Zoosyst. Evol. 100 (1) 2024, 291-308 | DOI 10.3897/zse.100.115696

yee
BERLIN

Taxonomic revision of Phoxinus minnows (Leuciscidae) from
Caucasus, with description of a new narrow-ranged endemic species

Oleg N. Artaev?, Ilya S. Turbanov!*3, Aleksey A. Bolotovskiy', Aleksandr A. Gandlin!,
Boris A. Levin!

1 Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Yaroslavl Region, 152742, Russia
2 AN. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky prosp., 33, 119071, Moscow, Russia
3 Cherepovets State University, Cherepovets, Vologda Region, 162600, Russia

https://zoobank. org/6F B4D4CA-3C7D-4014-9955-63E23637794C

Corresponding authors: Oleg N. Artaev (artaev@gmail.com); Boris A. Levin (borislyovin@gmail.com)

Academic editor: Nicolas Hubert # Received 13 November 2023 # Accepted 27 February 2024 Published 20 March 2024

Abstract

Taxonomic revision of Phoxinus from the Caucasus revealed two distinct species. One species, P. colchicus, was known from east-
ern drainage of Black Sea, but was recorded also in the middle reach of the Kuban (Sea of Azov basin), for the first time. The Kuban
population represents a genetically unique sub-lineage of P. colchicus. Its ancestors might have colonized the Kuban system through
the event of ancient river capture. Another species inhabits only the Adagum River basin in the lower Kuban and represents a new
narrow-ranged endemic species — Phoxinus adagumicus sp. nov. According to mtDNA phylogeny (COI and cytb), P. adagumicus
Sp. nov. represents deeply divergent and one of the two early branched lineages of the genus Phoxinus being distant to other species
(min. p-distance = 0.074) including geographical neighbors — P. chrysoprasius from Crimean Peninsula and P. colchicus from the
Caucasus. The new species differs from most Phoxinus species by frequently occurring single-row pharyngeal teeth (modal formula
5—4). The narrow geographic range (ca. 55 km in length and 15—20 km in width) and high anthropogenic load on local water systems
suggests the new species is under threat and needs protection.

Key Words

Caucasus, DNA barcoding, endemics, freshwater fish, taxonomy

Introduction in the numbers of newly described species, and taxonom-

ic and geographic range revisions (Palanda¢ic¢ et al. 2015,

Minnows of the genus Phoxinus Rafinesque, 1820 are
small freshwater fish in the family Leuciscidae Bonaparte,
1835, which prefer rheophilic environment and are wide-
spread in Eurasia from northern Spain eastward to the
Anadyr and Amur drainages in Russia and China. Before
the implementation of genetic methods to the taxonomy,
the genus Phoxinus was represented by a few species de-
spite its wide geographic range (Kottelat 1997). Although
detailed morphological studies still contribute to identifi-
cation of new species in Phoxinus (Kottelat 2007; Bianco
and De Bonis 2015), introduction of genetic methods to
Phoxinus taxonomy has resulted in a significant increase

2017, 2020; Vucié et al. 2018; Denys et al. 2020; Turan
et al. 2023). In particular, recent genetic studies have re-
vealed: 1) deep genetic divergences within the genus, and
ii) presence of numerous yet undescribed species. On the
other hand, discovered patterns of genetic diversity do
not always correspond to the morphology-based classifi-
cation (Palandaéic¢ et al. 2017).

The Caucasus is a mountainous transcontinental region
between the Black and Caspian seas. It is considered a sig-
nificant Pleistocene refugium and a hotspot (1.e., the Cau-
casus Biodiversity Hotspot) of endemism for both plants
and animals (Gadzhiev et al. 1979; Kolakovskiy 1980;

Copyright Artaev, O.N. 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.

292 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

Nakhutsrishvili et al. 2015; Mahmoud et al. 2019; Parvizi
et al. 2019; Myers et al. 2000; Gandlin et al. 2022), that
remains insufficiently studied from an evolutionary stand-
point. The freshwater fish fauna of the Caucasus is rich,
highly endemic (up to 68%; Naseka 2010), ecologically
diverse, important for fisheries, and despite permanent
interest is still significantly underexplored (Kuljanishvili
et al. 2020). During the last decade, 15 new species have
been described from the Caucasus Biodiversity Hotspot:
one species from each of the genera Rhodeus Agassiz,
1832 (Esmaeili et al. 2020), Benthophilus Eichwald, 1831
(Kovaci¢ et al. 2021), Cobitis Linnaeus, 1758 (Vasil’eva
et al. 2020), and Gobio Cuvier, 1816 (Turan et al. 2016);
two species from each of the genera Capoeta Valenci-
ennes, 1842 (Levin et al. 2019a; Roman et al. 2022) and
Oxynoemacheilus Banarescu et Nalbant, 1966 (Ci¢ek et
al. 2018; Freyhof et al. 2021); three species in the genus
Salmo Linnaeus, 1758 (Turan et al. 2022); and four spe-
cies in the genus Ponticola Iljin, 1927 (Vasil’eva et al.
2015; Eagderi et al. 2020; Zarei et al. 2022a, b; Epitashvili
et al. 2023). Species status of some other taxa was re-eval-
uated (e.g., Barbus rionicus Kamensky, 1899 — Levin et
al. 2019b; Sevan trout complex Salmo ischchan Kessler,
1877 — Levin et al. 2022; Proterorhinus nasalis (De Filip-
pi, 1963) — Zarei et al. 2022c), or new species for Cauca-
sus were recorded due to increase of knowledge on the
geographic distribution of some species described outside
the region — e.g., Alburnoides samiii Mousavi-Sabet,
Vatandoust & Doadrio, 2015 (Levin et al. 2018).

Minnows of the genus Phoxinus are distributed in the
western part of the Caucasus, Black and Azov seas drain-
age and absent in the eastern part of the Caucasus — in
the Caspian Sea basin (Berg 1949; Naseka 2010). Wide-
ly distributed P. colchicus Berg, 1910 is known from the
Eastern drainage of Black Sea — from the Chorokh Riv-
er system in south (Turkey and Georgia) to small rivers
nearby Novorossiysk in north (Russia) (ca. 550 km along
coastline). The Kuban River basin (Azov Sea drainage) 1s
locally inhabited by the Phoxinus sp., whose taxonomy
remains ambiguous for a long time. The Kuban species
was referred to P. phoxinus (Linnaeus, 1758) (Malyatsky
1930; Berg 1949; Sukhanova and Troitskiy 1949; Reshet-
nikov et al. 2003), but recent molecular studies (Palan-
dacic¢ et al. 2017, 2020) have shown that they form in-
dependent phyletic lineages. In addition, the geographic
range of the Kuban Phoxinus lineage appears to be dis-
continuous (Artaev et al. 2021). Some populations were
found in the system of the Adagum River (50-130 m
a.s.l.), a left tributary of Lower Kuban, while others are
located in a narrow riverine segment of the Belaya River
(280-580 m a.s.l.), a large left tributary of the Kuban Riv-
er. Therefore, a discontinuous geographic range as well as
difference in the altitude of the identified habitats ques-
tion the taxonomic identity of these populations.

Using an integrative morphological and molecular frame-
work, this study aims to assess and revise the taxonomic di-
versity and distribution of Phoxinus spp. from the Caucasus,
with a special focus on the Kuban basin populations.

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Materials and methods
Sampling

Fishes were collected by the authors from different lo-
calities using frame and seine nets (mesh size 6-8 mm)
(Fig. 1; Suppl. material 1). Fishes were euthanized in a
solution of clove oil and photographed in an aquarium
with artificial lighting using a Nikon D5300 camera
(Nikon Corporation, Tokyo, Japan) equipped with a Nik-
kor 60 mm f/2.8G lens (Nikon Corporation, Tokyo, Ja-
pan). Fin clips (pectoral or pelvic) were collected from
some specimens (DNA-vouchers) and fixed in 96% eth-
anol for subsequent DNA extraction in the laboratory.
Most of the collected specimens were preserved in 10%
formalin (form.), whereas some smaller specimens were
preserved in 96% ethanol for molecular analysis. Subse-
quently, formalin-fixed specimens were washed out in
running water and transferred to 70% ethanol for long-
term storage.

The types (holotype, part of paratypes), additional and
comparative material are deposited in the Fish collection
of the Papanin Institute for Biology of Inland Waters of
Russian Academy of Sciences, Borok, Russia (IBIW_
FS); the rest of the paratypes are kept in the Zoological
Institute of the Russian Academy of Sciences, Saint Pe-
tersburg, Russia (ZISP) and the Zoological Museum of
the Moscow University, Moscow, Russia (ZMMU).

Morphological studies

Morphological analyses included Phoxinus spp. materials
from fifteen localities (No. 1—2, 4, 6-8, 10, 12, 14, 16-19
and 21—22 on Fig. 1; Suppl. material 1). Morphological
methods followed Bogutskaya et al. (2019, 2023). In par-
ticular, 28 morphometric measurements (Suppl. material
2), 17 meristic and two qualitative characters (Suppl. ma-
terial 3), were investigated. Morphological abbreviations
of the examined characters are explained in Suppl. mate-
rial 4. Morphometric measurements were taken from the
left side of the body using a digital caliper to the nearest
0.1 mm by one operator for the purposes of consistency, as
recommended by Mina et al. (2005). Meristics (except for
axial skeleton) and type of breast scalation (Bogutskaya et
al. 2019) were assessed using material stained in an etha-
nol solution of alizarin red S (Taylor and Van Dyke 1985
with modifications), followed by short exposure to 1—2%
potassium hydroxide and preservation in 70% ethanol.
Sex was determined by the size of the pectoral fins.
External meristics were counted on the left side. The total
number of the pectoral and pelvic-fin rays was counted
on the left fins. Scales above lateral line were counted
between lateral line and base of first unbranched ray of
dorsal fin; scales below lateral line were counted between
lateral line and base of first unbranched ray of anal fin. In
both cases, lateral line scales were not taken into account.
The number of anterior gill rakers of the first gill arch

Zoosyst. Evol. 100 (1) 2024, 291-308

293

@ P.adagumicus sp. nov.

© P.chrysoprasius

@ Pcolchicus

Figure 1. Map of localities of Phoxinus spp. sampled for this study. Localities are designated in Suppl. material 1.

was counted on the left and right sides of the specimens.
The number of the scale rows was counted on the left
and right breast patches and average value was taken ac-
cording to Bogutskaya et al. (2019). Counts of meristic
characters (except for the axial skeleton) and assessment
of qualitative characters were done using a MC-2-ZOOM
stereomicroscope (Micromed, Saint Petersburg, Rus-
sia). Counts of vertebrae and pterygiophores followed
Naseka (1996) and Bogutskaya et al. (2019) based on
radiographs made by a PRDU (v. II) X-Ray equipment
(ELTECH-Med, St. Petersburg, Russia). Images of pha-
ryngeal teeth were taken using a JEOL JSM-6510LV
scanning electron microscope (Jeol, Tokyo, Japan).

Measurement indexes were statistically processed in
Microsoft Excel. Comparison of multiple samples was
carried out using the Kruskal—Wallis test followed by the
Dunn’s post hoc test with Bonferroni correction [rstatix
(Kassambara 2020) and tidyverse (Wickham et al. 2019)
packages in R version 4.3.1 (Ihaka and Gentleman 1996)].
Differences between sexes were tested using the Mann—
Whitney U test in Past 4.12b (Hammer and Harper 2001).
Principal component analysis (PCA) was performed us-
ing the gefortify (Tang et al. 2016) package in R.

Molecular analyses
Molecular analyses included Phoxinus spp. samples from

thirteen localities (No. 3-11, 15, 17, 20 and 22 on Fig. 1)
(Suppl. material 5).

DNA was isolated by salt-extraction (Aljanabi and
Martinez 1997) from ethanol-fixed tissues. Two mito-
chondrial markers were analyzed. Mitochondrial cyto-
chrome c oxidase subunit I (COI) barcode region was
amplified using M13-tailed primer cocktail: FishF2_ tl:
5'-TGT AAA ACG ACG GCC AGT CGA CTA ATC ATA
AAG ATA TCG GCA C-3', FishR2_ tl: 5'-CAG GAA
ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA
ATC AGA A-3', VF2_t1l: 5'-TGT AAA ACG ACG GCC
AGT CAA CCA ACC ACA AAG ACA TTG GCA C-3'
and FRid_tl: 5'-CAG GAA ACA GCT ATG ACA CCT
CAG GGT GTC CGA ARA AYC ARA A-3' (Ivanova et
al. 2007). PCR conditions for COI followed protocols
from Ivanova et al. (2007). In addition, mitochondri-
al cytochrome b fragment was amplified by PCR using
the following primers: GluF: 5'-AACCACCGTTGTAT-
TCAACTACAA-3' and ThrR: 5'-ACCTCCGATCTTC-
GGATTACAAGACCG-3' (Machordom and Doadrio
2001). PCR amplifications were performed using Evro-
gen ScreenMix-HS under conditions described by Levin
et al. (2017).

Sequencing of the PCR products, purified by ethanol
and ammonium acetate (3 M) precipitation, was conduct-
ed using the Applied Biosystems 3500 DNA sequencer
(Thermo Fisher Scientific, USA) with forward sequenc-
ing primer M13F 5'-GTA AAA CGA CGG CCA GT-3'
and reverse sequencing primer M13R-pUC 5'- CAG
GAA ACA GCT ATG AC-3' (Geiger et al. 2014).

DNA chromatograms were checked for errors in
FinchTV 1.4.0 (Rothganger et al. 2006), and the DNA

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294

sequences were aligned using the ClustalW algorithm in
MEGA7 (Kumar et al. 2016). Phylogenetic analysis was
performed on COI (567 bp) and cytb (1089 bp) concate-
nated sequences. In addition to the 29 newly determined
COI and cytb sequences in this study, 294 concatenated
sequences of all available Phoxinus spp. were mined from
the GenBank (derived from the studies of Imoto et al.
2013; Xu et al. 2014; Palandacéic¢ et al. 2015, 2017, 2020;
Ramler et al. 2016; Schonhuth et al. 2018; and unpublished
works). Three outgroups representing genera Pseudaspius,
Rhynchocypris, and Oreoleuciscus were selected accord-
ing to the previous phylogenetic studies (Palanda€ic et al.
2015, 2020). (Suppl. material 5). Only unique haplotypes
were used in downstream phylogenetic analyses.

The Bayesian phylogenetic analysis was performed in
a Bayesian statistical framework implemented in BEAST
v.1.10.4. (Hill and Baele 2019) with 2’x10 MCMC gen-
erations (10% burn-in) and parameters sampled every
2000 steps. The substitution models by codon position for
Bayesian analysis were selected in PartitionFinder v.2.1.1
(Lanfear et al. 2016) with the greedy algorithm (Lanfear
et al. 2012) (Suppl. material 6).

Maximum likelihood phylogenies were inferred us-
ing IQ-TREE v.2.2.0 (Nguyen et al. 2015) in PhyloSuite
v1.2.3 (Zhang et al. 2020; Xiang et al. 2023) under Edge-
linked partition model for 1000 ultrafast (Minh et al. 2013)
bootstraps. ModelFinder v.2.2.0 (Kalyaanamoorthy et al.

Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

2017) in PhyloSuite v.1.2.3 was used to select the best-fit
partition model (Edge-linked) using AICc criterion (Sup-
pl. material 6).

The average intra-group as well as the average pairwise
intergroup p-distances using concatenated COI+cytb se-
quences data set were calculated using the MEGA7 pro-
gram (Kumar et al. 2016) with 1000 bootstrap replicas.

Results

Phylogenetic placement and genetic distance

Phylogenetic Bayesian tree of the genus Phoxinus shows
that Phoxinus adagumicus sp. nov. has its own cluster
representing one of the earliest branches with a position
between the earliest branch of Phoxinus (P. tumensis and
Phoxinus sp. from Far East) and large clade represented
the other species from Europe (Fig. 2). Although ML-tree
shows some differences in topology, the early branch-
ing of Phoxinus adagumicus sp. nov. compared to the
most of European species is retained (Suppl. material 7).
Phoxinus adagumicus sp. nov. shows the lowest genet-
ic distance to P. cf. morella (Leske, 1774) from basins
of the North and Baltic seas and adjacent upper reaches
of the Danube (p-distance = 0.074+0.006) (Table 1), al-
though it is almost equally close to P. marsilii Heckel,

Table 1. Genetic p-distances between species or groups of Phoxinus spp. for concatenated COI and cytb mtDNA sequences. The av-
erages of interspecies distances are given below diagonal, the standard errors are given above diagonal; the intraspecies divergence

is given in a diagonal in bold.

s 3 aa of >

ap 8 Sx 8S. 83

Sf 2 8 8 Ss as

a: SS tie we

a:

P. adagumMicus Sp. nov. 0.007. 0.007 0.007 0.007 0.006
P. chrysoprasius 0.084 0.001 0.006 0.006 0.006
P. colchicus (Black Sea) 0.089 0.073 0.005 0.002 0.007
P. colchicus (Kuban basin) 0.091 0.074 0.012 0.001 0.007
P. csikii (Clade 5a) 0.086 0.072 0.085 0.086 0.006
P. csikii (Clade 5b) 0.085 0.071 0.084 0.084 0.019
P. karsticus 0.090 0.073 0.091 0.092 0.074
P. krkae 0.089 0.071 0.082 0.085 0.063
P. lumaireul (Clade 1a) 0.081 0.068 0.078 0.078 0.047
P. lumaireul (Clade 1b) 0.082 0.068 0.079 0.080 0.046
P. lumaireul (Clade 1c) 0.078 0.068 0.079 0.078 0.047
P. lumaireul (Clade 1d) 0.083 0.066 0.081 0.081 0.046
P. lumaireul (Clade 1e) 0.079 0.067 0.079 0.079 0.047
P. lumaireul (Clade 1f) 0.082 0.064 0.077 0.077 0.043
P. marsilii 0.075 0.066 0.074 0.076 0.071
P. cf. morella 0.074 0.066 0.072 0.070 0.074
P. phoxinus 0.086 0.077 0.082 0.084 0.078
P. septimaniae 0.086 0.071 0.078 0.078 0.067
P. sp. (Amur basin) 0.090 0.079 0.090 0.086 0.090
P. sp. (Clade 2) 0.078 0.062 0.077 0.078 0.044
P. sp. (Clade 3) 0.081 0.069 0.083 0.083 0.045
P. sp. (Clade 4) 0.080 0.067 0.078 0.080 0.043
P. sp. (Clade 8) 0.089 0.070 0.079 0.079 0.077
P. strandjae 0.077 0.066 0.081 0.081 0.043
P. strymonicus 0.081 0.065 0.078 0.078 0.047
P. tumensis 0.092 0.076 0.089 0.090 0.075

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a a aw av a~ aw aw
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0.048 0.073 0.060 0.007 0.002 0.002 0.004 0.003
0.047 0.073 0.059 0.021 0.013 0.002 0.003 0.003
0.046 0.070 0.058 0.019 0.017 0.004 0.003 0.003
0.045 0.070 0.064 0.028 0.028 0.026 0.004 0.003
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0.045 0.065 0.052 0.041 0.039 0.038 0.041 0.041
0.042 0.069 0.061 0.046 0.046 0.043 0.043 0.043
0.044 0.070 0.056 0.043 0.043 0.041 0.044 0.045
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Zoosyst. Evol. 100 (1) 2024, 291-308

Table 1. Continued.

Pr © =

aS #68 re 3 =

Soe ee 6 4S. ae mnee

Eee msl fo ges

a > ie a: “

a:
P. adagumicus Sp. nov. 0.006 0.005 0.006 0.006 0.006
P. chrysoprasius 0.006 0.006 0.006 0.006 0.006
P. colchicus (Black Sea) 0.006 0.006 0.006 0.006 0.006
P. colchicus (Kuban basin) 0.006 0.006 0.006 0.006 0.006
P. csikii (Clade 5a) 0.005 0.006 0.006 0.006 0.006
P. csikii (Clade 5b) 0.005 0.006 0.006 0.006 0.005
P. karsticus 0.005 0.006 0.006 0.006 0.006
P. krkae 0.005 0.006 0.005 0.006 0.006
P. lumaireul (Clade 1a) 0.003 0.005 0.006 0.006 0.006
P. lumaireul (Clade 1b) 0.003 0.005 0.005 0.006 0.005
P. lumaireul (Clade 1c) 0.003 0.005 0.006 0.006 0.006
P. lumaireul (Clade 1d) 0.003 0.006 0.006 0.006 0.006
P. lumaireul (Clade 1e) 0.003 0.006 0.006 0.006 0.006
P. lumaireul (Clade 1f) 0.000 0.005 0.006 0.006 0.006
P. marsilii 0.067 0.004 0.006 0.006 0.006
P. cf. morella 0.065 0.065 _ 0.006 0.006
P. phoxinus 0.076 0.072 0.069 0.001 0.005
P. septimaniae 0.064 0.066 0.064 0.059 0.003
P. sp. (Amur basin) 0.085 0.078 0.085 0.088 0.085
P. sp. (Clade 2) 0.039 0.066 0.065 0.070 0.068
P. sp. (Clade 3) 0.044 0.068 0.065 0.075 0.070
P. sp. (Clade 4) 0.041 0.068 0.066 0.076 0.069
P. sp. (Clade 8) 0.073 0.073 0.069 0.073 0.069
P. strandjae 0.037 0.064 0.068 0.072 0.067
P. strymonicus 0.037 0.065 0.063 0.073 0.069
P. tumensis 0.077. 0.081 0.079 0.088 0.080

1836 (0.075+0.005) from the Danube basin (Black Sea
drainage) and rivers of Baltic Sea basin. The new species
is not close in genetics to geographically neighbor species
— P. colchicus and P. chrysoprasius (Pallas, 1814) (Ta-
ble 1). At the same time, another population of Phoxinus
sp. from the Kuban basin, inhabiting the middle reach of
Belaya basin, is characterized by unique haplotypes and
has a minimum distance (0.012+0.002) to P. colchicus
from the Black Sea basin (Fig. 2) and currently is con-
sidered a divergent population of P. colchicus (Fig. 2).
Intraspecies divergence of Phoxinus adagumicus sp. nov.
(0.007) is comparable to such in other species (Table 1).

Systematics

Class Actinopterygii Klein, 1885
Order Cypriniformes Bleeker, 1859
Family Leuciscidae Bonaparte, 1835
Genus Phoxinus Rafinesque, 1820

Phoxinus adagumicus sp. nov.
https://zoobank.org/67F76C90-DC80-48CE-974B-B4A 3DEB39263
Figs 3, 4

Phoxinus phoxinus — Berg 1949: 590 (Khabl, Abin, Shibik, Psebeps
rivers and Abrau Lake); Sukhanova and Troitskiy 1949: 154, 164—
165 (Khabl, Adagum, Shibik, Abin and Ayuk rivers); Emtyl et al.
1994: 137-141, figs 1-2 (Ayuk and Dyurso rivers); Reshetnikov et

295

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0.083 0.004 0.004 0.004 0.005 0.004 0.004 0.006
0.087 0.036 0.002 0.004 0.005 0.004 0.005 0.006
0.086 0.033 0.035 0.001 0.006 0.005 0.005 0.006
0.081 0.065 0.067 0.072 0.001 0.005 0.006 0.006
0.085 0.035 0.039 0.042 0.067 — 0.004 0.006
0.086 0.038 0.044 0.041 0.069 0.038 — 0.007
0.073 0.074 0.077 0.075 0.084 0.078 0.079 —

al. 2003: 301-302 (Kuban River); Otrishko and Emtyl 2013a: 20
(Kuban River).

Phoxinus phoxinus kubanicum Emtyl et Ivanenko, 2002 unavailable
name: 90-92, fig. 69 ex Berg 1949 not P. adagumicus sp. nov. (Psek-
ups River basin; Aphips, I, Ubin, Abin, Adagum rivers and Abrau
Lake); Otrishko and Emtyl 2013a: 20 (Kuban River).

Phoxinus phoxinus kubanicus [sic] — Karnaukhov 2020: 76-77 (Ada-
gum and Psebeps rivers).

Phoxinus kubanicus [sic] — Otrishko and Emtyl 2013b: 22 (Adagum
River); Otrishko and Emtyl 2013c: 69-70 (Adagum, Ubin, Aphips
and Ayuk rivers).

Phoxinus colchicus — Kottelat and Freyhof 2007: 226 (southern tribu-
taries of lower Kuban).

Phoxinus sp. — Artaev et al. 2021 (Zybza, Il, Psyzh, Shibik and Neberd-
Jai rivers).

Phoxinus sp. Kuban Clade 19 — Palandaéi¢ et al. 2020: figs 1-2 (Kuban
River); Bogutskaya et al. 2023: 6, Fig. 2 (Adagum River).

Type material. Holotype, IBIW_FS_ 385, female, (57.5
SL mm, Genbank Accession numbers OR713923 - COI,
PP351730 - cytb), Russia, Krasnodar Krai, Kuban Riv-
er drainage, Pryamaya Shchel River (Adagum River
drainage) upstream Nizhnebakanskaya, 44.8538°N,
37.8417°E, 22 May 2023, IS. Turbanov leg. Paratypes:
3 females, 2 males (IBIW_FS_ 386), SL 45.7—51.1 mm,
3 females, 2 males (ZISP 57031), SL 42.2-51.1 mm, 3
females, 2 males (ZMMU P-24612), SL 45.3-51.7 mm,
all from the same locality and date as holotype.
Additional material. Suppl. material 1.

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296 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

aes ———— Pseudaspius hakonensis
Syme] i i i Se | ay | —e Rhynchocypris lagowskii
Sidewess — Oreoleuciscus potanini

P. sp., Amur basin

*/*

P. tumensis, Sea of Japan basin

a P. adagumicus sp. nov., Kuban basin

P. septimaniae, Adriatic Sea basin, North Sea basin,
Mediterranean Sea basin

*/-

P. colchicus, Black Sea basin, Sea of Azov basin
(Kuban)

P. cf. morella, North Sea basin
sl {| P. chrysoprasius, Salgir basin
a <| P. karsticus, Adriatic Sea basin

sp. (Clade 8), Adriatic Sea basin

0
o
s

P. krkae, Adriatic Sea basin

P. csikii (Clade 5a), Danube basin

P. csikii (Clade 5b), Danube basin,
Mediterranean Sea basin

P. sp. (Clade 2), Adriatic Sea basin, Danube basin

P. sp. (Clade 2), Danube basin

"|p sp. (Clade 4), Danube basin

94/0.87

*
;
woo~S a_———

P. sp. (Clade 3), Danube basin

x P. strandjae, Black Sea basin

P. strymonicus, Strymon basin

98/0.97 P. lumaireul (Clade 1d), Danube basin

P. lumaireul (Clade 1f), Danube basin
99/0.84

P. lumaireul (Clade 1e), Danube basin

P. lumaireul (Clades 1a, 1b, 1c), Adriatic Sea basin,

99/" Danube basin

~
x =
*
~~
LL
BR KS\

94/0.93 P. lumaireul (Clade 1b), Danube basin

P. lumaireul (Clade 1b), Danube basin

P. lumaireul (Clade 1b), Adriatic Sea basin,
Danube basin

co
Ww)
—
*
4
~ Oo
~o
Oo
\
+

P. lumaireul (Clade 1b), Danube basin

99/-
P. lumaireul (Clade 1a), Adriatic Sea basin

We

P. lumaireul (Clade 1a), Adriatic Sea basin,
Danube basin

/\

0.03 97/0.99

Figure 2. BI consensus tree of concatenated COI and cytb mtDNA sequences representing all available Phoxinus species in Gen-
bank combined with our data set. Number of some clades is given according to the study of Palanda¢ic¢ et al. (2020). Species from
the Kuban basin are highlighted with color. Bootstrap values/posterior probabilities above 80/0.8 are shown; asterisks represent of
100/1 bootstrap/posterior probabilities values. Scale bar is in expected substitutions per site. The nodes with multiple specimens
were collapsed to a triangle, with the horizontal depth indicating the level of divergence within the node.

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Zoosyst. Evol. 100 (1) 2024, 291-308

297,

B
Figure 3. Holotype of Phoxinus adagumicus sp. nov. IBIW_FS_385): A. General appearance of preserved specimen; B. Radiograph.

Comparative material. Suppl. material 1.

Material used in the genetic analysis. Suppl. material 5.

Etymology. The new species is named after the Adagum
River, left tributary of the lower reach of the Kuban River,
where the species occurs; adagumicus — an adjective.

Diagnosis. Phoxinus adagumicus sp. nov. 1s distin-
guished from geographically close species (P. chrysopra-
sius and P. colchicus) by the presence and predominance
of specimens with single-row pharyngeal teeth on one or
both sides and a combination of characters, none of which
is unique, as follows: head depth at nape 54.1-64.8% HL
(mean 60.5), and head depth through eye 45.3—51.6% HL
(mean 48.0); head length 2.7—3.7 (mean 3.1) times caudal
peduncle depth in females and 2.7—3.2 (mean 2.9) times
in males; body width at dorsal-fin origin 1.4-2 (mean
1.6) times caudal peduncle depth in females and 1.3—1.6
(mean 1.4) times in males; mean number of scale rows
on left and right breast patches 3-9 (mean 6.1); scales
below lateral line 8—14 (mean 11.8); number of circumpe-
duncular scales 37-50 (mean 41.5); 3-6" type of breast
scalation (mode 4" type).

Description. The general appearance of P. adagumic-
us Sp. NOV. is Shown in Figs 3-4. Morphometric measure-
ments for the holotype and type series with level of sig-
nificance of sex-related differences are given in Table 2;
meristic and qualitative characters for specimens from the
type locality are given in Table 3. Primary morphological
data for specimens from the type locality (holotype, para-
types and additional material) are given in Suppl. materi-
al 2; meristic and qualitative characters of P. adagumicus
sp. nov. and other Phoxinus spp. are given in Suppl. mate-
rial 3; morphometric measurements of P. adagumicus sp.

nov., P. chrysoprasius and P. colchicus and their compar-
ison are given in Suppl. material 4.

Morphometrics (Table 2, Suppl. materials 2, 4). The
new species has a medium size — the maximum SL is
59.4 mm (male from the II River). The new species has
elongated head — head depth at nape (% HL) (holotype:
62.2, paratypes: 58.5—63.9, add. material 54.1—64.8); and
head depth through eye (% HL) (holotype: 49.8, para-
types: 46.4—50.0, add. material 45.3-51.6). Head length
3.0 times caudal peduncle depth in the holotype, 2.8—3.2
in the paratypes, and 2.7—3.7 in the add. material.

Meristics (Table 3, Suppl. material 3). Dorsal fin with
3 (sometimes 2, rarely 4) unbranched and 7’ branched
rays. Anal fin with 3 unbranched and 7% (rarely 6/2)
branched rays. Pectoral fin with 14-18 rays, often 16-17.
Pelvic fin with 8 rays (rarely 7 or 9). Caudal fin with 19
rays (sometimes 18, rarely 20). Number of dorsal procur-
rent caudal-fin rays 9-13, often 10-12. Number of ventral
procurrent caudal-fin rays 7-12, often 9-10.

The most common pharyngeal teeth formulae are 5—4
(n= 11), 54.1 (n= 6), 1.54 (n=5) and 1.54.1 (n= 7)
(Fig. 5, Table 4). Total number of vertebrae in the holotype
41, 39-41 in the paratypes, and 39-42 in the add. materi-
al, commonly 40 or 41. Number of abdominal vertebrae
in the holotype 22, 22—23 in the paratypes, and 21—24 in
the add. material, commonly 22 or 23. Number of caudal
vertebrae in the holotype 19, 17—19 in the paratypes, and
16-19 in add. material, commonly 18. Number of pre-
dorsal abdominal vertebrae in the holotype 15, 14-15 in
the paratypes, and 14—16 in the add. material, commonly
15. Number of anal-fin pterygiophores in front of the first
caudal vertebrae in the holotype 3, 3-6 in the paratypes,

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Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

Ae per _
At WHO kik tok Lae
at ot HES
ys "J ae es

ie 5s CHR abe pre ence

a

Figure 4. Live coloration of Phoxinus adagumicus sp. nov. from the Zybza River. Side and ventral views are of the same specimens
(IBIW_FS_339). Fish of spawning or post-spawning coloration were caught on 8" May 2022.

and 3-7 in the add. material, commonly 4 or 5. Difference
in the number of abdominal and caudal vertebrae in the
holotype 3, 3-6 in the paratypes, and 2—7 in the add. ma-
terial, commonly 4 or 5.

Total number of scales in the lateral series 74-94, mean
84.5. Lateral line incomplete and discontinuous. Relative
number of total lateral-line (pored) scales in specimens
of 41-57 mm SL varies greatly from 26 to 92%, mean

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58.5%. Number of scale rows on breast patches 3-6,
commonly 4. Number of circumpeduncular scales 37-44,
mean 41.3. Number of scales above lateral line 16—22,
mean 18.4. Number of scales below lateral line 9-14,
mean 11.8.

Gill rakers (in series from the type locality) on the first
left arch 7—8 (mode 8), on the first right arch 7—9 (modes
7, 8, and 9) (Suppl. material 2).

Zoosyst. Evol. 100 (1) 2024, 291-308

299

Table 2. Morphometric measurements of Phoxinus adagumicus sp. nov. (type series) (primary data see in Suppl. material 2); p* -
difference between females (including the holotype) and males, Mann—Whitney U test: ns (p > 0.05), + (p < 0.05), ++ (p < 0.01).

Characters Holotype (female) Females, n=10 Males, n=6 p*
mean range SD mean range SD

Standard length (SL, in mm) 57.5 48.9 42.2-57.5 3.2 47.1 45.3-51.1 2.5
In percentage of standard length (% SL)
Body depth at dorsal-fin origin 20.0 21.1 19.4-23.4 1.3 20.5 19.1-22.2 1.2 ns
Body width at dorsalfin origin 12.7 13.4 12.6-14.9 0.8 13.5 12.9-14.2 0.5 ns
Minimum depth of caudal peduncle 8.1 8.7 8.0-9.2 0.4 9.5 8.7-10.0 0.6 ns
Caudal peduncle width 8.3 8.6 7.6-10.1 0.7 9.2 8.7-9.7 0.4 ns
Predorsal length 55.2 bys2 55.2-60.5 1.6 55.4 53.9-57.0 1.1 +
Postdorsal length 34.1 33.9 32.9-35.6 0.9 33.0 31.9-34.3 1.0 ns
Prepelvic length 47.3 50.9 47.3-54.8 1.6 49.1 46.6-51.1 1.5 +
Preanal length 65.1 67.9 65.1-70.8 1.3 65.7 63.2-69.1 2.4 ns
Pectoral — pelvic-fin origin length 23.8 25.8 23.8-27.2 1.2 23.7 22.4-24,3 0.7 ++
Pelvic — anal-fin origin length 19.0 19.0 17.8-20.7 0.9 18.0 16.5-19.3 1.2 ns
Caudal peduncle length 23.5 21.7 20.1-23.5 1.0 23.1 22.3-24.7 0.8 +
Dorsal-fin base length 11.8 11.8 11.1-12.4 0.5 12.2 11.1-13.1 0.8 ns
Dorsal-fin depth 20.6 20.8 18.8-22.4 1.2 22.2 21.5-23.8 0.9 +
Anal-fin base length 9.2 10.5 9.2-11.5 0.5 10.8 9.6-11.3 0.6 ns
Anal-fin depth 18.6 19.6 17.7-21.3 1.1 21.0 20.5-21.6 0.4 ++
Pectoral-fin length 18.3 19.0 17.2-21.9 1.5 21.1 20.1-21.6 0.6 +
Pelvic-fin length 14.1 14.4 12.5-15.9 1.1 16.2 15.1-17.2 0.7 ++
Head length (HL) 24.0 26.5 24.0-27.9 0.9 27.4 25.4-28.7 1.3 ns
Head depth at nape 14.9 16.1 14.9-17.1 0.6 16.7 16.2-17.3 0.4 ns
Maximum head width 12.6 13.8 12.6-14.8 0.7 13.9 13.0-14.8 0.7 ns
Snout length 7/1 8.3 7.3-9.4 0.7 8.2 7.4-9.3 0.7 ns
Eye horizontal diameter 6.3 7.2 6.3-7.7 0.3 6.8 6.4-7.1 0.3 +
Interorbital width 8.6 9.1 8.3-10.0 0.5 9.3 8.6-9.9 0.5 ns
In percentage of head length (% HL)
Maximum head width 52.5 52.0 46.6-54.4 2.8 50.7 45.4-54.9 3.1 ns
Snout length 31.9 31.2 28.9-33.6 1.8 30.0 27.6-32.5 1.8 ns
Head depth at nape 62.2 60.8 58.5-63.0 1.7 61.0 58.5-63.9 2.0 ns
Head depth through eye 49.8 48.2 46.4-50.0 1.5 48.4 47.4-49.2 0.7 ns
Eye horizontal diameter 26.3 27.2 26.3-28.5 0.7 24.9 23.8-25.7 0.7 ++
Postorbital distance 51.8 45.6 41.9-51.8 1.8 48.0 46.6-49.2 1.3 ns
Interorbital width 35.8 34.4 31.0-36.7 1.6 34.1 32.1-35.4 1.3 ns
In percentage of caudal peduncle length
Minimum depth of caudal peduncle 34.3 40.1 34.3-43.8 2.9 41.3 38.1-44.9 3:0 ns
In percentage of body depth
Head length 120.0 126.0 116.7-139.9 7.9 133.9 128.2-143.4 5.6 +
In percentage of interorbital width
Eye horizontal diameter 73.4 79.3 71.8-90.1 5.0 73.3 71.0-75.6 1.6 +
Ratios
Interorbital width/eye horizontal diameter 1.4 1.3 1.1-1.4 0.1 1.4 1.3-1.4 0.0 +
Snout length/eye horizontal diameter 1.2 1.1 1.0-1.2 0.1 1.2 1.1-1.3 0.1 ns
Head depth at nape/eye horizontal diameter 2.4 2.2 2.1-2.4 0.1 2.4 2.3-2.6 0.1 ++
Head length/caudal peduncle depth 3.0 3.1 2.8-3.2 0.1 2.9 2.8-3.1 0.1 +
Length of caudal peduncle/caudal peduncle depth 2.9 2.5 2.3-2.9 0.2 2.4 2.2-2.6 0.2 ns
Pectoral fin length/pectoral — pelvic-fin origin distance 0.8 0.7 0.6-0.9 0.1 0.9 0.9-0.9 0.0 ++
Predorsal length/head length 2.3 2.2 2.1-2.3 0.1 2.0 1.9-2.1 0.1 ++
Body width at dorsal-fin origin/caudal peduncle depth 1.6 1.5 1.4-1.7 0.1 1.4 1.3-1.5 0.1 +

Qualitative characters. Pectoral fins do not reach be-
ginning of pelvic fins, except for a few males (ca. 7%
in total). In most specimens (ca. 70%), the tip of upper
lip above the horizontal level of lowest point of the eye,
in some specimens (ca. 25%) at the level, and in 5% of
the specimens below the level. Origin of the anal fin is
mainly behind the vertical of the posterior insertion of the
dorsal fin. (ca. 53%), often at the vertical (39%), rarely
ahead (8%). Free margin of the dorsal fin mainly straight
or slightly convex, anal fin slightly convex or rarely
slightly concave. 3-6" type of breast scalation (mode

A" type).

Coloration. Live coloration of females outside the
Spawning period 1s brown, gray or light golden hues (see
Fig. 4). In males, the color is similar, but with a great-
er dominance of golden or greenish hues. In spawning
coloration of females, golden hue increases signifi-
cantly and the coloration becomes more contrasting,
in general. The spawning coloration of males is also
more contrasting, with dominance of green color with
variations towards yellow-green or black-green. The
operculum stains are blue and the suboperculum is yel-
low in both sexes, but this is much more pronounced
in males. Red hues appear in spawning coloration and

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300 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

Figure 5. Most frequent variants of formula of pharyngeal bones of Phoxinus adagumicus sp. nov.: A. Single-row formula 5—4 (in
33% of specimens); B. Double-row formula 1.5—4.1 (in 21% of specimens). Scale bar: 0.5 mm.

are concentrated at the base of the pelvic, pectoral and
anal fins, as well as around the mouth. The specimens
preserved in formalin had a yellowish color, which is
somewhat darker with a brown tint in the upper parts.

Sexual dimorphism. Significant differences were ob-
served in 18 out of 41 morphometric characters (Table 2).
In addition to some classical sex characteristics in Phoxi-
nus minnows (e.g., narrower pectoral fins and less bright
colors in females), females of the new species generally
have shorter anal and pelvic fins, smaller eye diameter,
and a higher ratio of predorsal length to head length.

Table 3. Meristics and scalation pattern of Phoxinus adagumic-
us sp. nov. from type series and additional material from the
type locality (primary data see in Suppl. material 2). Additional
material from non-type localities is given in Suppl. material 3.

Nos. Characters mean range SD n
1 Total number of scales in lateral series (sql) 84.9 81-90 3.0 7
2 Total number of lateralline (pored) scales (It) 44.2 24-67 17.7 6
s) Number of pored scales in first complete (non- = 29.7 19-57 15.0 7
interrupted) section of lateral line (Ics)
4 Relative number of total lateral line scales, 05 O3- O02 6
quotient IIt:sql (Iltr) 0.8
5 Mean number of scale rows on left and right 64 67 O04 7
breast patches (BrPScale)
6 Number of circumpeduncular scales (cps) 41.1 39-43 15 7
7 Scales above lateral line (ScAboveLL) 17.3. 15-20 16 7
8 Scales below lateral line (ScBelowLL) 11.7. 10-13 1.0 6
9 Pattern of scalation on the breast and anterior 5 4-6 7
belly (cstyp)
10 Total number of pectoral fin rays (P) 16.6 15-18 1.0 7
11 Total number of pelvic fin rays (V) 8.0 8-8 7
12 Number of branched dorsal fin rays (with 1/2)(D) 7.0 7-7 7
13. Number of branched anal fin rays (with 1/2) (A) 7.0 7-7 7
14 Number of rays in caudal fin (C) 18.9 18-20 04 7
15 Total number of vertebrae (tv) 40.4 39-42 0.8 23
16 Number of abdominal vertebrae (abdv) 22.6 21-24 0.7 23
17 Number of caudal vertebrae (caudv) 17.9 17-19 0.7 23
18 Number of predorsal abdominal vertebrae 14.8 14-16 0.5 23
(preDv)

19 Number of anal fin pterygiophores in front of the 4.5 3-6 0.8 23
first caudal vertebrae (preAp)

20 ~ Difference between numbers of abdominal and 4.6 3-7 1.1 23
caudal vertebrae (dac)

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Table 4. Frequency of different pharyngeal teeth formulas in
Phoxinus adagumicus sp. nov., P. colchicus and P. chrysoprasius.

tuauaNtNdandaa
Shedies. OL AS Oa ek ek ae ain
P ee fg tH Ho wo t+ HH

dd dA ANN AN
P.adagumicus 11 5 6 1 0 7 1 21 #0 1 +40 =O 33

Sp. nov.
P.chrysoprasius 0 O08 O 0 0 0 0 0 0 1 #12 2 «15
P. colchicus ORO. 20-250 mel

bt
w
oO
bm
w
ol
hb
pan
ol

Taxonomic remarks. The presence of Phoxinus min-
now in the left lower tributaries of the Kuban has been
documented since the first half of the 20" century (Maly-
atsky 1930; Berg 1949; Sukhanova and Troitsky 1949),
and all previous researchers have attributed this popu-
lation to P. phoxinus. Emtyl and Ivanenko (2002) used
the name ‘Phoxinus phoxinus kubanicum sp. nov.’ for
the minnows from Trans-Kuban rivers (Aphips, II, Ubin,
Abin, and Adagum) and Lake Abrau (Black Sea basin).
Even though the name is accompanied by a comparative
description, it cannot be considered as valid because it
does not comply with the criteria stipulated in art. 16.4
of the International Code of Zoological Nomenclature
(Ride et al. 1999) for species-group names proposed af-
ter 1999, as it is not accompanied by an explicit preser-
vation of a holotype or syntypes for the nominal taxon
(art. 16.4.1.) and a statement of deposit in a collection
(art. 16.4.2.) (Bogutskaya et al. 2023). The names P.
Pphoxinus kubanicum, P. phoxinus kubanicus [sic], and
P. kubanicus [sic], after an attempt to describe this spe-
cies (Emtyl and Ivanenko 2002), were subsequently used
several times (Otrishko and Emtyl 2013a, 2013b, 2013c;
Karnaukhov 2020); however, according to Bogutskaya et
al. (2023), an ICZN commissioner, Nikita Kluge (pers.
comm. ), taxonomists Pyotr Petrov (pers. comm.) and Bo-
ris Kataev (pers. comm.), they cannot be considered as
available names.

It is worth noting that the original description of
‘P. phoxinus kubanicum’ does not correspond to the

Zoosyst. Evol. 100 (1) 2024, 291-308

morphological diagnosis of minnows from the Adagum
basin rivers obtained in this research. For example, the
two-row formula (2.5—4.2) of the pharyngeal teeth is in-
dicated (Emtyl and Ivanenko 2002), but here, none out
of 33 examined specimens had such a formula (Table 4).
Also, it is necessary to emphasize that the given image of
‘P. phoxinus kubanicun’ (fig. 69 in Emtyl and Ivanenko
2002) does not relate to this species but 1s borrowed from
the monograph of L.S. Berg (1949, fig. 447) and refers
to the minnows inhabiting Lovozero (Northern Russia),
which most probably belong to Phoxinus sp. clade 17 of
Palandaéi¢ et al. (2020). Thus, ‘P. phoxinus kubanicum’
should be considered as an unavailable name.

Type locality. Pryamaya Shchel River (44.8538°N,
37.8417°E) upstream of Nizhnebakanskaya, Krasnodar
Krai, Russia. A tributary of the Bakanka River — Ada-
gum River — Kuban River — Sea of Azov.

Distribution and habitats. An endemic species living
in the northwestern Caucasus in the Adagum River basin,
a tributary of the Kuban (Fig. 1). The species has a rath-
er limited range with only 55 km between most distant
known occurrences. The species has been found only in
small streams located in mountainous and foothill areas
— ina zone of a width about 15—20 km along the northern
slope of the western part of the Main Caucasian Range.
Example of habitat for this species (Abin River) is shown
in Fig. 6. Habitat of P adagumicus sp. nov. in other parts
of the Kuban basin and on the Black Sea coast of the Cau-
casus (Lake Abrau) indicated according to literature data
(Berg 1949; Sukhanova and Troitskity 1949; Emtyl et al.
1994; Emtyl and Ivanenko 2002; Karnaukhov 2020) was
not confirmed by our research and may require additional
study (see Discussion).

Morphological comparisons. PCA of morphomet-
ric characters shows that P. adagumicus sp. nov. is more
overlapping with P. chrysoprasius than with P. colchicus
(Fig. 7). The highest loadings for P. adagumicus sp. nov.

a

301

are: head length/caudal peduncle depth, body width at
dorsal-fin origin/caudal peduncle depth and eye horizon-
tal diameter (% interorbital width). Sex differences in all
three species are divergent in the second component.

The occurrence of single-row pharyngeal teeth, fre-
quent in P. adagumicus sp. nov. (Fig. 5, Table 4), is rare
in the genus Phoxinus. In comparison with the geograph-
ically neighboring species, P. colchicus and P. chryso-
prasius, P. adagumicus sp. nov. has unique formulas of
pharyngeal teeth: 5—4, 1.5-4, 5-4.1, 5—5.1 and 1.5—5.1.
According to our data, P. colchicus and P. chrysoprasius
do not show single-row formulae even on one side, while
in P. adagumicus sp. nov. formula 5—4 is found in 1/3 of
all studied specimens. In P. colchicus and P. chrysopra-
sius, formula 2.5—4.2 is modal, but this 1s absent among
individuals of P. adagumicus sp. nov. Single-row pharyn-
geal teeth were indicated as a unique feature for P. apol-
lonicus Bianco et De Bonis, 2015 from the basin of Lake
Skadar in Montenegro (Bianco and De Bonis 2015).

Compared to P. chrysoprasius from the rivers of the
Crimean Peninsula (Bogutskaya et al. 2023; this study),
P. adagumicus sp. nov. has a more elongated head — head
depth at nape (% HL) 54.1-64.8, mean 60.5 (vs. 58.9—
70.7, mean 64.0) in both sexes; larger eyes relative to
head height in females — head depth at nape/eye horizon-
tal diameter 2.1—2.6, mean 2.3 (vs. 2.4-3.4, mean 2.7)
(Suppl. material 4); less anal fin pterygiophores in front
of the first caudal vertebrae (3—7, mean 4.4 vs. 4-8, mean
5.4); less total number of scales in the lateral series (74—
94, mean 84.5 vs. 78-104, mean 90.5); less total number
of scales in the lateral series (pored) (21-81, mean 58.5
vs. 46-84, mean 70.0); less mean number of scale rows
on left and right breast patches (3-9, mean 6.1 vs. 6-11,
mean 8.0); less number of circumpeduncular scales (37—
44, mean 41.3 vs. 41-55, mean 46.2); and less number
of scales below lateral line (8-14, mean 11.8 vs. 11-16,
mean 13.8) (Suppl. material 3).

NUE ae

ae

Figure 6. Abin River (44.7210°N, 38.2051°E; 18 Aug. 2016) — example of biotope of Phoxinus adagumicus sp. nov.

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302 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

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Figure 7. PCA of morphometric characters for Phoxinus spp. under comparison and loading plot showing how strongly each char-
acter influences principal components. Specimens of P. colchicus from the Kuban River basin (Khamyshinka River, a tributary of

Belaya River) are encircled by black color.

Compared to P. colchicus from the Khamyshinka Riv-
er (Belaya River drainage, Kuban River basin), Adygea,
Russia and the Dyurso, Khotetsai, Dzubga and Pshenaho
rivers (Black Sea coast of the Caucasus), Krasnodar Kra1,
Russia (this study), P. adagumicus sp. nov. has lower cau-
dal peduncle — minimum depth of caudal peduncle (% SL)
7.1-10.2, mean 9.1 (vs. 9.6-13.1, mean 11.2) and mini-
mum depth of caudal peduncle (% length of caudal pedun-
cle) 31.3—53.7, mean 40.6 (vs. 42—58.6, mean 49.5); more
elongated head — head depth at nape (% HL) 54.1-64.8,
mean 60.5 (vs. 61.4—74.7, mean 66.6); caudal peduncle
depth in head length 2.7—3.7, mean 3.0 times (vs. 2.1—2.8,
mean 2.4 times) (Suppl. material 4); less mean number of
scale rows on left and right breast patches (3-9, mean 6.1
vs. 5-11, mean 8.3); 3-6" types of scalation pattern of
the breast and anterior belly with predominance of 4" type
(vs. 310", 13" and 14" types with predominance of 6"
type); and less number of scales below lateral line (8-14,
mean 11.8 vs. 11-17, mean 13.4) (Suppl. material 3).

Compared to P. csikii from the Danube River basin,
Montenegro and Bulgaria (Bogutskaya et al. 2019, 2023),
P. adagumicus sp. nov. has a smaller number of anal-fin

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pterygiophores in front of the first caudal vertebrae (3-7,
mean 4.4 vs. 4-8, mean 6.7); and 36" types of scalation
pattern of the breast and anterior belly with predominance
of 4" type (vs. 3-9" and 11" types with predominance of
7" type) (Suppl. material 3).

Compared to P. abanticus Turan, Baycelebi, Ozulug,
Gaygusuz et Aksu, 2023 from the Lake Abant basin in Tur-
key (Turan et al. 2023), P. adagumicus sp. nov. has scales
on the breasts in both sexes (vs. absence of scales on the
breast in males); 15—24 scales above lateral line (vs. 11-14
scales); and 18—20 rays in caudal fin (vs. 15—16 rays).

Compared to P. septimaniae Kottelat, 2007 from the
Herault River, France (Bogutskaya et al. 2019), P. adagu-
micus sp. nov. has more total number of vertebrae (39-43,
mean 40.4 vs. 37-41, mean 39.3); less number of anal-fin
pterygiophores in front of the first caudal vertebrae (3-7,
mean 4.4 vs. 4-7, mean 5.4); and 36" types of scalation
pattern of the breast and anterior belly with predominance
of 4" type (vs. 12-14" types with predominance of 8"
type) (Suppl. material 3).

Compared to P. /umaireul (Schinz, 1840) clades la
and 1b from rivers in the Adriatic and Black Sea basins

Zoosyst. Evol. 100 (1) 2024, 291-308

in Italy, Slovenia, and Croatia (Bogutskaya et al. 2019),
P. adagumicus sp. nov. has a smaller number of anal-fin
pterygiophores in front of the first caudal vertebrae (3-7,
mean 4.4 vs. 3-8, mean 5.5 and 3-6" types of scala-
tion pattern of the breast and anterior belly with predom-
inance of 4" type (vs. 2"—7" types with predominance of
3 type) (Suppl. material 3).

Compared to P. krkae Bogutskaya, Jelic, Vucic, Jelic,
Diripasko, Stefanov et Klobuéar, 2019 from the Krka
River, Croatia (Bogutskaya et al. 2019), P. adagumicus
sp. nov. has more total number of vertebrae (39-43, mean
40.4 vs. 37-40, mean 38.4); more number of abdominal
vertebrae (21—24, mean 22.5 vs. 21-22, mean 21.6); more
number of caudal vertebrae (16-18, mean 18 vs. 15-18,
mean 16.8); more number of predorsal abdominal verte-
brae (14-16, mean 14.8 vs. 13-15, mean 14.0)and 3-6"
types of scalation pattern of the breast and anterior belly
with predominance of 4" type (vs. 3-7" types with pre-
dominance of 5" and 6" types) (Suppl. material 3).

Compared to P. marsilii Heckel, 1836 from the Dan-
ube River basin, Austria and Croatia (Bogutskaya et al.
2019, 2023), P. adagumicus sp. nov. has 36" types
of scalation pattern of the breast and anterior belly with
predominance of 4" type (vs. 3-8" types with predomi-
nance of 6" type) (Suppl. material 3).

Compared to P. strandjae from the rivers of the Black
Sea basin, Bulgaria and the rivers of the Marmara Sea,
Turkey (Bogutskaya et al. 2019, 2023), P. adagumicus sp.
nov. has a smaller number of anal-fin pterygiophores in
front of the first caudal vertebrae (3—7, mean 4.4 vs. 4-8,
mean 5.6); and 3-6" types of scalation pattern of the
breast and anterior belly with predominance of 4" type
(vs. 3-12" types with predominance of 6", 7", 9" and
11" types) (Suppl. material 3).

Discussion

This study clarified the taxonomy, morphology, genet-
ics, and distribution of the Phoxinus minnows inhabiting
the Caucasus including the Kuban basin, a large river-
ine system in the Northern Caucasus that is the richest
in endemic species compared to fish fauna in remaining
European Russia (Abell et al. 2008). Two distinct species,
P. adagumicus sp. nov. and P. colchicus, were identified.
Geographic ranges of P. adagumicus sp. nov. and P. col-
chicus in the Kuban basin are separated from each other
by at least 145 km in a straight line, and also differ in al-
titude that is ranging within ca. 50-130 m above sea level
for P. adagumicus sp. nov. and within ca. 280-580 m for
P. colchicus. Phoxinus adagumicus sp. nov. was found
only in the Adagum basin, while another species, inhabit-
ing the middle reach of the Belaya River, refers to P. col-
chicus — species widely distributed in the rivers draining
the east coast of the Black Sea. Phoxinus adagumicus
Sp. nov. represents deeply divergent lineage that 1s ear-
lier branched than other European species. Therefore,
P. adagumicus sp. nov. may represent a relic lineage of

303

European minnows, the ancestors of which were among
first colonizers of Europe from the East.

The population of P. colchicus in the Belaya River sys-
tem is apparently a result of the past river capture event.
The Kuban population of P. colchicus is the single popula-
tion recorded outside the Black Sea basin. The uniqueness
of haplotypes from the Kuban basin may indicate rather
long isolation of this population or be a result of founder
effect. The upper reaches of the Belaya River system share
watershed with upper reaches of the rivers Shakhe, Sochi
and Achipse belonging to the Black Sea basin. River cap-
tures with naturally translocated fish individuals between
the Kuban and Black Sea tributaries might be a common
phenomenon. For example, the recent discovery of the
Black Sea populations of Barbus tauricus Kessler, 1877 in
some tributaries of the Lower Kuban might be a result of
past colonization through main channel of the Kuban River
or be an event of river captures (Levin et al. 2019b).

Large morphological variation and overlap between
species are a challenge for Phoxinus taxonomy. However,
Phoxinus adagumicus sp. nov. differs from other closely-re-
lated or geographically neighboring species tn pharyngeal
teeth formula, having reduced number of teeth and rows.
The Phoxinus spp. usually have two-rowed pharyngeal
teeth (Berg 1949) while P. adagumicus sp. nov. represents
one-rowed formula as strictly dominating over two-rowed.
Remarkably, one-rowed teeth formula was found in only
one another species — P. apollonicus from the basin of Lake
Skadar in Montenegro (Bianco and De Bonis 2015), which
was suggested to be synonymized with P. karsticus Bianco
et De Bonis, 2015 (Palanda¢ic¢ et al. 2017). A decrease in
the number of teeth (4.2 and 4.1) is also observed in an-
other Western Balkan species — P. ketmaieri Bianco et De
Bonis, 2015, synonymized subsequently with P. /umaireul
(Palanda¢éi¢ et al. 2017). Traditionally, the phylogenetic
value of pharyngeal tooth numbers and their organization
(formula) was high (Chu 1935; Tao et al. 2019) but some
recent studies showed great phenotypic plasticity of this
character from one side (Shkil et al. 2010; Bolotovskiy
and Levin 2011) as well as inheritance of the teeth row
numbers from another side (Shkil’ and Levin 2008). Nev-
ertheless, in our opinion, future studies should pay more
attention to the pharyngeal teeth formula since it is often
neglected in the current taxonomical studies.

The geographic distribution of P. adagumicus sp.
nov. might be wider than its known distribution range in
the Adagum River basin. Emtyl et al. (1994) stated that
minnows from the rivers Dyurso (Black Sea basin) and
Ayuk (tributary of the Psekups River, Kuban basin) are
different from “common minnow” (authors did not use
Latinian name of species using common name instead,
we assume that this is some Phoxinus from the European
part of the USSR) and “Colchis minnow” (which refers to
P. colchicus). The habitat of minnows in the Ayuk basin
was noted earlier (Sukhanova and Troitskiy 1949), where
it was identified as ‘P. phoxinus’. This species was pre-
viously reported for the Dyurso River (Luzhnyak 2003).
Our examination of the Phoxinus material from this river

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304 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

and nearby (Ozereyka River), only confirmed the pres-
ence of P. colchicus. Emtyl and Ivanenko (2002), when
described *P. phoxinus kubanicum’ , indicated Lake Abrau
in the Black Sea basin as the habitat for this species in ad-
dition to the rivers of the Kuban basin. The Phoxinus was
recorded in the lake previously (Malyatsky 1930; Berg
1949), but current studies could not confirm its presence
in both Lake (Luzhnyak 2003) and its tributary, the Abrau
River (our field survey in 2023). What Phoxinus species
lived there is unknown, but given the presence of P. col-
chicus in adjacent rivers, one may suggest that this is the
same species. In addition, Emtyl and Ivanenko (2002)
noted the following locations of the Kuban Phoxinus
outside the Adagum and Psekups basins: the Aphips and
Ubin Rivers. It is worth noting that the Aphips River ba-
sin including its left tributary, the Ubin River, are located
between the Adagum and Psekups basins (Fig. 1). At the
same time, Phoxinus was never recorded in the Aphips
basin by our numerous field surveys during 2015-2023,
or by earlier researchers (Sukhanova and Troitskiy 1949),
which also pointed to a large gap in the distributional
range between the Adagum and Psekups basins. From
all of the above, we can conclude that the entire or the
main range of P. adagumicus sp. nov. is located within the
Kuban River in its sub-basin — the Adagum River system.
The taxonomic status of Phoxinus from the Ayuk River in
the Psekups basin, should be clarified in future research.

We have to consider other peculiarities of the distribu-
tion of P. adagumicus sp. nov. in the Kuban basin in the
light of recent report on its finding (clade 19 by Palan-
dacic¢ et al. 2020) in the Belaya River (tributary of the
Kuban). Clade 19 was also reported by Bogutskaya et al.
(2023) for “Adagum River at Krymsk, Kuban drainage”.
Habitat of the Phoxinus in the Belaya basin is confirmed
by our studies but according to genetic results (Fig. 2) and
morphological analysis (Fig. 7), this population belongs
to P. colchicus. The latter is common in the Belaya River
and its tributaries at least from Khamyshki vil. down to
Tulsky town along ca. 60 km (Artaev et. al 2021). Thus,
we have to conclude that either two species of the genus
Phoxinus coexist in the Belaya basin, or the coordinates
in Palandaéi¢ et al. (2020) are indicated incorrectly. We
are inclined to the second opinion.

Acknowledgements

The authors are very grateful to Andrey Pashkov and
Sergey Reshetnikov for bibliographic assistance; to Max-
im Shapovalov, Maxim Saprykin, Alexey Motorin, Alex-
ey Kutuzov, Dmitry Karabanov, Sergey Arefyev, Marina
Levina, and Ilya Zabaluev for their help in sampling of
material; to Danila Melentev for his help in molecular
laboratory; to a member of the International Commission
on Zoological Nomenclature — Nikita Kluge, and taxon-
omists Pyotr Petrov and Boris Kataev for their valuable
advice on zoological taxonomy and nomenclature. We
are very grateful to Dr. Fatah Zarei and two anonymous
reviewers for their valuable comments on this paper. The

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study was supported partially by the Russian Science
Foundation (grant 23-14-00128).

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307

Supplementary material |

Additional material on Phoxinus adagumicus
sp. nov. and comparative material on
Phoxinus chrysoprasius and P. colchicus

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: docx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppl1

Supplementary material 2

Primary morphological data of Phoxinus
adagumicus sp. nov. from type locality
(Pryamaya Shchel River)

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: xlsx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppl2

Supplementary material 3

Meristic and qualitative characters

of Phoxinus adagumicus sp. nov.,

P. chrysoprasius, P. colchicus and other
Phoxinus species published in the literature

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: xlsx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppl3

zse.pensoft.net

308 Artaev, O.N. et al.: Taxonomic revision of Phoxinus minnows (Leuciscidae) from Caucasus

Supplementary material 4

Morphometrics of Phoxinus adagumicus sp.
nov., P. chrysoprasius, P. colchicus and its
comparison

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: xlsx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacom-
mons.org/licenses/odbl/1.0/). The Open Database
License (ODbL) is a license agreement intended
to allow users to freely share, modify, and use this
Dataset while maintaining this same freedom for oth-
ers, provided that the original source and author(s)
are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppl4

Supplementary material 5
Material for genetic studies

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: xlsx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacom-
mons.org/licenses/odbl/1.0/). The Open Database
License (ODbL) is a license agreement intended
to allow users to freely share, modify, and use this
Dataset while maintaining this same freedom for oth-
ers, provided that the original source and author(s)
are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppI5

zse.pensoft.net

Supplementary material 6

The best partition schemes generated
by ModelFinder v.2.2.0 (ML) and
PartitionFinder v.2.1.1 (BI)

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: docx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zse.100.115696.suppl6

Supplementary material 7

ML phylogenetic tree of concatenated COI
and cytb mtDNA sequences representing all
available species in Genabnk combined with
our data set

Authors: Oleg N. Artaev, Ilya S. Turbanov, Aleksey A.
Bolotovskiy, Aleksandr A. Gandlin, Boris A. Levin

Data type: docx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.115696.suppl7