Zoologischer Anzeiger 272 (2018) 73–80

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Zoologischer Anzeiger
journal homepage: www.elsevier.com/locate/jcz

Research paper

Comparative body shape variation of the European grayling Thymallus

thymallus (Actinopterygii, Salmonidae) from wild populations and
hatcheries
Aleksandar Bajić a , Vida Jojić b , Aleš Snoj c , Branko Miljanović a , Oleg Askeyev d ,
Igor Askeyev d , Saša Marić e,∗
a
Department of Biology and Ecology, Faculty of Science, University of Novi Sad, Trg D. Obradovića 2, 21000 Novi Sad, Serbia
b
Department of Genetic Research, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade,
Serbia
c
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia
d
Institute for Problems of Ecology and Mineral Wealth, Tatarstan Academy of Sciences, Daurskaya 28, 420087 Kazan, Tatarstan Republic, Russian
Federation
e
Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski Trg 16, 11001 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: We employed geometric morphometric techniques to investigate external (body) morphology of Euro-
Received 12 July 2017 pean grayling (Thymallus thymallus) populations from the wild and hatchery facilities. Wild graylings
Received in revised form 8 December 2017 were representative of Balkan and Caspian phylogenetic clades, whereas hatchery-reared specimens
Accepted 19 December 2017
originated from Balkan and Adriatic phylogenetic clades. Individuals of T. thymallus from the Adriatic phy-
Available online 20 December 2017
logenetic clade were the largest, followed by those from the Balkan phylogenetic clade, while graylings
Corresponding Editor: Alexander Kupfer
from the Caspian phylogenetic clade were the smallest. Graylings from hatchery facilities were larger
than graylings from the wild. Body shape variation in T. thymallus coincides with genetic differentiation
Keywords:
Allometry
of the analyzed populations, whereas it is less influenced by difference in environment they experience in
Geometric morphometrics wild and captive habitats. Although hatcheries can generate large numbers of individuals, some of which
Morphological variation will have an extreme phenotype, the variance in body shape was similar in captive and wild populations.
Shape Allometric relations were different between specimens from the wild and from hatchery facilities, as
Size well as among those belonging to different phylogenetic clades. Allometric analyses performed sepa-
rately for the wild and hatchery-reared populations revealed significant effect of allometry and similar
trends in size-related shape variation among populations from different phylogenetic clades. We found
that phenetic relationships among the studied wild grayling populations inferred from non-allometric
body shape variation better reflected their phylogenetic relationships than equivalent data from hatchery
populations.
© 2017 Elsevier GmbH. All rights reserved.

1. Introduction morphology and osteology provided important information about

Graylings (subfamily Thymallinae) are salmonid fishes from
the genus Thymallus found across Eurasia and North America. The
distribution of European grayling (Thymallus thymallus Linnaeus,
1758) extends from France and England in the west, throughout
northern Europe, including Scandinavia, and across northern Rus-
sia to the Ural Mountains near the River Kara in the east (Kottelat
and Freyhof, 2007; Northcote, 1995). Earlier studies of external
∗ Corresponding author.
E-mail address: sasa@bio.bg.ac.rs (S. Marić).
salmonid taxonomy (Shaposhnikova, 1975), including the genus
Thymallus (Knizhin et al., 2006; Norden, 1961). Moreover, the fish
body shape is the product of multiple different processes (swim-
ming behavior, feeding aptitude, social and physical environment)
and morphometric characters play an important role in the iden-
tification of intraspecific variation, i.e. can be used as a tool to
distinguish between salmonid populations adapted to different
environmental conditions (Pulcini et al., 2013). Besides studies on
specimens from the wild (Bush and Adams, 2007; Garduño-Paz
et al., 2012; Janković, 1960; Marić et al., 2011a; Nicieza, 1995;
Shaposhnikova, 1964; Surre et al., 1986), many authors have inves-
tigated how the environments experienced by cultured salmonids

https://doi.org/10.1016/j.jcz.2017.12.005
0044-5231/© 2017 Elsevier GmbH. All rights reserved.
74 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80
in hatcheries affect their morphology (von Cramon-Taubadel et al.,
2005; Fleming et al., 1994; Hard et al., 2000; Pulcini et al., 2013;
Taylor, 1986).
Although Janković (1960) conducted a comprehensive morpho-
logical examination of T. thymallus populations from the Adriatic
and Black Sea basins and Shaposhnikova (1964) evaluated those
from the Caspian basin, the majority of investigations on Euro-
pean grayling have focused on genetic structure and descriptions of
the phylogenetic lineages/clades (Gum et al., 2005, 2009; Koskinen
et al., 2000; Marić et al., 2011b, 2012,2014; Meraner and Gandolfi,
2012; Stamford and Taylor, 2004; Sušnik et al., 2001; Weiss et al.,
2002). Based on the control region of mitochondrial DNA, Weiss
et al. (2002) revealed two well-supported phylogenetic clades
within the Danubian basin, one highly divergent clade in the Adri-
atic basin, and one large, diverse group representing most other
European grayling populations. Furthermore, Marić et al. (2012,
2014); reported two additional clades, Balkan from the Danubian
drainage of the western Balkans, and Caspian from the middle Volga
and upper Ural drainages of the Caspian basin. Besides considerable
genetic divergence of Adriatic from other graylings, vast genetic
diversity of European grayling throughout its whole distribution
range has been reported (Marić et al., 2011b, 2014; Meraner and
Gandolfi, 2012; Sušnik et al., 2001; Weiss et al., 2002).
Herein, we employed geometric morphometric techniques to
investigate external (body) variation of European grayling popu-
lations from the wild and from hatchery facilities. Wild graylings
were representative of Balkan and Caspian phylogenetic clades,
whereas hatchery-reared specimens originated from Balkan and
Adriatic phylogenetic clades. Our first goal was to provide a general
picture of body size and shape variation of the analyzed popula-
tions related to both phylogenetic origin (Balkan/Caspian/Adriatic)
and environmental (wild/captive) conditions. Since the pheno-
type of fish can diverge greatly in captivity, i.e. hatcheries can
generate large numbers of individuals, some of which will have
an extreme phenotype (Stringwell et al., 2014), we expected to
detect a higher level of among-individual variance in body shape
in hatchery-reared populations than in wild ones. Therefore, as a
second research goal, we examined within-population variation in
T. thymallus populations from the wild, as well as in those from
hatchery facilities. Finally, salmonids exhibit extensive plasticity
in overall body shape (Beacham, 1990; von Cramon-Taubadel et al.,
2005; Currens et al., 1989). Thus, by comparing morphometric and
genetic distances in both groups of T. thymallus populations (wild
and hatchery-reared), we aimed to identify how well relationships
among the studied populations observed from their body shape
variation reflect those identified from molecular phylogenetic data.
2. Material and methods
2.1. Specimens and data collection
The analyzed sample (Table 1, Fig. 1) comprised 54 wild and
59 hatchery-reared adult T. thymallus individuals. Specimens from
the wild were caught using a landing net and angling from the
rivers Lim (Black Sea basin and Balkan phylogenetic clade), Kana
and Bugurla (Caspian Sea basin and Caspian phylogenetic clade).
Graylings from hatchery facilities came from the following river
stocks: Soča (Adriatic Sea basin and Adriatic phylogenetic clade),
Una and Sava Bohinjka (Black Sea basin and Balkan phylogenetic
clade). Just after capture they were euthanized, photographed and
sexed by gonad inspection. The age of individuals was determined
from their scales (microscope Leica MZ75 with × 2 objective; www.
leica-microsystems.com). All individuals were 2+ years old, except
for four specimens (aged 3+ ) from the Kana population. Images of
specimens from the left body side were taken with a Sony DSC-
W110 digital camera fixed on a stand. The camera lens was set to
be perpendicular to the mid-lateral body line (Fig. 2).
2.2. Geometric morphometrics and statistical analyses
Variations in size and shape of the body were examined using
methods of landmark based geometric morphometrics (Bookstein,
1991; Dryden and Mardia, 1998; Rohlf and Marcus, 1993). Fourteen
two-dimensional landmarks (Fig. 2) were recorded with tpsDig
software (Rohlf, 2015a, 2015b) and centroid size (CS) as the size
variable was calculated. Landmark configurations were superim-
posed using a generalized Procrustes analysis (GPA) to remove
effects of differences in size, position and orientation (Rohlf and
Slice, 1990). After alignment of the raw landmark coordinates by
GPA, Procrustes coordinates as shape variables were extracted.
Procrustes coordinates contain complete (overall) shape variation,
which also includes the influence of allometry, i.e. the relationship
between shape and size.
For each population, descriptive statistics (means, standard
deviations and standard errors) of centroid size (CS) were calcu-
lated and graphically presented. To check for body size differences
among the studied populations, analysis of variance (ANOVA)
was performed with CS as the dependent variable and popula-
tion as the independent variable. Subsequently, we conducted
the post hoc Tukey honest significant difference test for unequal
sample sizes. To explore body shape variation of the analyzed
populations related to phylogenetic (Balkan/Caspian/Adriatic) and
environmental (wild/captive) origins, we used Principal Compo-
nent Analysis (PCA). Besides inter-population variation, this PCA
can provide a visual inspection into within-population varia-
tion in body shape, both among individuals and between sexes.
Additionally, we quantified within-population variation by among-
individual variance in shape according to the standard formula for
a variance: Varshape = ˙Pdj 2 /n-1, where Pdj is the Procrustes dis-
tance of individual j from the population mean shape and n is
the sample size for a population. Procrustes distance is the mea-
sure of shape difference between two forms (Bookstein, 1991).
Comparisons between shape variances of the analyzed popula-
tions were done using F-test, followed by Bonferroni adjustment.
To test for homogeneity of allometric slopes among phylogenetic
clades and between wild and hatchery groups of populations, we
performed two separate multivariate analyses of covariance (MAN-
COVAs) with Procrustes coordinates as dependent variables and
log-transformed CS (log CS) as the covariate. Phylogenetic and envi-
ronmental origin was used as the categorical factor in first and
second MANCOVA, respectively.
To estimate inter-population allometric effect on overall shape
variation, in each group of populations (wild and hatchery), we per-
formed multivariate regression of Procrustes coordinates onto log
CS using a permutation test with 10,000 iterations under the null
hypothesis of independence between size and shape (Edgington,
1995; Good, 1994). Allometry tests were based on pooled within-
population regression. Then, we conducted multivariate analysis of
covariance (MANCOVA) with Procrustes coordinates as dependent
variables, population as the categorical factor and log-transformed
CS (log CS) as the covariate to test for homogeneity of allomet-
ric slopes among populations. If MANCOVA showed no significant
interaction of population and log CS, i.e. if populations did not differ
in allometric slopes, we could use residuals from the regression of
shape on size to examine the non-allometric component of shape
variation in subsequent analysis (Drake and Klingenberg, 2008;
Klingenberg, 2009).
Discriminant Function Analysis (DFA) was done to compare the
non-allometric component of shape variation between pairs of
the analyzed populations. Thus, we obtained Procrustes distances
(observed by pairwise comparisons of the analyzed populations
 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80 75

Table 1
List of T. thymallus specimens (m – males, f – females) analyzed in this study.

Origin Population Coordinates Drainage Basin (m, f)

Wild Lim 43◦ 35 53” N Drina → Sava → Danube Black Sea 17 (7, 10)
19◦ 22 54” E
Bugurla 52◦ 36 27” N Yushatyr → Sakmara → Ural Caspian Sea 17 (9, 8)
56◦ 02 44” E
Kana 55◦ 47 27” N Belaya → Kama → Volga Caspian Sea 20 (15, 5)
57◦ 34 45” E
Hatchery Soča 46◦ 18 29” E Soča Adriatic Sea 18 (10, 8)
13◦ 29 09” N
Sava Bohinjka 46◦ 19 16” N Sava → Danube Black Sea 21 (16, 5)
14◦ 03 44” E
Una 44◦ 31 56” N Sava → Danube Black Sea 20 (9, 11)
16◦ 06 52” E

Fig. 1. Analyzed populations of T. thymallus (filled symbols – wild populations, open symbols – hatchery populations; triangles – populations from the Balkan phylogenetic
clade (1–Lim, 5 – Sava Bohinjka and 6–Una), circles – populations from the Caspian phylogenetic clade (2–Bugurla and 3–Kana), square – population from the Adriatic
phylogenetic clade (4–Soča).

Fig. 2. Landmarks collected on the T. thymallus body. 1–anterior edge of maxilla, 2 – center of the eye, 3–posterior edge of maxilla, 4 – first dorsal scale, 5 – posterior overlap
of opercle and subopercle, 6 – anterior base of pectoral fin, 7 – anterior base of dorsal fin, 8–anterior base of ventral fin, 9 – posterior base of dorsal fin, 10 – anterior base of
anal fin, 11–anterior base of adipose fin, 12 – posterior base of anal fin, 13–posterior base of adipose fin, 14 – last scale in lateral line. This specimen is from the river stock
Sava Bohinjka (date of capture: 14. december 2012; size: Ltot = 29 cm, CS = 34.27; age: 2+ years; sex: male).

non-allometric mean shape), their statistical significance, as well as
non-allometric shape changes between the examined populations.
The statistical significance of the observed Procrustes distances
was assessed using a permutation test (with 10,000 permutation
runs) under the null hypothesis of equal population means. Sub-
sequently, Bonferroni correction was made. Non-allometric shape
differences between population mean shapes were visualized by
wireframe graphs (Klingenberg, 2013). Additionally, phenetic rela-
tionships among the studied grayling populations were compared
with their phylogenetic relationships, i.e. previously observed mor-
phometric distances between pairs of the analyzed populations
(Procrustes distances) were compared with their genetic distances.
76 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80

Fig. 3. Mean centroid size (CS) for T. thymallus body of the analyzed populations with standard error (SE) and standard deviation (SD). Soča (mean: 40.17; min-max:
34.86–45.26), Sava Bohinjka (mean: 32.00; min-max: 28.22–38.99), Una (mean: 34.18; min-max: 29.07-39.21), Lim (mean: 30.50; min-max: 27.39-33.04), Bugurla (mean:
18.67; min–max: 16.29–21.16), Kana (mean: 20.78; min-max: 16.44–25.71).

As genetic distances we used both allelic sharing distances (DAS ) for

microsatellite marker data and between group mean distances cal-
culated from control region mtDNA sequences. These sequences
were previously detected in the analyzed populations (GenBank
accession numbers: Soča – JX099344; Sava Bohinjka – JX099336
and JX099337; Una – JX099343; Bugurla – JX144732; Kana –
KF280207 and KF280208) (Marić et al., 2012, 2014) based on the
Kimura two-parameter model (Kimura, 1980)).
Descriptive statistics, ANOVA, Tukey test, F-test and MANCOVA
were conducted using Statistica v. 5.1 (StatSoft Inc., 1997). The pro-
gram MEGA 6 (Tamura et al., 2013) was used for calculation of
between group mean genetic distances. All other analyses were
done in MorphoJ v. 1.06d software (Klingenberg, 2011).

3. Results

For each studied T. thymallus population, descriptive statistics

(means, standard deviations and standard errors) of centroid size
(CS) are graphically presented in Fig. 3. When comparing body
size in relation to phylogenetic origin of populations, bodies of T.
thymallus from the Adriatic phylogenetic clade (Soča population)
were the largest, followed by those from the Balkan phylogenetic
clade (Una, Sava Bohinjka and Lim populations), while graylings
from the Caspian phylogenetic clade (Kana and Bugurla popula-
tions) were the smallest. When comparing body size in relation
to environmental conditions, graylings from hatchery facilities
were larger than graylings from the wild. Analysis of variance
(ANOVA) for centroid size revealed significant population differ-
ences (F5,107 = 216.38, P < .0001). However, the post hoc Tukey’s test
for unequal sample sizes disclosed statistically significant size dif-
ferences for all pairwise comparisons (P < .001), except for those
between: Sava Bohinjka and Una populations (P = .0525), Sava
Bohinjka and Lim populations (P = .4482) and Bugurla and Kana
populations (P = .1098).
As evident from the PCA scatter plot of overall body shape
variation (Fig. 4), PC1 (accounting for 38.2% of the variation) sepa- Fig. 4. PCA (Principal Component Analysis) of T. thymallus overall body shape varia-
rated T. thymallus populations belonging to the same phylogenetic tion. (A) Specimens from the same phylogenetic clade are joined by outline polygons
clade (Fig. 4A), whereas PC2 (accounting for 17.1% of the variation) (left – Adriatic, middle – Balkan, right – Caspian). (B) Specimens from the same envi-
ronmental conditions are joined by outline polygons (up – wild, down – hatcheries).
tended to separate hatchery-reared grayling specimens from wild
 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80
Table 2
Multivariate analysis of covariance (MANCOVA) for T. thymallus body shape with
log-transformed CS (log CS) as the covariate and phylogenetic (phylogeny) and
environmental (environment) origin as the categorical factor.
␭Wilks df1 , df2 F P
Phylogeny 0.3776 48, 168 2.20 0.0001
Log CS 0.4277 24, 84 4.68 0.0000
Phylogeny x Log CS 0.3729 48, 168 2.23 0.0001
Environment 0.3366 24, 86 7.06 0.0000
Log CS 0.1427 24, 86 21.52 0.0000
Environment x Log CS 0.3399 24, 86 6.96 0.0000
ones (Fig. 4B). In addition, PCA scatter plot (Fig. 1 in Supplementary
material) showed that males and females from the same popu-
lation grouped together, justifying the use of pooled sexes in all
subsequent analyses.
Estimated among-individual body shape variances of the
analyzed populations (from the highest to the lowest value)
were: Kana (Varshape = 0.000756), Soča (Varshape = 0.000474), Lim
(Varshape = 0.000451), Bugurla (Varshape = 0.000418), Sava Bohinjka
(Varshape = 0.000322) and Una (Varshape = 0.000315). Although F-
test showed somewhat higher level of within-population variation
in wild Kana population compared to hatchery-reared Sava Bohin-
jka (P = 0.0328) and Una (P = 0.0320) populations, after Bonferroni
correction these differences were insignificant (for visual inspec-
tion into within-population variation in body shape see Fig. 1 in
Supplementary material).
The results of multivariate analyses of covariance (MANCOVAs)
are summarized in Table 2. Significant interaction between log CS
and categorical factor indicated heterogeneity of allometric slopes
both among phylogenetic clades and between groups of wild and
hatchery-reared graylings. Thus, due to different allometric slopes
for wild and captive populations, further analyses were performed
separately for these two groups of populations.
Using multivariate regression of the Procrustes coordinates
onto log CS we found that the allometric effect was signifi-
cant (wild: P = 0.0001; hatchery: P < 0.0001) and accounted for
9.83% and 7.46% of overall shape variation in wild and hatch-
ery populations, respectively. Multivariate analysis of covariance
(MANCOVA) showed no significant interaction between log CS and
population (wild: ␭Wilks = 0.1847, F48,50 = 1.38, P = 0.1297; hatchery:
␭Wilks = 0.2630, F48,60 = 1.19, P = 0.2625) indicating homogeneity of
allometric slopes between populations.
All pairwise comparisons of the analyzed wild grayling popu-
lations (Fig. 5A) revealed statistically significant differences in the
non-allometric component of shape variation. However, the values
of Procrustes distances, together with visualized non-allometric
shape differences between population mean shapes, pointed to
the smallest body shape differences between the Caspian (Bugurla
and Kana) populations (Pd = 0.0163). In comparison to fish from
the Bugurla population, specimens from Kana are characterized by
slightly longer heads, shorter dorsal and adipose fins and more slen-
der anterior trunk regions. Additionally, the Procrustes distance
observed between Lim and Bugurla (Pd = 0.0328) was somewhat
greater than that calculated between Lim and Kana (Pd = 0.0314)
populations. However, as evident from the wireframe diagrams
given in Fig. 5A, similar patterns of non-allometric body shape vari-
ation exist for the Balkan and both Caspian populations. Namely,
graylings from the Balkan population have generally deeper trunks,
higher heads at the level of the operculum and caudal peduncles
moved downward compared with those from the Caspian popu-
lations. It is also noticeable that specimens from the Balkans have
the anterior base of the ventral fin moved posteriorly and the ante-
rior base of the anal fin displaced anteriorly making these two fins
closer together. All pairwise comparisons of the analyzed hatch-
77
Table 3
Allelic sharing distances (DAS ) for microsatellite marker data (above the diago-
nal) and between group mean distances calculated from control region mtDNA
sequences (below the diagonal) for wild and hatchery T. thymallus.
Wild Lim Bugurla Kana
Lim – 0.895 0.918
Bugurla 0.010 – 0.280
Kana 0.011 0.002 –
Hatchery Una Sava Bohinjka Soča
Una – 0.484 0.746
Sava Bohinjka 0.006 – 0.572
Soča 0.032 0.031 –
ery grayling populations (Fig. 5B) unveiled statistically significant
differences in the non-allometric component of shape variation
as well. The greatest difference between population mean non-
allometric shapes was identified for the Soča vs. Una comparison
(Pd = 0.0387), whereas the smallest Procrustes distance was found
between the Soča and Sava Bohinjka populations (Pd = 0.0295).
According to the visualized non-allometric shape changes (Fig. 5B),
graylings from the Adriatic phylogenetic clade (Soča population)
are characterized by considerably higher trunks and shorter adi-
pose fins in comparison to those from the Balkan phylogenetic clade
(Una and Sava Bohinjka populations). However, when compared
to specimens from the Sava Bohinjka population, those from the
Soča population have more robust heads, whereas when compared
to Una specimens, Soča grayling heads are shorter and deeper in
their bases. Additionally, graylings from the Una population have
the slenderest bodies. The most obvious body shape differences
between Una and Sava Bohinjka populations were the elongated
heads and shallower trunks of the Una specimens.
As already mentioned, morphometric distances, i.e. Procrustes
distances, are presented in Fig. 5, while genetic distances, i.e.
allelic sharing distances (DAS ) for microsatellite marker data and
between group mean distances for mtDNA sequences are given
in Table 3. For both wild and hatchery populations, phylogenetic
relationships among them observed from both kinds of genetic dis-
tances correspond to each other in a one-to-one manner (Table 3).
However, phenetic relationships among the studied wild grayling
populations inferred from non-allometric body shape variation bet-
ter reflected their phylogenetic relationships than the data for
hatchery-reared populations. Namely, within the group of wild
grayling populations, specimens from the same phylogenetic clade
(Caspian), i.e. graylings from Kana and Bugurla populations are
genetically and morphologically the most similar (Table 3; Fig. 5A).
In contrast, within the group of hatchery populations, specimens
from the same phylogenetic clade (Balkan), i.e. graylings from Una
and Sava Bohinjka populations are genetically but not morphologi-
cally the most similar (Table 3; Fig. 5B). At the morphological level,
the most similar were graylings from Soča (Adriatic phylogenetic
clade) and Sava Bohinjka (Balkan phylogenetic clade) populations
(Fig. 5B).
4. Discussion
After applying geometric morphometric methods, statistically
significant body size differences among the studied T. thymallus
populations were uncovered. Although the larger size of specimens
from the Adriatic phylogenetic clade (Soča population) and those
from the Balkan phylogenetic clade (Una and Sava Bohinjka popu-
lations) could be the outcome of their origin from hatcheries, the
same pattern of body size differences exists between Balkan and
Caspian populations from the wild (Fig. 3). Namely, individuals
from the Balkans (Lim) were larger than those from two Caspian
populations (Kana and Bugurla). More importantly, all T. thymallus
78 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80
Fig. 5. Non-allometric body shape changes between the analyzed T. thymallus populations from (A) the wild and (B) hatcheries with Procrustes distances (Pd) and their
statistical significance (P) after Bonferroni adjustment. Shape changes (magnified three times) are presented in the form of wireframe diagrams.
specimens from the Balkan phylogenetic clade (Lim, Sava Bohin-
jka and Una populations), despite being wild or hatchery-reared,
have similar size (Fig. 3). Moreover, previous studies on the mor-
phological variation of T. thymallus populations from the Adriatic
and Black Sea basins (Janković, 1960), and from the Caspian basin
(Shaposhnikova, 1964), using traditional morphometrics, reported
comparable results. Balkan population graylings aged 2+ and 3+
were characterized by an average body length of about 24 and
30 cm, respectively (Janković, 1960), whereas specimens of the
same age from Caspian populations were considerably smaller,
with an average body length of about 18 and 24 cm, respectively
(Shaposhnikova, 1964).
In contrast to many phylogenetic studies of the European
grayling conducted in past decades, morphological investigations
of this species are rare. Janković (1960) found considerable mor-
phological diversity among Balkan populations of T. thymallus,
including the Soča population from the Adriatic basin. On the
other hand, Shaposhnikova (1964) showed that grayling popula-
tions from the Kama and Urals drainages of the Caspian basin were
morphologically very similar. Discriminant multivariate analysis
of meristic and morphometric characteristics clearly distinguished
three neighbouring French populations of T. thymallus from the
Rhone drainage basin (Surre et al., 1986). In line with previous
morphological investigations in T. thymallus (Janković, 1960; Surre
et al., 1986), our results for body shape variation point to the
existence of intraspecific phenotypic structuring. We also found
that body shape variation in T. thymallus coincides with genetic
differentiation of the analyzed populations, whereas it is less influ-
enced by difference in environment they experience in wild and
captive habitats. Considering within-population variation related
to natural and rearing conditions, no difference in the variance
of body shape between wild and captive populations was found.
However, allometric relations were different between specimens
from the wild and from hatchery facilities, as well as among
those belonging to different phylogenetic clades. On the other
hand, allometric analyses performed separately for the wild and
hatchery-reared populations, besides significant effect of allome-
try on body shape variation, revealed similar trends in size-related
shape variation among representatives (populations) of different
phylogenetic clades.
Body shape variation in fishes could concur with phylogenetic
relationships reconstructed from molecular data. Thus, a geomet-
ric morphometric study of 31 species of darters (a diverse clade
of small, mostly benthic stream fishes endemic to North Amer-
ica) revealed a significant positive association between body shape
differences and phylogenetic interrelatedness (Guill et al., 2003).
However, body shape variation in fishes could be the result of
phenotypic plasticity and high phenotypic plasticity is common
in many fish (Smith and Skúlason, 1996). Thus, salmonids exhibit
largescale plasticity in overall body shape (Beacham, 1990; von
Cramon-Taubadel et al., 2005; Currens et al., 1989). Moreover,
Stringwell et al. (2014) emphasized that in hatcheries any pheno-
typic shifts will be mostly due to phenotypic plasticity, in contrast
to natural conditions where changes in trait means will probably
result from both plasticity and selection. Consistent with the afore-
mentioned statement, we found that for wild T. thymallus phenetic
relationships among the studied populations inferred from non-
allometric body shape variation better reflected their phylogenetic
relationships than in the group of hatchery populations. In contrast
to the group of wild grayling populations where specimens from the
 A. Bajić et al. / Zoologischer Anzeiger 272 (2018) 73–80
same phylogenetic clade are genetically and morphologically the
most similar (Kana and Bugurla populations from Caspian phylo-
genetic clade), among the hatchery grayling populations specimens
from the same phylogenetic clade are genetically, but not morpho-
logically the most similar (Sava Bohinjka and Una populations from
Balkan phylogenetic clade). Genetic and morphological similarity
between Kana and Bugurla populations suggests a recent river con-
nection between adjacent Kama and Ural watersheds (Marić et al.,
2014). Within the group of domesticated T. thymallus populations,
graylings from the Soča and Sava Bohinjka populations were mor-
phologically the most similar. Some of the earlier studies designed
to characterize the genetic structure of Adriatic grayling restricted
to the Soča river system reported that this population contains
hybrid individuals with a varying proportion of autochthonous
genes (Marić et al., 2012; Sušnik et al., 2001, 2004). Contrary to
Sava Bohinjka and Una populations (characterized by indigenous
origin), the Soča drainage had been stocked and introgressed with
the nearby northern Sava grayling population from Slovenia. Spec-
imens from the Soča population analyzed in our study were under
the Adriatic grayling conservation programme in Slovenia, which
includes genotyping and selection of individuals with up to about
60% of Adriatic genes. This fact could explain the morphological
resemblance of graylings from the Soča and Sava Bohinjka popula-
tions.
Without going into details of already described body shape
diversity between the analyzed T. thymallus populations (Fig. 5), we
wish to emphasize the following. According to Pulcini et al. (2013),
in comparison to wild salmonid phenotypes, domesticated ones
are characterized by deeper heads, trunks and caudal peduncles,
longer dorsal and anal fins and general reduction in body stream-
lining. An almost identical pattern of body shape variation was
detected in our study between graylings from the Soča popula-
tion (Adriatic phylogenetic clade) and those from the Sava Bohinjka
and Una populations (Balkan phylogenetic clade). However, the fact
that specimens from Soča, Sava Bohinjka and Una are all hatchery-
reared excludes environmental origin (wild/hatchery) as a factor
driving these shape differences. The pattern of body shape vari-
ation observed between the Adriatic and two Balkan populations
is clearly different from that described between the two Balkan
populations (Sava Bohinjka and Una). This supports findings by
Janković (1960) and Sabbadini (2000), who described Soča grayling
as morphologically distinct from Danube drainage populations,
suggesting that grayling from the river Soča is racially differen-
tiated from other graylings from the western Balkans. Moreover,
based on the mtDNA control region, Sušnik et al. (2001) found
that T. thymallus populations from two rivers in the Adriatic basin
(Soča and Sesia) differed from the Danubian and Atlantic genetic
lineages by 3.39–3.92%. This divergence was greater than that for
different salmonid species, as defined by Berg and Ferris (1984).
Meraner and Gandolfi (2012) later described sixteen new mtDNA
haplotypes of T. thymallus in the North Adriatic basin of the Rivers
Po and Adige. Therefore, for Adriatic grayling native to Northern
Italy and Western Slovenia, Bianco (2014) reactivated the name,
T. aeliani (Valenciennes, 1848), which was previously used for
endemic grayling from Lake Maggiore. However, a more compre-
hensive study, including populations from all phylogenetic lineages
of European graylings, is needed for complete understanding of the
morphological diversity of this species and its relationship with
phylogeny.
Acknowledgements
This study was supported by the Ministry of Education, Science
and Technological Development of the Republic of Serbia (Grant No.
173045) and the Slovenian Research Agency. Many thanks go to D.
79
Jesenšek, who provided grayling specimens from the River Soča,
T. Strgar for specimens from the River Sava Bohinjka, R. Tahirić for
specimens from the River Una and R. Berović who collected grayling
samples from the River Lim. We thank two anonymous reviewers
for their helpful comments.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.jcz.2017.12.005.
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