Morphological comparison of bleaks (Alburnus, Cyprinidae) from the Adriatic Basin with the description of a new species

Abstract

3 National Museum, Václavské náměstí 68, 115 79 Prague 1, Czech Republic 4 Museo Nacional de Ciencias Naturales, C/ José Gutiérrez Abascal 2, 28006 Madrid, Spain 5 Institute of Fisheries, Zoology and Water Protection, Faculty of Agronomy, University of Mostar, Biskupa Čule 10, 88000 Mostar, Bosnia and Hercegovina 6 Department of Natural Sciences, U.b. Učakar 108, Abstract. The morphometric, meristic and phenotypical characters of the members of the genus Alburnus from the Adriatic Basin were analyzed on specimens from 11 localities, representing eight watersheds. The number of gill rakers, the number of lateral line scales, the number of branched anal fi n rays and the coverage of the ventral keel by scales have the greatest signifi cance in differentiating between species. Signifi cant morphological differences exist between the Alburnus population from Lake Lugano (type locality for Alborella maxima Fatio, 1882) and all the remaining investigated populations. Alburnus from the Neretva River drainage is described as a new species and it is distinguished from other species of the genus Alburnus by the following combination of characters: 13 1 / 2 -15 1 / 2 branched anal fi n rays; 17-26 gill rakers; 40-48 lateral line scales; fi rst anal fi n ray below branched dorsal rays 6-10 or located up to one scale after the last dorsal fi n ray; ventral keel exposed for at least 2 / 3 of a distance between the pelvic fi n base and the anal aperture. The status of the Neretva River basin populations as a separate species was corroborated by genetic analyses, with p-distance between newly described species and A. arborella from the Zrmanja River 1.8-2.2%. by different authors (see Vuković & Ivanović 1971, Dimovski & Grupče 1975, Šorić 1980, Oliva et al. 1988) what made the taxonomy of the bleaks quite complicated and puzzling. Based on the works of Bogutskaya (1997) and Bogutskaya & Naseka (2004), as well as the systematics of Kottelat & Freyhof (2007), genera Alburnus and Chalcalburnus were merged into single genus, Alburnus. Recently, an intensive taxonomic work on the genus Alburnus has lead to a description of several new species from the Black and Aegean Sea Basins, mostly belonging Folia Zool. – 59 (2): 129– 141 (2010) Introduction Although bleaks (genus Alburnus) are widely spread in the waters of the Adriatic watershed, their taxonomy, systematic and actual distribution are still not well understood. Until recently, it was considered that only two widely distributed species inhabit the European waters (Kottelat 1997): Alburnus albidus (Costa, 1838) in the Adriatic Basin and Alburnus alburnus (Linnaeus, 1758) in the other parts of Europe. However, numerous subspecies, morphs and varieties of the later species were recognized

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Morphological comparison of bleaks (Alburnus,
Cyprinidae) from the Adriatic Basin with the
description of a new species
Ivana BUJ1, Jasna VUKIĆ2, Radek ŠANDA3, Silvia PEREA4, Marko ĆALETA1,
Zoran MARČIĆ1, Ivan BOGUT5,7, Meta POVŽ6 and Milorad MRAKOVČIĆ1
1 Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia;
e-mail: ivaradic@biol.pmf.hr
2 Department of Ecology, Faculty of Science, Charles University, Viničná 7, 128 44 Praha 2, Czech Republic
3 National Museum, Václavské náměstí 68, 115 79 Prague 1, Czech Republic
4 Museo Nacional de Ciencias Naturales, C/ José Gutiérrez Abascal 2, 28006 Madrid, Spain
5 Institute of Fisheries, Zoology and Water Protection, Faculty of Agronomy, University of Mostar, Biskupa
Čule 10, 88000 Mostar, Bosnia and Hercegovina
6 Department of Natural Sciences, U.b. Učakar 108, Sl-1000 Ljubljana, Slovenia
7 Institute of Special Zootechniques, Faculty of Agriculture, Josip Jujar Strossmayer University of Osijek,
Trg Sv. Trojstva 3, 31000 Osijek, Croatia
Received 17 October 2008; Accepted 12 October 2009
Abstract. The morphometric, meristic and phenotypical characters of the members of the genus Alburnus
from the Adriatic Basin were analyzed on specimens from 11 localities, representing eight watersheds. The
number of gill rakers, the number of lateral line scales, the number of branched anal fi n rays and the coverage
of the ventral keel by scales have the greatest signifi cance in differentiating between species. Signifi cant
morphological differences exist between the Alburnus population from Lake Lugano (type locality for
Alborella maxima Fatio, 1882) and all the remaining investigated populations. Alburnus from the Neretva
River drainage is described as a new species and it is distinguished from other species of the genus Alburnus
by the following combination of characters: 131/2-151/2 branched anal fi n rays; 17-26 gill rakers; 40-48 lateral
line scales; fi rst anal fi n ray below branched dorsal rays 6-10 or located up to one scale after the last dorsal fi n
ray; ventral keel exposed for at least 2/3 of a distance between the pelvic fi n base and the anal aperture. The
status of the Neretva River basin populations as a separate species was corroborated by genetic analyses, with
p-distance between newly described species and A. arborella from the Zrmanja River 1.8-2.2%.
Key words: taxonomy, east Adriatic Basin, Neretva River basin
by different authors (see Vuković & Ivanović 1971,
Dimovski & Grupče 1975, Šorić 1980, Oliva et al.
1988) what made the taxonomy of the bleaks quite
complicated and puzzling. Based on the works
of Bogutskaya (1997) and Bogutskaya & Naseka
(2004), as well as the systematics of Kottelat &
Freyhof (2007), genera Alburnus and Chalcalburnus
were merged into single genus, Alburnus. Recently,
an intensive taxonomic work on the genus Alburnus
has lead to a description of several new species from
the Black and Aegean Sea Basins, mostly belonging
Folia Zool. – 59 (2): 129– 141 (2010)
Introduction
Although bleaks (genus Alburnus) are widely
spread in the waters of the Adriatic watershed, their
taxonomy, systematic and actual distribution are still
not well understood. Until recently, it was considered
that only two widely distributed species inhabit the
European waters (Kottelat 1997): Alburnus albidus
(Costa, 1838) in the Adriatic Basin and Alburnus
alburnus (Linnaeus, 1758) in the other parts of
Europe. However, numerous subspecies, morphs
and varieties of the later species were recognized

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to the Alburnus chalcoides (Gueldenstaedt, 1772)
and A. mento (Heckel, 1836) species complexes
(Freyhof & Kottelat 2007a, b, Özulug & Freyhof
2007a, b). Nowadays, 20 different species of bleaks
are recognized in European freshwater systems
(Kottelat & Freyhof 2007).
The region of eastern Adriatic Coast has been
proved as a hotspot of ichthyological diversity for
several cyprinid genera, like Telestes, Scardinius or
Delminichthys (Kottelat & Freyhof 2007). However,
the taxonomic status of the Alburnus populations in that
area has not been particularly studied yet. Four Alburnus
species (A. albidus, A. arborella Bonaparte, 1841,
A. belvica Karaman, 1924, and A. scoranza Bonaparte,
1845) are reported to inhabit the waters of the Adriatic
Basin in Italy, Switzerland, Slovenia, Croatia, Bosnia
and Herzegovina, Montenegro, Macedonia, Albania
and Greece (Kottelat & Freyhof 2007). Contrary to the
situation with A. alburnus from the central and northern
parts of Europe, the morphological data about many
populations of bleaks in the Adriatic Basin are very
scarce. That is especially true for the populations of
A. arborella, which has the widest distribution range of
all Periadriatic bleaks, occurring from northern Italy to
the Neretva River drainage (Kottelat & Freyhof 2007).
No other cyprinid from the Adriatic Basin – with the
exception of Phoxinus lumaireul (Schinz, 1840) and
Squalius squalus (Bonaparte, 1837), which probably
represent complexes of several species (Kottelat &
Freyhof 2007) has so wide distribution range. The
aim of this work was to morphologically compare the
populations of bleaks from the Adriatic Basin in order
to check for morphological differences between species
and to revise their taxonomic status. In order to ascertain
the taxonomic status of populations from the Neretva
River drainage, mitochondrial DNA analyses were also
conducted, because they have proved to be a useful
tool in identifying cryptic diversity among populations,
especially when phenotypic plasticity is high among
species and diagnostic characters are diffi cult to fi nd.
Material and Methods
The specimens were collected by electrofi shing
from 11 localities belonging to nine river systems
(Fig. 1). The examined material is listed at the end
of this section and in species description. Besides
the populations from the east Adriatic Coast, which
are supposed to belong to the A. arborella (from
the Vipava River in the Soča River drainage, Lake
Butoniga in the Istrian peninsula, the Zrmanja River
in the northern Dalmatia; and Lakes Baćinska and
Kuti as well as the Mušnica River from the Neretva
River drainage), we have also analyzed the specimens
of A. scoranza from the Crni Drim and Zeta Rivers in
the Ohrid-Drin-Skadar drainage and the Mat River,
as well as A. belvica from Lake Prespa. Furthermore,
the specimens from Lake Lugano (type locality of
Alborella maxima Fatio, 1882) were included in
the study. Three types of morphological characters
(meristic, morphometric, and phenotypic) were
examined on specimens fi xed in 5% solution of
formaldehyde and preserved in 70% ethanol. Meristic
characters included the number of unbranched and
branched fi n rays in dorsal, anal, pelvic (ventral),
pectoral and caudal fi n. The last two branched rays in
dorsal and anal fi ns, that are articulating on a single
pterygiophore, are noted as ‘11/2’. The number of
gill rakers was counted on the fi rst gill arch. The
number of scales in the lateral line was counted in the
complete lateral line, from the anterior scale next to
the operculum to the posterior one on the caudal fi n.
A total of 24 morphometric characters was measured
using an electronic caliper to the nearest 0.01 mm:
Fig. 1. The map of the Adriatic Basin. collecting
localities. 1 – Lake Lugano, 2 – Vipava River, 3 – Lake
Butoniga, 4 – Zrmanja River, 5 – Zeta River, 6 – Mat
River, 7 – Crni Drim River, 8 – Lake Prespa. Frame A
shows the Neretva River drainage.
total length (TL), standard length (SL), head length
(c), distance between head tip and anal aperture
(pan), preanal distance (aA), preventral distance
(aV), prepectoral distance (aP), predorsal distance
(aD), caudal peduncle length (lpc), length of dorsal
(lD), anal (lA), caudal (lC), pectoral (lP) and pelvic
(ventral) fi n (lV), distance between pelvic (ventral)
fi ns and anal aperture (Van), head depth (hc),
maximum body depth (H) and the caudal peduncle
depth (or the minimum body height – h), head width

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(laco), maximum body width (lac), distance between
eyes (io), eye diameter (o), preorbital distance (prO),
postorbital distance (poO). The percentage ratios of
morphometric characters in relations to SL, c and
H were analyzed. The morphometric ratios of the
populations from northern Adriatic and the Neretva
River drainage, that were supposed to belong to the
same species, were compared using the analysis of
variance (ANOVA) with the aim to test the statistical
signifi cance of the difference between the means of
each morphometric ratio at a signifi cance level of
α = 0.05. Principal component analysis (PCA) was
employed to determine whether there is a separation
of the individuals from different populations of
A. arborella, based on their morphometric ratios. All
statistical comparisons were made on standardized
body measures, e.g. on body ratios. The Statistica
6.0 software package was used for data analyses. The
holotype as well as the paratypes of newly described
species were included in morphometric analyses. In
addition to meristic and morphometric characters,
overall external morphology was examined.
In order to affi rm the taxonomic status of Alburnus
populations from the Neretva River drainage, mtDNA
analyses were conducted on 10 specimens (two
A. scoranza specimens from the Zeta River; four
specimens of A. arborella from the Zrmanja River;
one specimen from the Mušnica River – holotype of
newly described species; and three specimens from the
Neretva River). Total genomic DNA was extracted from
fresh or deep-frozen muscle tissues using a standard
extraction product (DNeasy tissue kit, Qiagen).
Polymerase chain reaction (PCR) amplifi cations
were performed in 50μl reaction volume containing
25μl of the HotStarTaq Master Mix (Quiagen), 2μl of
each primer and 4μl of template DNA. The complete
mitochondrial cytochrome b (cyt b) gene was amplifi ed
using primers L15267 and H16526 (Brito et al. 1997)
and the following temperature regime: 15 min at
95ºC; 35 cycles of 30 s at 94ºC, 30 s at 50ºC and 90 s
at 72ºC; 7 min at 72ºC. Sequencing was carried out by
Macrogen Service Centre (Seoul, South Korea) with
the internal primers. All sequences have been deposited
in the GenBank under following accession numbers:
GU479865-GU479874.
Pairwise comparisons of uncorrected sequence
divergence (p-distances) in the cyt b gene were
analyzed using Mega version 3.1 (Kumar et al.
2004). Two methods of phylogenetic inference were
employed: maximum parsimony (MP) and maximum
likelihood (ML), as implemented in PAUP (v 4.0b10,
Swofford 2002). For MP analysis, the heuristic search
mode with 100 replicates was used, with randomized
input orders of taxa, and tree bisection-reconnection
(TBR) branch swapping with all codon sites and
nucleotide substitutions types weighted equally. ML
analysis was performed under the heuristic search
option using the TBR branch swapping algorithm.
As the optimal model of sequence evolution, the
Tamura-Nei plus Gamma model (TrN + G) was
selected by hierarchical likelihood ratio (hLRTs) tests
using the Modeltest software (version 3.06, Posada &
Crandall 1998). Nonparametric bootstrapping (1000
pseudoreplicates, 10 addition-sequence replicates)
was used to assess the branch support. Sequence of
Leucaspius delineatus (Briolay 1998) was used as
outgroup and previously published sequences of
A. alburnus (Briolay 1998, Saitoh et al. 2006), A.
macedonicus, A. thessalicus and A. belvica (Zardoya
& Doadrio 1999) were also included in the analyses.
List of examined material
Institutional abbreviations: MNCN – Museo Nacional
de Ciencias Naturales, Madrid; NMP – National Mu-
seum, Prague; PMF – Faculty of Science, Zagreb.
All specimens from the Neretva River basin are listed
in the species description.
Alburnus belvica: MNCN 121113-121128, 16 spec.,
SL 71.2-109.7 mm, Lake Prespa, Greece
Alburnus arborella: PMF ALZR2-21, 20 spec.,
SL 54.5-100.1 mm, Zrmanja River, Croatia; PMF
ALBU4-25, 22 spec., SL 39.9-92.2 mm, Butoniga
reservoir, Mirna River drainage, Istrian peninsula,
Croatia; PMF ALVI1-10, 10 spec., SL 55.9-90.1 mm,
Vipava River, Soča River drainage, Slovenia
Alburnus maxima: NMP uncatalogued, 7 spec.,
SL 116-128.6 mm, Lake Lugano, Italy
Alburnus scoranza: NMP P6p 84/2007, 8 spec.,
SL 63.2-90.0 mm, Crni Drim River in village
Dobovjani, FYROM, N 41°15.219´ E 20°39.196´,
elevation 688 m, 26th July 2006; NMP P6V 81887-
81892, 6 spec., SL 63.8-75.9 mm, Mat River in
village Milot, Albania, N 41°41´16.2´´ E 19°42´11.7´,
25th August 2004; NMP P6V 80608-80610, 3 spec.,
SL 84.5-107.0 mm, Zeta River in village Glava Zete,
Montenegro, N 42°39.904´ E 18°59.830´, 15th July 2003
Results and Discussion
General appearance of the investigated bleaks
is quite similar, while the morphometric, some
meristic, and also some phenotypic features differ
between the species. The variability of all – meristic,
morphometric and phenotypic characters – can also
be noted between the different populations of the

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Table 1. Meristic characters assessed for the investigated Alburnus species. N – the number of specimens
used for assessment of all meristic characters, with the exception of the gill rakers count; n – number of
specimens on which the number of gill rakers was counted.
same species, probably depending on the ecological
factors variation in different localities.
Table 1 summarizes the results of meristic
investigations. The uniformity of the number of rays
in all fi ns, with the exception of anal fi n, as well as
the variability of the number of gill rakers and lateral
line scales among the species, and their importance
for a taxonomic identifi cation of Alburnus species
were already reported (Dimovski & Grupče 1975,
Šorić 1980, Freyhof & Kottelat 2007a, b, Kottelat
& Freyhof 2007). Although the numbers observed
by above mentioned authors are not completely
the same as obtained in this investigation, they fall
generally within the mentioned ranges, with some

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exceptions (populations from Lake Lugano and the
Neretva River drainage).
A. belvica is easily distinguished from all other
investigated species by having higher number of gill
rakers (29-32 vs. max. 26 in the remaining species)
as well as the higher number of lateral line scales
(52-57 vs. max. 54 in the remaining species).
A. scoranza differs from the remaining investigated
species by the following combination of meristic
characters: the number of lateral line scales 46-53,
the number of gill rakers 15-20, and the number of
branched anal fi n rays 131/2-141/2.
A. albidus, not examined in this study, differs from
all other Adriatic bleaks by lower number of anal fi n
rays (11-131/2) (Kottelat & Freyhof 2007) vs. 131/2-
151/2 found in other species examined in this study.
Noteworthy is the differentiation of specimens from
Lake Lugano, which is the type locality for Alborella
maxima (Fatio, 1882), currently treated as synonym
of Alburnus arborella (Kottelat & Freyhof (2007)
mention only A. arborella for northern Italy). The
following combination of meristic characters: the
number of lateral line scales 49-54, the number of
gill rakers 18-24 and the number of branched anal
fi n rays 131/2-151/2 clearly differentiate the population
from Lake Lugano not only from the remaining
investigated populations, but also from all the other
European Alburnus species. The only species with
similar (but not the same) meristic characters is
A. scoranza. However, the ventral keel exposure
(see later) is a character that differentiates between
them. It is also notable that the maximum size of
investigated specimens from Lake Lugano (TL up
to 159 mm) was largest among all the investigated
populations. Only A. scoranza and A. belvica from
large Lakes Skadar, Ohrid and Prespa reach the
similar size, up to 180 mm TL (Dimovski & Grupče
1971, Ivanović 1973). A. arborella (including the
populations from the Neretva River basin) and
A. albidus (Bianco 1980, Kottelat & Freyhof 2007)
seem to attain smaller maximum size.
The populations from the northern Adriatic Basin and
those from the Neretva River drainage differ from
the remaining investigated populations by the lower
number of lateral line scales (40-48). However, we
have found a different range of number of gill rakers
for A. arborella specimens from Butoniga, Zrmanja
and Vipava (14-22) than for those from the Neretva
River basin (17-26).
The coverage of ventral keel by scales also differs
between species. In A. scoranza the whole ventral
keel, from the pelvic fi n base to the anal aperture,
is exposed. In A. arborella from the Butoniga
Lake, Zrmanja and Vipava Rivers, as well as in the
populations from the Neretva River drainage, the
ventral keel is exposed for at least 2/3 of the length
between the pelvic fi n base and the anal aperture,
although usually the anterior part of ventral keel is
very thin and it only peers between the scales, while
its caudal part before the anal aperture is wider.
Contrary to data of Economidis (1986) and Kottelat
& Freyhof (2007), most A. belvica specimens had
the ventral keel completely covered with scales and
only very rarely it was exposed for up to 3 scales in
front of the anus. The specimens from Lake Lugano
had the ventral keel exposed for 1-3 (exceptionally 5)
scales in front of the anal aperture.
The character often used for diversifi cation of
Alburnus species is also the position of the anal fi n.
In the investigated A. arborella populations (from
the Butoniga Lake, Zrmanja and Vipava Rivers) the
beginning of the anal fi n was located below 4-9th
branched dorsal ray, very rarely immediately behind
the last dorsal fi n ray. In the populations from the
Neretva River drainage the fi rst anal ray was located
below 6-10th or behind the last dorsal ray (up to one
scale behind). In the specimens from Lake Lugano as
well as in A. scoranza specimens, the anal fi n origin
was located below 6-9th or immediately behind the
last dorsal ray. In A. belvica the fi rst anal fi n ray was
located usually for 1-11/2 scales behind the base of
the last dorsal ray. However, we also found a small
number of specimens where the anal fi n originated
immediately after the base of the last dorsal fi n ray
or only 1/2 scale behind it. In A. albidus the anal
fi n begins below or behind base of last dorsal ray
(Kottelat & Freyhof 2007). Thus, these results
suggest that this character is highly overlapping
among the Adriatic bleaks.
Besides the number of gill rakers, their length is also
sometimes used as a diagnostic character (Kottelat
& Freyhof 2007). However, since we examined
more than one population of some species, we were
able to conclude that the length of gill rakers varies
between different populations of the same species,
probably depending on the specifi c ecological
factors or a diet of a certain population and cannot
be used for the differentiation of the species. Namely,
in the A. arborella population from the Zrmanja
River, the maximum gill raker length (measured at
the angle between upper and lower gill arch limb)
was for about 30% smaller than the maximum
gill fi laments length (measured at the same point),
while in the populations from Butoniga Lake and

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the Neretva River drainage the maximum gill raker
length was smaller, about 50% of the maximum gill
fi laments length. In the population from Baćinska
Lakes, the largest gill rakers were almost as long
as the largest gill fi laments, while in the specimens
from the Mušnica River they were for about 50%
smaller. However, we have found differences in the
morphology of gill rakers that might be species-
specifi c. In A. arborella populations from the
northern Adriatic basin (Vipava, Butoniga, Zrmanja)
the gill rakers appeared as thin, fl exible projections
with a hook at the top and with a slightly serrated
inner side. In the populations from the Neretva River
drainage, they were more solid and not as thin as in
A. arborella, but they were also slightly serrated on
the inner side. In A. scoranza the gill rakers were
both thin and short, but without serration. The gill
rakers of the Lake Lugano population ended with a
large, pointed buckled hook. Moreover, the gill rakers
in the middle part of a gill arch were often forked, a
character found in a few specimens of A. belvica and
not in any other species.
Table 2 comprises morphometric data for the
investigated species. The morphometric ratios were
mostly similar and with great overlap between
different species. Nevertheless, fi ve morphometric
ratios pan/SL, aA/SL, aV/SL, hco/H and h/lpc were
found signifi cantly different (ANOVA, p < 0.05)
between populations supposed to belong to
A. arborella, but not among populations from the
northern Adriatic basin (Vipava, Butoniga, Zrmanja)
nor from those from the Neretva River basin (Lakes
Kuti and Baćinska, Mušnica). Furthermore, lpc and
lA in relation to SL as well as prO, poO and hc in
relation to c, vary between populations from the
northern Adriatic area and those from the Neretva
River drainage (Table 2), although the differences are
not signifi cant.
Fig. 2 represents the result of the PCA analysis
that included specimens from the populations that
were until now considered as A. arborella (Lake
Lugano; Lake Butoniga, Vipava and Zrmanja
Rivers; Lakes Baćinska and Kuti and Mušnica River
from the Neretva River drainage). The separation
of the specimens from Lake Lugano is obvious.
However, the remaining populations are not so
clearly separated although grouping of the specimens
from Butoniga Lake and the Zrmanja and Vipava
Rivers on one hand, and those from Baćinska and
Kuti Lakes and the Mušnica River on other hand,
is apparent, but with overlapping between these
two groups. It suggests that although A. arborella
and Alburnus from the Neretva River drainage
are quite similar in their morphometric ratios and
there is a certain overlap, they demonstrate some
differences in morphometric features. Furthermore,
there is no separation of the specimens from different
populations belonging to the same geographic area,
which suggests that they are more similar to each
other then to the remaining investigated populations.
General characteristics of external morphology show
great similarities between investigated populations.
All the investigated Alburnus species have elongated
body with a pointed head, forked caudal fi n and
slightly superior mouth. The colouration of all the
investigated species (preserved specimens) is also
quite similar; the dorsal part of the body and head
bears a dark stripe, below it on each side there
is a lighter area, then a blue or grey stripe and the
ventral part is usually whitish to yellow. However,
some differences in colouration exist between the
species. A. scoranza and the populations from the
Neretva River drainage have darker upper half of the
body, while in A. arborella from the Zrmanja and
Vipava Rivers and Butoniga Lake, the dorsal half of
the body is lighter. In live specimens of A. arborella
a greenish colouration can often be seen on top of the
blue stripe, while in the populations from the Neretva
River drainage, there is usually a red tone.
Although morphological differences between bleaks
from the northern Adriatic Basin and those from the
Neretva River drainage are not very pronounced,
phylogenetic analyses corroborated their taxonomic
value and separation of specimens from two
geographically isolated areas in two independent
lineages. The entire cyt b gene (1140 bp) was
obtained from 10 specimens. The sequences showed
59 variable sites, 44 of them being parsimoniously
informative. Uncorrected
divergence (p-distance) between specimens from
the Zrmanja River and those from the Neretva
River drainage ranged between 1.8 and 2.1%, with
a mean value of 1.9%. Among the haplotypes from
the Zrmanja River the p-distance was between 0.1
and 0.3% (mean = 0.2%), while for those from the
Neretva River basin it was 0.4-0.7% (mean = 0.5%).
Both employed methods of phylogenetic inference
resulted in similar topology of the phylogenetic
trees. MP analysis revealed one tree (tree length = 244,
consistency index (CI) = 0.7951, homoplasy index
(HI) = 0.2049, retention index (RI) = 0.7547,
rescaled consistency index (RC) = 0.5992). ML
analysis also resulted in a single tree (-Ln likelihood
= 2725.92080). The only difference between the
pairwise sequence

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Table 2. Morphometric ratios assessed for the investigated Alburnus species. N = the number of specimens
used for morphometric analyses.

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Table 2. continued

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tree obtained by ML analysis and that recovered by
MP analysis is the position of A. belvica lineage,
that, however, has no importance in revealing the
taxonomic status of the Neretva River drainage
populations. Both employed phylogenetic methods
have verifi ed the genetic distinctiveness of the
Alburnus from the Neretva River drainage (Fig. 3).
There is a clear separation of the haplotypes from the
Neretva River drainage and they form an independent
lineage in the phylogenetic tree. This separation and
clustering is very well supported by MP and ML
bootstrap values.
Based on morphological differences as well as
genetic distinctiveness, we conclude that the
bleaks from the Neretva River drainage represent
a separate species. Furthermore, we have found great
morphological differences between the population
from Lake Lugano and all the remaining investigated
populations suggesting that A. maxima should
be considered as a valid species. Nevertheless,
a detailed study is needed to compare the populations
of A. arborella throughout its rage, from central Italy
to the Zrmanja River basin, especially with respect to
the lacustrine populations from northern Italy.
Alburnus neretvae Buj, Šanda et Perea, sp. nova (Fig. 4)
Holotype: NMP P6V 83 139, 101.1 mm SL,
Bosnia and Herzegovina, the Mušnica River by the
village Avtovac in Gatačko polje, N 43°08.290´,
E 18°34.343´, elevation 960 m, 12th July 2006, leg.
Šanda R., Kohout J. & Šedivá A., Fig. 4.
Paratypes: NMP P6V 83 140 and 83 141, 106.7 and
65.8 mm SL, respectively, the same data as for the
holotype; PMF BAC 1-5, 5 spec., 88.0-118.8 mm SL,
Croatia, Lakes Baćinska jezera, 27th June 1997, leg.
Mrakovčić M., Schneider D., Mustafi ć P. & Vajdić S.
Additional material: NMP P6p 84/2007, 10 spec., SL
58.8-108.9 mm, same data as holotype; PMF BAC 6,
8, 10, 14, 15, 17 and 20, 7 spec., SL 71.3-84.0 mm,
same data as paratypes PMF BAC 1-5; PMF KUT
1-14, 14 spec., SL 50.2-88.1 mm, Lake Kuti, Croatia,
27th June 1997, leg. Mrakovčić M., Schneider D.,
Mustafi ć P. & Vajdić S.
Diagnosis: Alburnus neretvae is distinguished from
other species of the genus Alburnus by the following
combination of characters: 131/2-151/2 branched anal
fi n rays; 17-26 gill rakers; 40-48 lateral line scales;
fi rst anal fi n ray below branched dorsal rays 6-10 or
located up to one scale after the last dorsal fi n ray;
Table 2. continued

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ventral keel exposed for at least 2/3 of the distance
between the pelvic fi n base and the anal aperture.
Description: D III 81/2-91/2, A III 131/2-151/2, P 14-17,
V I 7-8, C 17, ll 40-48 scales, 1-3 located on the
caudal fi n. Body is medium sized (62 up to 129 mm
TL; 50 up to 109 mm SL; morphometric characters
are summarized in Table 2), elongated, slightly
to moderately high and moderately compressed.
The maximum body depth is between the pectoral
and ventral fi ns. The head is elongated and slightly
pointed. The head depth is 47-59% of the head length
and 49%-73% of a maximum body depth. The mouth
is quite large and slightly superior. The lower lip
has a small, pointed projection, in the middle. The
lateral line is complete, with the beginning located
in the level of the upper third of the eye, than it is
descending to the end of the ventral fi ns, where it
reaches the lowest point. Afterwards, the lateral line
is slightly rising until the caudal peduncle. Pectoral
fi ns are ending 1/2-5 scales before the beginning of
the pelvic fi ns. Pelvic fi n end is located 1/2-5 scales
before the anal aperture. Ventral keel is exposed for
at least 2/3 of the distance between the pelvic fi n base
and anal aperture; however, usually it is wider just
before the anal aperture (up to 3 scales before). The
fi rst anal fi n ray is most often located below the last
dorsal fi n ray or immediately after it, but its position
varies. It can also be positioned below 6-10th dorsal
fi n ray or up to one scale behind the base of the last
dorsal fi n ray. The maximal size of gill rakers varies
between the populations from reaching the half of the
size of the gill fi laments (in the population from the
Mušnica River) to being almost as long as the gill
fi laments (in the population from Baćinska Lakes),
all measured at the angle between the upper and
lower limb of the fi rst gill arch.
Fig. 2. Plot of the factor scores for factors 2 and 3 of all morphometric ratios for six investigated populations
previously supposed to belong to A. arborella. L – Lake Lugano population; Az – A. arborella population from
the Zrmanja River; Ab – A. arborella population from Lake Butoniga; Av – A. arborella population from the
Vipava River; Nb – A. neretvae, sp. nova from Lakes Baćinska; Nk – A. neretvae, sp. nova from Lake Kuti;
Nm – A. neretvae, sp. nova from the Mušnica River.

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Ventral parts of the body are silvery (white or yellow
in preserved specimens) with few very small dark
spots located below the blue line and on the pectoral
and ventral fi ns.
Comparativere marks: A. neretvae differs from
A. belvica by the number of gill rakers (17-26 in
A. neretvae vs. 29-32 in A. belvica) and the number
of lateral line scales (40-48 in A. neretvae vs. 52-57
in A. belvica). A. neretvae can be distinguished from
A. scoranza by the number of gill rakers (17-26 in A.
neretvae vs. 15-20 in A. scoranza) and by the lateral
line scales number (40-48 in A. neretvae vs. 46-53 in
A. scoranza). A. neretvae is different from A. albidus
by the number of branched anal fi n rays (131/2-151/2
in A. neretvae vs. 11-131/2 in A. albidus, Kottelat &
Freyhof 2007) and the number of gill rakers (17-26
in A. neretvae vs. 13-18 in A. albidus, Kottelat &
Freyhof 2007). A. neretvae differs from A. arborella
by the number of gill rakers (17-26 in A. neretvae
vs. 14-22 in A. arborella) and by the position of the
fi rst anal fi n ray (in A. neretvae located below 6-10th
or up to one scale behind the last dorsal ray, while
in A. arborella it is located below 4-9th branched
dorsal ray, only exceptionally immediately behind
the last dorsal fi n ray). A. neretvae is different from
A. alburnus by the number of branched anal fi n rays
(131/2-151/2 in A. neretvae vs. 17-201/2 in A. alburnus,
Kottelat & Freyhof 2007), the number of lateral line
scales (40-48 in A. neretvae vs. 48-51 in A. alburnus,
Kottelat & Freyhof 2007) and the position of the
anal fi n origin (in A. neretvae located below 6-10th
branched dorsal fi n ray or up to one scale behind the
base of the last dorsal fi n ray, while in A. alburnus
it is located below 4-5th branched dorsal fi n ray,
Kottelat & Freyhof 2007).
Fig. 4. Alburnus neretvae, holotype – NMP P6V 83 139, 101.1 mm SL, the Mušnica River (Bosnia and
Herzegovina).
Fig. 3. Phylogenetic tree derived from the ML analysis
of the investigated Alburnus specimens. Numbers
at nodes represent ML and MP bootstrap values.
Legend: ZET1 and ZET2 – haplotypes of A. scoranza
from the Zeta River; NER1, NER2 and NER3 –
haplotypes of Alburnus specimens from the Neretva
River in Metković; NER4 – haplotype of the holotype
of the newly described species; ZRM1, ZRM2, ZRM3
and ZRM4 – haplotypes of A. arborella from the
Zrmanja River.
Colouration: Dorsal part of the body and head is
dark brown to black, with a brownish to dark reddish
colouration below. Underneath that there is a blue
line (dark blue to dark grey in preserved specimens).

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Distribution: Endemic to the Neretva River drainage
in Croatia and Bosnia and Herzegovina. Recorded in
Lakes Kuti and Baćinska Lakes, the Neretva River
in Metković, the Bregava River, wetland Hutovo
blato, waters of karstic fi elds Rastoke and Jezero
near Vrgorac, in the Mušnica River (Gatačko polje),
the Trebišnjica River (Popovo polje) and Tihaljina/
Trebižat River system (Fig. 5). It occurs also in the
Buna River (Kosorić & Vuković 1966) and in lower
and middle section of the Neretva River, from the
mouth of the River Buna downstream to the mouth
into the sea (Kosorić & Vuković 1966, Kosorić et al.
1989, Mrakovčić et al. 1995). After formation of the
systems of the artifi tial reservoirs on the upper and
middle part of the Neretva River, it was recorded
also in the reservoir Salakovac, aproximately 20 km
upstream of Mostar (Škrijelj 2002).
Etymology: Named after the Neretva River (the
Croatian name), because it is endemic to the Neretva
River drainage.
Acknowledgements
IB, MĆ, ZM and MM thank the Croatian Ministry
of Science, Education and Sport for fi nancial
support (Project nos. 119-1782739-1233 and 119-
0000000-3184) RŠ and JV received support from
the SYNTHESYS projects at Museo Nacional de
Ciencias Naturales (ES-TAF-1187 and 3770),
fi nanced by EC Research Infrastructure Action
under the FP6 “Structuring the European Research
Area” Programme and the Leadership Conservation
Programme (project Survey of endangered fi sh
species of the Morača River system). IB was also
supported by the ‘For women in science’ programme
of the Croatian UNESCO Representation and L’oreal
ADRIA. RŠ was also partially supported by the
project MK00002327201 and internal grant of the
National Museum in Prague.
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