating would suggest the revi-
sion of the inferred divergence time for the entire genus Salmo, and we
suggest there is not yet enough evidence for such revision.
It is likely that the divergence of Ponto- Caspian trout was associ-
ated with glacial cycles. The landscape model of the last glacial max-
imum (Gavashelishvili & Tarkhnishvili, 2016) suggests that the river
courses throughout the most of the Caucasus and Asia Minor were
shorter than in present time, which means that the upper currents of
these rivers would likely not be suitable for trout reproduction. A large
continuous refugium was located at the southeastern coast of the
Black Sea (Gavashelishvili & Tarkhnishvili, 2016; Van Andel & Tzedakis,
1996) and, hence, the brown trout populations could have survived
there during glacial periods. The area at the southeastern coast of the
Black Sea had climate and forest landscapes most similar to the inter-
glacial period; trout habitats of that area (mostly in the basin of Coruh
[Chorokhi] and other rivers draining into the Black Sea along its south-
eastern coast) were least affected by climatic fluctuations. This is the
area of the highest genetic diversity of the Ponto- Caspian trout, home
also to the basal lineages of this clade. The split between this lineage
andtheEuphrates/PersianGulflineagehappenedinearlyPleistocene.
It is possible that some parts of the upper reaches of the Euphrates
and Coruh (Chorokhi) changed their courses, causing isolation of some
mountain populations, hence reinforcing the divergence of fish from
the drainages of the Black Sea and the drainage of the Euphrates; a
similar process explains the presence of two distinct lineages of trout
in upper reaches of the Danube (Schenekar etal., 2014). Indeed,
only the largest rivers of the northern Eurasia retained their courses
through the entire Pleistocene (Shahgedanova, 2002).
The populations from the Black Sea were captured during the
consequential glacial cycles in small isolated streams flowing into the
southeastern part of the sea, and in interglacial periods, probably used
the sea as a transit area, dispersing through different rivers of the
samedrainage.Oneoftheselineagesmightgainsomemorphological
peculiarities and lose ability to switch to anadromy, the form recently
described as S. rizeensis(Turanetal.,2009).Otherfourmajorlineages
haveapparently maintained this ability.Oneofthosedispersedinto
the Caspian drainage (and probably into the Aral Sea drainage), and
three others remained in the Black Sea drainage. The most recent con-
junction between the Black and Caspian Seas, through the lowland
drainages north of the Caucasus Mountains, could have happened as
late as 160 kya (Gelembiuk, May, & Lee, 2006) and even 9 kya (Reid
&Orlova,2002).However,overmillionyearsofisolationbetweenthe
Black Sea lineages and the Caspian lineage of brown trout (according
to a more likely estimate) suggest that the conjunction periods have
been strongly reduced since earlier time. Although there are differ-
ences between average body proportions of trout from the Black
and Caspian catchment (Figure 7), the morphological overlap is high.
Morphological differences between the fish from the basin of Terek
and Mtkvari (Kura) are stronger and fixed.
ThesplitbetweenthefourBlackSeaclades,includingtheDanube
clade, occurred shortly after the initial split and could be associated
with increasing time of glacial cycles and decreasing temperature
duringtheglacialmaximainmid-Pleistocene (Imbrie etal., 1993). In
conclusion, Pleistocene glacial cycles and the latest transgressions be-
tween the Black and Caspian Seas are the main reasons of separation
and genetic diversification of Ponto- Caspian lineages of brown trout.
4.2 | Riverine and lacustrine versus anadromous
mode of life—obligatory or facultative?
It has never been established to what extent anadromy is an inherit-
able feature of individual evolutionary lineages of brown trout. Even
in better studied American rainbow trout, there are multiple gaps of
knowledge concerning this question (Kendall et al., 2014). In the ear-
liest treatises (e.g., Sabaneev, 1875) Atlantic salmon and Black Sea
salmon were clumped together under the name Salmo salar, although
the author mentioned that the salmon from the Caspian Sea drain-
age might belong to a different species, Caspian salmon (S. caspius).
Conversely, for brown trout from the rivers and streams of Europe
and the Caucasus, including drainages of the Black and Caspian Seas,
Sabaneev (1875) used the name Salmo fario, considering it a purely
freshwater form.
Field studies conducted in 1920s revealed that the transition
between the riverine and anadromous life mode occurs easily. Berg
(1959) suggested that an individual trout may occasionally change
the riverine life mode to an anadromous one during its life cycle.
However, the question remains—to what extent is anadromy a ge-
netic feature typical for only some lineages of brown trout? It has
been shown for other salmonids, for example, Oncorhynchus mykiss
(Kendall et al., 2014; Nichols, Edo, Wheeler, & Thorgaard, 2008) and
S. salar(Perrier,Bourret,Kent,&Bernatchez,2013)thatchangebe-
tween the life modes is associated with a complex genetic mecha-
nism, and these are heritable. Turan et al. (2009) showed that within
the basin of Coruh (Chorokhi), the two forms coexist, forms which
he named S. rizeensis and S. coruhensis, the former purely riverine
and the latter anadromous. This study suggested that, indeed, the
mitochondrial haplogroup of S. rizeensis was never found in fish
caught in the Black Sea. Simultaneously, the individuals from the
Black Sea (as well as a single Caspian salmon included in this anal-
ysis), although they differ from the freshwater fish morphologically
(Figures 7 and 8), belong to various branches within a monophy-
letic Ponto- Caspian clade, which has S. rizeensis as the sister clade.
Anadromous life mode presents in brown trout from the catchment
ofAtlanticOcean (S. trutta s. str.). It is unknown for S. marmoratus
12
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NINUA et Al.
and Anatolian S. platycephalus (most closely related to the Ponto-
Caspian forms), Balkan S. ohridanus, or S. obtusirostris. The absence
of anadromy of these forms may be related to a high salinity of the
seas connecting to the rivers populated by these species (Thunell
& Williams, 1989). We hypothesize that the reappearance of anad-
romy in brown trot from the Black Sea and Caspian catchments is
related with low salinity of these seas.
4.3 | Taxonomic inference
The trout from the Caspian Sea basin (Caspian trout) is a monophyletic
lineage. Nominal S. caspius from the drainage of Mtkvari (Kura) and
southern Caspian, on one hand, and nominal S. ciscaucasicus from the
Terek (Tergi) River are monophyletic. Moreover, the river forms of this
fish are morphologically distinct from each other: Nominal S. ciscauca-
sicus has shorter snout, and this feature is nearly diagnostic. Black Sea
trout have on average shorter trunk than nominal S. caspius, and nar-
rower body, although these differences cannot be used for diagnosis
between the two lineages.
We suggest using the valid names S. caspius for brown trout from
the rivers flowing into the Caspian Sea from the south and southwest,
and S. ciscaucasicus for that found in the Terek River and other rivers
flowing into the Caspian from the north and northwest. Meanwhile,
the taxonomic position of lacustrine S. ischchan and S. ezenami needs
further analysis.
The anadromous individuals from the Black Sea catchment, de-
scribed as S. coruhensis by Turan et al. (2009), were shown not to
represent a monophyletic lineage with respect to both the riverine
and anadromous fish from Georgia, as well as to the fish from the
Danube drainage.Turanetal. (2009) showed some morphological
differences between the anadromous fish from Coruh catchment
and S. labrax from the northern part of the Black Sea drainage.
However, the latter were not investigated genetically, and their
characteristic features (lower number of gill rakers and different
body measurements) may be associated with body size of fish,
smaller than that of other nominal species. The number of rakers
may increase with body size of fish (Ross, Martínez- Palacios, Aguilar
Valdez, Beveridge, & Chavez Sanchez, 2006), and moreover, run-
ning PCA without excluding size would obviously harvest the first
componentdependentlargelyonbodysize(Thorpe&Leamy,1983),
an extremely variable characteristic in fish. Hence, there is not suf-
ficient evidence of morphological separation between S. labrax and
S. coruhensis. Consequently, we suggest a conservative approach,
and all individuals from the watercourses of Coruh (Chorokhi),
Kintrishi,Rioni,Enguri,Danube,andthose caughtin theBlackSea
(except S. rizeensis, a narrow- ranged purely riverine form) we con-
sider conspecific at this stage, and use the priority name S. labrax
(Black Sea salmon).
We should emphasize the findings reported here are based
on sequences from a mitochondrial cytochrome b fragment, and
might be refined if nuclear sequence data were added to the anal-
ysis. However, based on a limited number of existing studies that
combine nuclear and mitochondrial sequence data from salmonids,
mitochondrial and nuclear sequences converge on the same clades
in genus Salmo (Crête- Lafrenière et al., 2012). Simultaneously, one
can expect patterns of incomplete lineage sorting and gene flow
between closely related lineages, similar to that revealed in Central
Europe as a result of microsatellite genotyping (Schenekar et al.,
2014), and obviously further analysis including improved sampling
and more DNA markerswill substantially improve ourknowledge
of western Eurasian trout and salmon phylogeny and population
structure.
ACKNOWLEDGMENTS
The project has been supported by the Ministry of Environment and
NaturalResources Protection of Georgia(2014/ #148) and internal
grant of Ilia State University. We appreciate Mariam Gabelaia who
assisted with morphometric measurements, Marine Murtskhvaladze
for assisting in the laboratory work. Cort Anderson corrected English
of the manuscript and made valuable comments on the text and the
analyses. We appreciate anonymous referee for comments on the
first draft of the paper.
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTIONS
LN conducted the field work, processed the samples for molecular
geneticanalysis,andtogetherwithDTanalyzedphylogenies.DTand
LN designed the study and prepared the manuscript. EG conducted
morphometricanalysisandassistedinDNAprocessing.
ORCID
David Tarkhnishvili http://orcid.org/0000-0003-1479-9880
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How to cite this article:NinuaL,TarkhnishviliD,GvazavaE.
Phylogeography and taxonomic status of trout and salmon
from the Ponto- Caspian drainages, with inferences on
European Brown Trout evolution and taxonomy. Ecol Evol.
2018;00:1–14. https://doi.org/10.1002/ece3.3884
