Zoosyst. Evol. 100 (1) 2024, 101-110 | DOI 10.3897/zse.100.113467

> PENSUFT.

pg tusrum ror
BERLIN

Description of a new species of Phoxinus from the Ergene River
(Aegean Sea Basin) in Tiirkiye (Actinopterygii, Leuciscidae)

Esra Baycelebi', Ismail Aksu!, Davut Turan!

1 Recep Tayyip Erdogan University, Faculty of Fisheries and Aquatic Sciences, 53100 Rize, Turkiye

https://zoobank. org/3 D001 C5D-C528-4CB4-BFE3-D1C08S&0DBB29

Corresponding author: Esra Bay¢elebi (doganeesra@gmail.com)

Academic editor: N. Hubert # Received 29 September 2023 # Accepted 21 December 2023 @ Published 26 January 2024

Abstract

Phoxinus radeki, a new species, is described from the Ergene River (Aegean Sea Basin). It is distinguished from Phoxinus species in
Turkiye and the adjacent area by having the scales of the breast, scaled but separated unscaled area anteriorly, short dark rectangular
blotches along the lateral line between the lateral line and belly yellowish in both males and females, body depth dorsal fin origin
16-21% SL, caudal peduncle depth 8—10% SL. Additionally, molecular results demonstrated that the new species differed from its
closest congeners with a mean genetic distance value of 3.08% (min. 2.82—max. 3.29) and moderately support values in cytochrome
b (Cyt b) gene partial sequences (1064 bp.). Further, the species delimitation analysis identified the new species as a single MOTU

independent of other Phoxinus species.

Key Words

Cyt b, Freshwater fish, minnows, taxonomy

Introduction

The genus Phoxinus Rafinesque, 1820 is widely distributed
in the Palaearctic region, from the Ebro drainage in Spain
eastward to the Anadyr and Amur drainages in Russia and
China (basins of the Atlantic, North and Baltic Seas, the Arc-
tic and the northern Pacific Ocean) (Kottelat 2007; Vuci¢ et
al. 2018; Bogutskaya et al. 2023; Turan et al. 2023). The
fish of the genus Phoxinus belong to the family Leuscis-
cidae and are commonly known as minnows. These small
freshwater fish are also found in various freshwater habitats,
including streams, rivers, lakes, and ponds, in various en-
vironments (Banarescu 1992; Kottelat and Freyhof 2007).
Phoxinus species are typically small fish, with most species
reaching lengths of only a few centimeters. They are omniv-
orous, feeding on various small aquatic invertebrates, algae
and plant matter (Billard 1997). Some species of Phoxinus
are popular among aquarium enthusiasts due to their small
size and attractive coloration Froese and Pauly (2023).

All European minnows were previously identified as
Phoxinus phoxinus (Linnaeus 1758). With the development
of molecular and morphological techniques, numerous

Phoxinus species’ designations as synonyms of P. phoxinus
(such as the list of synonyms published by Kottelat 2007)
started to be questioned (Palanda¢ic¢ et al. 2015, 2017, 2020;
Vucic et al. 2018; Bogutskaya et al. 2019, 2023; Denys et
al. 2020; Turan et al. 2023). The first study on this was by
Kottelat (2007), who described three species from Greece
and southern France. Following are some studies: Four new
Phoxinus species were described by Bianco and De Bonis
(2015) from Italy and the Western Balkans. Molecular evi-
dence points to a multispecies Phoxinus (Cyprinidae) com-
plex in the Western Balkan Peninsula, as reported by Paland-
acic¢ et al. (2015). Moreover, Vucic¢ et al. (2018) investigated
The Western Balkans’ distribution of Eurasian minnow.
Again, Palanda¢éic¢ et al. (2017) used molecular information
in their taxonomic investigations to assign Phoxinus species
to genetic lineages. The latest studies, P krkae Bogutska-
ya et al. (2019), described a new minnow from Croatia and
initially identified it as a molecular clad and Palanda€cic¢ et
al. (2020) researched museum collections to help with the
genetic evaluation of species introductions in freshwater
fishes (Cyprinidae: Phoxinus species complex): European
minnows through time. In 2020, Denys et al. (2020) revised

Copyright Baycelebi, E. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

102

Phoxinus in France and described two new species (Te-
leostei, Leuciscidae) and also created zones of coloration
in Phoxinus. Finally, Turan et al. (2023) described a new
species from Turkiye. Presently, the minnows of the genus
Phoxinus include about 25 species (Eschmeyer et al. 2023;
Froese and Pauly 2023; Turan et al. 2023). The geography
of Turkiye has become the hub for diversifying many spe-
cies and taxa currently recognized. Recent genetic-based
studies on this topic demonstrate Turkiye’s contribution to
the planet’s biodiversity (Geiger et al. 2014; Bektas et al.
2019, 2020). Despite this rich biodiversity, the genus Phoxi-
nus does not have a wide distribution in Turkiye.

The genus is represented by three species in Turkiye:
Phoxinus abanticus, P. colchicus, P. strandjae. Ergene
populations in the Thrace (in Turkiye) were defined as
Phoxinus strandjae in previous studies (Ci¢ek et al. 2023;
Ozulus et al. 2023). These populations have never been
studied in detail and we were able to collect them from
six different places. Here, based on the morphological
characters and a molecular data set, we describe a new
Phoxinus species from the Ergene, a tributary of the
Meric¢ River, Aegean Sea Basin, Turkiye.

Materials and methods

Fish sampling and measurements

Fish were collected by Samus 1000 pulsed DC electro-fish-
ing equipment, at thirty-five sampling sites carried out be-
tween 2006 and 2017. After anaesthesia, specimens were
fixed in 5% formaldehyde and stored in 70% ethanol or di-
rectly fixed in 96% ethanol. Measurements were made with
a dial calliper and recorded to 0.1 mm. All measurements

Bulgaria

Baycelebi, E. et al.: Phoxinus radeki, a new species

were made point to point. Methods for counts and measure-
ments follow Kottelat and Freyhof (2007). Standard length
(SL) was measured from the tip of the upper lip to the end
of the hypural complex. The length of the caudal peduncle
was measured from behind the base of the last anal-fin ray
to the end of the hypural complex, at mid-height of the cau-
dal-fin base. The last two branched rays articulating on a
single pterygiophore in the dorsal and anal fins were count-
ed as “1/4”. Its body measurements were standardized by
individuals’ SL, and the measurements taken from the head
region were standardized by individuals’ head length.

The map in Fig. | was created using the Qgis software,
version 3.22, available at http://diva-gis.org. Occurrence
data in the map (Fig. 1) are based on the authors’ material.
The drawings were made using a Wacom Intuos comic
brand drawing tablet, Adobe illustrator and Adobe Photo-
shop programs (Fig. 7).

Abbreviations used: SL, standard length. HL, Head
length. SD, standard deviation, Collection codes: FFR,
Recep Tayyip Erdogan University Zoology Museum of
the Faculty of Fisheries, Rize.

DNA isolation, amplification and sequencing

Total DNA from ethanol-preserved tissue of Phoxinus
specimens was isolated with the DNeasy Blood & Tissue
Kit (Qiagen, Hilden, Germany) following the manufactur-
er’s instructions. The Cytochrome b (Cyt 5) gene of ver-
tebrate mitochondrial DNA was amplified using the prim-
ers AIbCF (5'-CAACTACAAGAACATGGCAAGCC-3')
and AlbCR (5'-CTTCGGATTACAAGACCGATGC-3’')
described by Bektas et al. 2019. The PCR protocol and
thermocycler conditions were performed according to

© P abanticus
OP strandjae
AP. strymonicus
@ P radeki

Figure 1. Distribution of Phoxinus species in the Aegean and Black Sea basins and Abant Lake.

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Zoosyst. Evol. 100 (1) 2024, 101-110

Turan et al. (2023). PCR products were purified using
the QIAquick PCR Purification Kit (Qiagen, Hilden,
Germany), and both directional sequencing of PCR
products was performed with the same primers used for
amplification at Macrogen Europe using an ABI PRISM
3730x1 Genetic Analyser and a BigDye Terminator 3.1
cycle sequencing ready reaction kit (Applied Biosystem).

Molecular data analyses

The Cyt 5b gene of seven specimens five specimens
of stream Buyukdere and two specimens of stream
Ahmetbey) from the type locality of the newly identified
species was sequenced for molecular comparison. First,
the chromatograms of the raw Cyt b sequences were ex-
amined with the program Bioedit 7.2.5 (Hall 1999) and
the detected errors were manually edited. The sequenc-
es were then matched with reference data stored in this
database using nBLAST (Basic Local Alignment Search
Tool) on GenBank. In order to determine the phylogenetic
position of the new species and to calculate the sequence
difference, the reference Cyt b sequences of the topotype
samples of the congener species were downloaded from
the Genbank. In the absence of sequences of topotype
samples of congeners taxa, correct sequences considered
representative of the species were included (Also see the
“Material used in molecular genetic analysis” section).

All sequences were aligned using the CLUSTAL-W
method (Thompson et al. 1994), trimmed from the ends
and converted to FASTA file. The final data set had 1064
nucleotide positions without insertion and deletion. Also,
the sequences were translated into protein sequence, and
the stop codon was not determined. The interspecific ge-
netic distances were calculated based on the uncorrected
p-distance in MEGA X version (Kumar et al. 2018). Phy-
logenetic relationships between the species were estimat-
ed using maximum likelihood (ML) algorithm in MEGA
X program and the bayesian (BI) algorithm in MrBayes
v3.2.1 program (Ronquist et al. 2012). ML tree was gen-
erated based on the GTR+I+G model such that the best-fit
evolution model was selected by the Akaike information
criterion (AIC) in jModeltest 0.1.1 (Posada 2008). The
ML tree was generated with 1000 bootstrap replicates to
estimate the phylogenetic relationships of the mtDNA lin-
eages. BI tree was generated according to the GTR+I+G
model that the best-fit evolution model was selected by
the Bayesian Information Criterion (BIC) in jModelTest
0.1.1. For BI, analyses were run for 110° generations
with Metropolis coupled Monte Carlo Markov Chains
(MCMC) sampled every 1000 generations. A conserva-
tive 25% of the trees were discarded as burn-in. To root
the all phylogenetic tree, the authors used A/burnoides
fasciatus, Alburnus alburnus, Squalius cephalus and
Rhynchocypris lagowskii (GenBank accession numbers:
MK860065, Bektas et al. 2019; MT394745, Bektas et al.
2020; JQ652365, Dubut et al. 2012; MG806688, Schon-
huth et al. 2018, respectively) as out-groups.

103

The species delimitation analysis was carried out us-
ing ASAP (Assemble Species by Automatic Partitioning;
Puillandre et al. 2012) method based on Cyt 6 data. To
implement the ASAP method, we used the Kimura 2-pa-
rameter (K2P) distances and transition/transversion ratio
(R:7.2) settings at the web address https://bioinfo.mnhn.
fr/abi/public/asap/. The transition/transversion ratio (R)
for the Cyt 5 data was calculated in MEGA X software.

Results

Molecular data analyses

We used molecular methods to test the validity of the new
species, Phoxinus radeki, identified in this study. The Cyt
b gene data of the new species were deposited at NCBI
(OR552423—OR552429) and its first genetic record was
created. In order to determine the phylogenetic position
of the new species, we included the morphologically valid
European Phoxinus species in our dataset and reconstruct-
ed phylogenies with ML and BI methods. Almost similar
topologies emerged in the results of both methods, and
the genus Phoxinus was monophyletic in both. The new
species was moderately supported by both methods (BI:
0.55; ML: 72% Fig. 8). The phylogenetic analysis placed
the new species as the sister species of P. abanticus.

The results based on genetic distance demonstrated
that Phoxinus strandjae was the closest neighbor spe-
cies to the new species with a mean p-distance of 3.08%
(min. 2.82—max. 3.29%), while the most distant species
was P. fayollarum with a mean p-distance of 8.73% (min.
8.46—max. 8.83%).

The species delimitation analysis according to the
ASAP method based on Cyt 6 data identified 15 MOTUs
(molecular operational taxonomic units) for 15 morpho-
logically valid Phoxinus species. This result had the best
ASAP score of 1.0 (p = 0.01) at a threshold distance of
0.017644. The new species, P. radeki, formed a single
MOTU independent of other Phoxinus species.

Phoxinus radeki sp. nov.
https://zoobank. org/1 AECCBFE-FS5AB-4E2D-916B-29582D7A98DA
Figs 2-4

Materials examined. Holotype. FFR 2327, 54 mm SL;
Turkiye: Kirklareli prov.: stream Biiytikdere about 6 km
west of Pinarhisar, 41.6337, 27.5994.

Paratypes. FFR 2301, 12, 45-71 mm SL. —FFR 2304,
20, 42-68 mm SL; —FFR 2314, 19, 38-46 mm SL; same
data as holotype. -FFR 2320, 22, 44-60 mm SL; Turki-
ye: Kirklareli prov.: stream Poyrali about 5 km west of
Pinarhisar, 41.6172, 27.5909. -FFR 2311, 1, 49 mm SL;
Turkiye: Kirklareli prov.: stream Ahmetbey at Sogurcak,
41.6345, 27.6540. —FFR 2326, 26, 44-65 mm SL; Tur-
kiye: Tekirda& prov.: Ergene River at Saray, 41.4257,
OTN I,

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104 Baycelebi, E. et al.: Phoxinus radeki, a new species

Figure 2. Phoxinus radeki FFR 2327: holotype, 54 mm SL, male; Turkiye: Ergene River.

Figure 3. Phoxinus radeki FFR 2326, from top: paratypes, 57-55 mm standard length (SL), possible males; Turkiye: Ergene River.

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Zoosyst. Evol. 100 (1) 2024, 101-110

Genetic material. FFR-DNA-Ph42-43-44-45-46; Tur-
kiye: Kirklareli prov.: stream Btytikdere about 6 km west
of Pinarhisar, 41.6337, 27.5994 (GenBank accession num-
bers: ©OR552425—OR552426—OR552427—OR552428—
ORS52429). —FFR-DNA-Ph31-32; Turkiye: Kurklareli
prov.: stream Ahmetbey at SoSurcak, 41.6345, 27.6540
(GenBank accession numbers: OR552423—OR552424).

Diagnosis. Phoxinus radeki is distinguished from the
Phoxinus species (P. strandjae, P. abanticus) in adjacent
basins as below. It is immediately distinguished from spe-
cies P. strandjae by body color and pattern (not spawning
period and immediately after fixation). Phoxinus radeki
has short dark rectangular blotches along the lateral line.
The area between the lateral line and belly is yellowish in
both males and females. There are irregularly-shaped black
spots on the upper part of the flank and no dark stripes on
the middle part of the flank in males. Phoxinus strandjae
(Fig. 5) has bars reaching from the dorsal to below the later-
al line, hyaline between the lateral line and belly in females,
blackish in males and no black spots on the upper part of
the flank. It further differs from P. strandjae by the scales of
the breast. Phoxinus radeki has breast scaled but separated
unscaled area anteriorly, P. strandjae has breast scaled con-
nected or scales not connected anteriorly (Fig. 7).

Phoxinus radeki is distinguished from species P. aban-
ticus by the presence of scales on the breast in males (vs.
absent, Fig. 7), a slenderer body (body depth dorsal fin or-
igin 16-21% SL vs. 22—25) and a slenderer caudal pedun-
cle (8—10% SL vs. 11-13). It further differs from P. aban-
ticus by having more lateral line scales (75—96 vs.60-69).

Description. The general appearance is shown in
Figs 2-4, and morphometric data are given in Table 1.

105

The maximum size is 71 mm SL. Body depth at dorsal-fin
origin 16%—21% SL.The dorsal body profile more con-
vex than the ventral profile. The head short, its length
24%-27% SL, upper profile straight or slightly convex on
the interorbital area and convex on the snout. The snout
short and its upper profile markedly convex. The mouth
terminal to slightly subterminal, the upper lip not project-
ing or slightly projecting beyond the tip of the lower lip.
The corner of the mouth reaches to level with the lower
margin of the eye or pupil.

Lateral line complete, with 75—96 scales, and almost
reaching to caudal-fin base; 9-15 scale rows between
lateral line and dorsal-fin origin; and 6—9 scale rows
between lateral line and anal-fin origin. Dorsal fin with
three simple 7’2 branched rays, outer margin straight or
slightly convex. Pectoral-fin with 16-18 rays, outer mar-
gin convex. Pelvic fin with seven to eight branched rays,
outer margin convex. Anal fin with three simple 62-714
branched rays, outer margin straight or convex. The cau-
dal fin, deeply forked.

Coloration (Not spawning period and immediately after
fixation): A short dark rectangular blotch along the lateral
line, between the lateral line and belly yellowish in both
males and females, irregularly shaped black spots on the up-
per part of the flank and no dark stipe or very faintly marked
narrow stripe on the middle part of flank in males. Dorsal
and caudal fins slightly gray or hyaline, pectoral, pelvic and
anal fins hyaline (except for some individuals). In some
specimens often 10—14 short brownish bars over-imposed
on the stripe. Black spot in the middle of the caudal-fin base.

Sexual dimorphism. Males with stronger and longer pec-
toral fins, and nuptial tubercles on the head, in both species.

Figure 4. Phoxinus radeki FFR 2311, from top: paratypes, 46 mm SL, male; 55 mm SL female; Turkiye: Stream Ahmetbey.

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106

Baycelebi, E. et al.: Phoxinus radeki, a new species

Figure 5. Phoxinus strandjae FFR 2312, from top: 62 mm SL male; 62 mm SL female; Turkiye: Rezve River.

Etymology. The species is named for Radek Sanda
(Prague) for his contribution to the knowledge of the ich-
thyofaunal of Europe. A noun in genitive, indeclinable.

Distribution. Phoxinus radeki is presently known
from the Ergene River (Ageaen Sea Basin) (Fig. 1). It
inhabits the cold, well-oxygenated waters of fast-flow-
ing mountain streams and large lowland rivers. A trans-
boundary river known as Meri¢-Ergene (Maritsa, Evros)
serves as a border between Turkiye, Greece, and Bulgar-
ia. It starts in Bulgaria and flows through Turkiye, where
it forms a 203 km-long border with Greece. It is the lon-
gest river in the Balkan Region. Before flowing into the
Aegean Sea, the river passes through Greece. Bulgaria
(upstream), Turkiye (downstream), and Greece (down-
stream) are the three riparian nations in the basin. The
remaining 35% of its catchment area is divided into 28%
and 7% within the borders of Turkiye and Greece, respec-
tively, while the remaining 65% of its catchment area is
contained within the boundaries of Bulgaria. The border
between Turkiye and Greece is formed by a piece of the
Meri¢ River’s lower course, while the remaining portion
flows in Turkiye. The river emerges into the Aegean Sea
from Saros. (https://floodmerg.tarimorman. gov.tr/en/
meric-ergene-river-basin). It is known that after the en-
try of the Ergene River into the system, the water quality
of the Meri¢ River decreased seriously and this situation
adversely affected the biota (Elipek et al. 2010; Guher et
al. 2011; Tokatl: 2020).

Discussion

Until 2023, only two Phoxinus species were known in Tir-
kiye which are P. colchicus from Coruh River (southeast

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of the Black Sea basin) and P. strandjae from the coastal
rivers southwest of the Black Sea basin in Thrace region
and Lake Sapanca drainage (Geiger et al. 2014; Bayc¢elebi
et al. 2015; Sag and Ozulu% 2015; Sari et al. 2019). Most
recently, Turan et al. (2023) described P. abanticus from
the Lake Abant drainage. The morphological characters of
Phoxinus radeki that distinguish it from P. abanticus and P.
strandjae species are given in the identification section. In
addition, Phoxinus radeki is distinguished from P. strymon-
icus, which is distributed in the Aegean basin, by having
fewer scale rows between anal-fin origin and lateral line
(7-9 vs.10—13) and a slenderer caudal peduncle (8—10%
SL vs. 10-12 Fig. 6). It further differs from P. strymonicus
by scales of breast. Phoxinus radeki has breast scaled but
separated unscaled area anteriorly, while P. colchicus and
P. strymonicus have breast scaled and scaled area connect-
ed anteriorly.

The COI marker has been frequently used to identify
many vertebrate and invertebrate species (Hebert et al.
2003; Hubert et al. 2008, 2015; Geiger et al. 2014). This
gene region was preferred in Phoxinus species in previ-
ous studies, but it was observed that it was less success-
ful in species delimitation for this genus compared to the
Cyt b gene (Palandaéi¢ et al. 2015, 2017, 2020; Vucié et
al. 2018; Corral-Lou et al. 2019). Especially within the
genus Phoxinus, genetic differences based on COI be-
tween neighboring species appear to be lower compared
to the Cyt b gene. (Palandaéi¢ et al. 2015, 2017, 2020).
Therefore, we performed our analyses based on the Cyt
b gene.

The findings of analyses based on the mitogenome’s
Cyt b gene proved that it diverged from valid Europe-
an Phoxinus species and developed an independent lin-
eage from them, supporting the morphometric findings.

Zoosyst. Evol. 100 (1) 2024, 101-110

107

Figure 7. Breast scale shape of a. Phoxinus radeki,b. P. strandjae and c. P. abanticus.

The genetic characterization of the new species was
carried out in the current investigation, and its first ge-
netic record was created. This could be important for
upcoming research on the genus Phoxinus. With the ini-
tial data of type samples, inaccurate identifications can
be avoided. Previous investigations have evaluated the
effectiveness of the mtDNA Cyt 6 gene in identifying
genetic lineages in the genus Phoxinus, and those inves-
tigations have produced positive outcomes (Palandaci¢
et al. 2015, 2017, 2020; Vucic et al. 2018; Corral-Lou et
al. 2019; Turan et al. 2023). Furthermore, our findings
agreed with those of previous investigations (Palandaci¢

et al. 2015, 2017, 2020; Vucic et al. 2018; Corral-Lou et
al. 2019; Turan et al. 2023).

Comparative material

Materials examined are already listed by Turan et al.
(2023), except the materials listed below:

Phoxinus strymonicus NMP6V88934-88954, — 17,

42-66 mm SL; Bulgaria: EleSnica River, upstream of
Vaksevo Strymon River basin, 42.1364, 22.8521.

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108 Baycelebi, E. et al.: Phoxinus radeki, a new species

OR552423/Turkiye: Kirklarell Buyukdere stream)
OR552429/Turkiye: Kirklareli Buyukdere stream)
Phoxinus radeki (OR552427/Turkiye: Kirklareli Buyukdere stream)
Phoxinus radeki (OR552426/Turkiye: Kirklareli Buyukdere stream)
Phoxinus radeki (OR552425/Turkiye: Kirklareli Buyukdere stream)
Phoxinus radeki (OR552423/Turkiye: Kirklareli Anmetbey stream)
Phoxinus radeki (OR552424/Turkiye: Kirklareli Anmetbey stream)
Phoxinus abanticus (OP313694/Turkiye: Lake Abant)
Phoxinus abanticus (OP313691/Turkiye: Lake Abant)
Phoxinus abanticus (0P313692/Turkiyve: Lake Abant)
Phoxinus abanticus (OP313690/Turkiye: Lake Abant)
Phoxinus abanticus (OP313693/Turkiyve: Lake Abant)
Phoxinus strandjae (MG&06685/Bulgaria: Rezowska River)
Phoxinus strandjae (OP313695/Turkiye: Kirklareli Mutlu stream)
Phoxinus strandjae (OP313696/Turkiye: Kirklareli Mutlu stream)
Phoxinus strymonicus (MG606686/Greece: Strymon Drainage)
Phoxinus lumaireul (KT16677 1/taly: Ticino)
Phoxinus csikii (KT166556/Serbia: Brestovacka River)
Phoxinus septimaniae (MMG606664/France: Tech River)
Phoxinus marsilii (KX673461/Austria: Hutteldorf Vienna River)
Phoxinus fayoillarum (M7T481984/Portugal: Sousa River)
I Phoxinus bigerri (MG681509/France: Adour River Gimat)
a Phoxinus phoxinus (MF408221/Germany: Lohmar Auelsbach creek North Sea)
Phoxinus dragarum (MK/8/814/France: Garonne River)
Phoxinus karsticus (KT166776/Bosnia and Herzegovina: Trebisnjica)
Phoxinus krkae (MG661277/Croatia: Brodic)
Phoxinus morella (MG&306683/Czech Republic: Vazovecky Potok River)
Rhynchocypris lagowshkii (MG&806688)
Alburnoides fasciatus (MK&60065)
6 Alburnus alburnus (M1394745)
62 Squalius cephalus (JQ652365)

Phoxinus radeki
Phoxinus radeki

[|=

100/1

72/0.35

97/0.99

73/0.99

90/0.93
62/0.89 ri
- 10.66

|

0.10
Figure 8. Maximum Likelihood (ML) phylogenetic tree reconstructed based on the Cyt 6 gene. ML and BI methods yielded the same
topologies, and therefore only the ML tree is indicated. The bootstrap values of ML and posterior probability values of BI are indi-
cated on nodes (ML/BI). The bootstrap and posterior probability values are indicated above nodes on tree if 50% and 0.5 or higher.

Table 1. Morphometry of Phoxinus radeki and P. strymonicus species. Mean values are given in parentheses.

P. radeki n=38 P. strymonicus n=19

Aegean Sea Aegean Sea
Ergene River Strymon River

Range SD H Range SD
Standard length (mm) 46-61 54 47-64
In per cent of standard length
Head length 24.4—-26.7 (25.7) 0.07 P55 23.9-28.3 (25.8) 0.12
Body depth at dorsal-fin origin 16.0-21.1 (18.8) 0.11 19.4 18.8-21.6 (20.0) 0.08
Caudal peduncle depth 8.1-10.2 (9.2) 0.05 9.9 9.7-12.0 (10.7) 0.05
Head width, (ant. margin of the eye) 30.4-39.9 (36.2) 0.20 39.6 31.6-49.7 (38.3) 0.04
Head width, (post. margin of the eye) 47.7-55.2 (50.6) 0.16 51.0 38.6-57.6 (51.5) 0.48
Head width, (at opercle) 48.8-58.1 (53.3) 0.26 o7.0 46.2-64.6 (57.2) 0.40
Head depth, at the interorbital region 44.2-53.1 (48.9) 0.21 O31 40.7-60.1 (50.4) 0.46
Head depth, (at occiput) 57.6-69.8 (64.1) 0.28 67.7 56.9-72.0 (64.5) 0.39
Eye diameter 20.6-30.5 (25.2) 0.20 26.7 24.7-31.7 (27.6) 0.19
Snout length 25.1-32.4 (29.0) 0.19 29.7 22.3-31.6 (28.1) 0.27
Interorbital width 23.9-30.1 (27.6) 0.16 28.7 27.1-42.7 (32.6) 0.43
Snout width at nostrils 24.6-35.9 (31.2) O23 29.8 26.4-39.0 (32.1) 0.36
Snout depth at nostrils 25.6-35.6 (30.9) 0.23 35.6 30.1-42.9 (36.2) 0.34
Predorsal length 52.2-57.7 (55.0) 0.11 54.0 52.3-57.0 (54.3) 0.15
Prepelvic length 44.0-50.2 (46.6) 0.16 44.5 41.3-46.8 (44.6) 0.16
Preanal length 61.8-67.1 (64.2) 0.13 62:3 58.2-65.0 (62.0) 0.20
Pectoral-fin origin to anal fin 36.7-44.0 (41.0) 0.19 39.7 37.7-44.6 (41.0) 0.20
Pectoralfin origin to pelvic fin 19.4-27.2 (23.1) 0.18 215 19.7-25.1 (22.3) 0.19
Pelvic-fin origin to anal fin 16.3-20.7 (17.8) 0.09 17.1 16.4-22.1 (18.6) 0.12
Caudal peduncle length 22.1-28.0 (25.3) 0.12 255 22.4-28.6 (26.1) 0.14
Dorsal fin height 17.0-24.3 (20.7) 0.14 21.7 17.9-23.3 (21.3) 0.15
Pectoral-fin length 16.7-22.3 (19.3) 0.15 20-7 15.6-23.4 (18.2) 0.17
Pelvic-fin length 13.1-19.6 (15.3) 0.14 16.6 13.0-17.5 (15.3) 0.13
Analfin length 18.9-23.2 (20.4) OAL 22.1 16.2-21.0 (18.9) 0.13
Upper caudalfin lobe 19.1-24.4 (21.8) 0.12 20.9 21.3-26.1 (23.6) 0.12

zse.pensoft.net

Zoosyst. Evol. 100 (1) 2024, 101-110
Material used in molecular genetic analysis

Phoxinus abanticus: _FFR-DNA-Ph26-27-28-29-30;
Turkiye: Bolu prov., Lake Abant, 40.6647, 31.4250
(GenBank accession numbers: OP313690—
OP313691—OP3 13692—OP313693—OP313694; Topo-
type samples; Turan et al. 2023).

P. strandjae: FFR-DNA-Ph34-36; Turkiye: Kirklareli
prov.: stream Mutlu (Rezve), 41.9422, 27.6202 (Gen-
Bank accession numbers: OP313695—OP313696;
Topotype samples; Turan et al. 2023). — Bulgar-
ia: Rezowska River (GenBank accession number:
MG806685; Topotype sample; Schonhuth et al. 2018).

P. strymonicus: Greece: Strymon Drainage (GenBank
accession number: MG806686; Topotype sample;
Schonhuth et al. 2018).

P. csikii: Serbia: Brestovacka River (GenBank accession
number: KT166556; Palanda¢éi¢ et al. 2015).

P. septimaniae: France: Tech River (GenBank accession
number: MG806684; Schonhuth et al. 2018).

P. lumaireul: Italy: Ticino, Po River (GenBank accession
number: KT166771; Topotype sample; Palandacic¢ et
al. 2015).

P. marsilii: Austria: Hutteldorf Vienna River (GenBank
accession number: KX673481; Topotype sample;
Ramler et al. 2016).

P. bigerri: France: Adour River Gimat (GenBank acces-
sion number: MG681509; Topotype sample; Vucic et
al. 2018).

P. karsticus: Bosnia and Herzegovina: Trebisnjica (Gen-
Bank accession number: KT166776; Topotype sam-
ple; Palandaéic¢ et al. 2015).

P. phoxinus: Germany: Lohmar Auelsbach creek North
Sea (GenBank accession number: MF408221; Palan-
dacic et al. 2017).

P. morella: Czech Republic: Vazovecky Potok River
(GenBank accession number: MG806683; Topotype
sample; Schonhuth et al. 2018).

P. krkae: Croatia: Brodic (GenBank accession number:
MG681277; Topotype sample; Vuci¢ et al. 2018).

P._ fayollarum: Portugal: Sousa River (GenBank accession
number: MT481984; Garcia-Raventos et al. 2020).

P. dragarum: France: Garonne River (MK787814; Topo-
type sample; Corral-Lou et al. 2019).

Acknowledgements

We are pleased to thank Jorg Freyhof (Berlin) and Radek
Sanda (Prague) for the loan of comparative material. We
are also grateful to Hazel Baytasoglu (Rize) for produc-
ing the drawing (Fig. 7).

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