Cytochrome b Sequencing as a Tool for Identification of Morphologically Similar Mediterranean Gobies Gobius incognitus and Gobius bucchichi (Actinopterygii: Gobiidae)

Abstract

Despite being one of the most speciose fish families in the Mediterranean Sea, knowledge about the diversity of gobies (Actinopterygii: Gobiidae) in this sea is still unsatisfactory, as documented by recent descriptions of a number of new species. Although very common in shallow water, Gobius incognitus Kovačić & Šanda, 2016, had escaped attention until 2016, when it was discovered. Due to its overall superficial morphological similarity, G. incognitus used to be confused with a much rarer species, Gobius bucchichi Steindachner, 1870, which was considered one of the most common shallow-water gobies in the Mediterranean Sea. In this work, we tested the suitability of the genetic data (mitochondrial gene encoding cytochrome b) for identifying and distinguishing between these two goby species, and assessed the congruency between the distribution records based on genetic data and those based on morphological identification. We analysed material of 304 specimens of G. incognitus and G. bucchichi from 49 localities covering a considerable part of the Mediterranean Sea, Black Sea, and the Atlantic Ocean near Gibraltar, representing 19 geographically well-separated areas. We detected 270 sequences of G. incognitus, and only 34 of G. bucchichi. In both species, a high haplotype variability was observed. The sequence species identity matched morphological identification for all specimens for which vouchers were available. The mean uncorrected p-distance between G. incognitus and G. bucchichi was 13%, while the mean intraspecific distances were much lower (0.63% and 0.68%, respectively). We found 79 fixed mutations between these two species. Data on distribution based on genetic identification are completely congruent with published results based on morphological identification. The results of this study support molecular methods as a reliable tool for distinguishing morphologically similar fish species, which is particularly useful when only tissue is available for determination.

Full text

Citation: ˇ
Cekovská, K.; Šanda, R.;
Ašenbrenerová, E.; Kassar, A.;
Zogaris, D.; Pappalardo, A.M.;
Tarkan, A.S.; Vasil’eva, E.; Santos, D.;
Vuki´c, J. Cytochrome b Sequencing as a
Tool for Identification of
Morphologically Similar
Mediterranean Gobies Gobius
incognitus and Gobius bucchichi
(Actinopterygii: Gobiidae). J. Mar.
Sci. Eng. 2023,11, 2289. https://
doi.org/10.3390/jmse11122289
Academic Editor:
Jose Pedro Andrade
Received: 30 October 2023
Revised: 22 November 2023
Accepted: 28 November 2023
Published: 2 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Journal of
Marine Science
and Engineering
Article
Cytochrome b Sequencing as a Tool for Identification of
Morphologically Similar Mediterranean Gobies Gobius
incognitus and Gobius bucchichi (Actinopterygii: Gobiidae)
Katarína ˇ
Cekovská1, Radek Šanda 2, Eva Ašenbrenerová2, Abderrahmane Kassar 3, Dimitris Zogaris 4,
Anna Maria Pappalardo 5, Ali Serhan Tarkan 6,7, Ekaterina Vasil’eva 8, David Santos 1,† and Jasna Vuki´c 1,*
1
Department of Ecology, Faculty of Science, Charles University, Viniˇcná7, CZ-128 44 Prague, Czech Republic;
chalupek@natur.cuni.cz (K. ˇ
C.); davidsantos226@gmail.com (D.S.)
2Department of Zoology, National Museum of the Czech Republic, VáclavskéNám. 68,
CZ-115 79 Prague, Czech Republic; radek.sanda@nm.cz (R.Š.); kyralova.e@seznam.cz (E.A.)
3LCVRM, Ecole Nationale Supérieure des Sciences de la Mer et de l’Aménagement du Littoral,
Campus Universitaire de Dély Ibrahim Bois des Cars, B.P. 19, Algiers 16047, Algeria; a.w.kassar@gmail.com
4Department of Ichthyology and Aquatic Environment, University of Thessaly, GR-38446 Volos, Greece;
zogarisd@gmail.com
5Department of Biological, Geological and Environmental Sciences, University of Catania, Via Androne 81,
95124 Catania, Italy; pappalam@unict.it
6Department of Basic Sciences, Faculty of Fisheries, Mu˘gla Sıtkı Koçman University, Mu˘gla 48000, Turkey;
serhantarkan@gmail.com
7Department of Ecology and Vertebrate Zoology, Faculty of Biology and Environmental Protection,
University of Lodz, PL-90136 Lodz, Poland
8Zoological Museum, Moscow State University, Moscow 121069, Russia; vas_katerina@mail.ru
*Correspondence: jasna.vukicova@natur.cuni.cz
†Current address: Marine and Environmental Sciences Centre (MARE), Faculty of Sciences,
University of Lisbon, PT-2750-374 Cascais, Portugal.
Abstract:
Despite being one of the most speciose fish families in the Mediterranean Sea, knowledge
about the diversity of gobies (Actinopterygii: Gobiidae) in this sea is still unsatisfactory, as docu-
mented by recent descriptions of a number of new species. Although very common in shallow water,
Gobius incognitus Kovaˇci´c & Šanda, 2016, had escaped attention until 2016, when it was discovered.
Due to its overall superficial morphological similarity, G. incognitus used to be confused with a much
rarer species, Gobius bucchichi Steindachner, 1870, which was considered one of the most common
shallow-water gobies in the Mediterranean Sea. In this work, we tested the suitability of the genetic
data (mitochondrial gene encoding cytochrome b) for identifying and distinguishing between these
two goby species, and assessed the congruency between the distribution records based on genetic
data and those based on morphological identification. We analysed material of 304 specimens of
G. incognitus and G. bucchichi from 49 localities covering a considerable part of the Mediterranean
Sea, Black Sea, and the Atlantic Ocean near Gibraltar, representing 19 geographically well-separated
areas. We detected 270 sequences of G. incognitus, and only 34 of G. bucchichi. In both species, a
high haplotype variability was observed. The sequence species identity matched morphological
identification for all specimens for which vouchers were available. The mean uncorrected p-distance
between G. incognitus and G. bucchichi was 13%, while the mean intraspecific distances were much
lower (0.63% and 0.68%, respectively). We found 79 fixed mutations between these two species. Data
on distribution based on genetic identification are completely congruent with published results based
on morphological identification. The results of this study support molecular methods as a reliable
tool for distinguishing morphologically similar fish species, which is particularly useful when only
tissue is available for determination.
Keywords: diversity; distribution; gobies; Mediterranean; genetic identification
J. Mar. Sci. Eng. 2023,11, 2289. https://doi.org/10.3390/jmse11122289 https://www.mdpi.com/journal/jmse

J. Mar. Sci. Eng. 2023,11, 2289 2 of 12
1. Introduction
Gobies (Gobiidae) are one of the most speciose fish families worldwide [
1
]. At the
same time, they are one of the most species-rich families of the Mediterranean marine
fishes [
2
]. Despite this fact, gobiid diversity in the Mediterranean Sea is still far from being
completely known. At the family level, the native gobiid fishes from the Mediterranean
Sea are considered either members of a single family, Gobiidae [
1
], or of two sister families,
Gobiidae and Oxudercidae [
3
]. The knowledge about the diversity of the gobies in the
Mediterranean Sea is still unsatisfactory, as documented by a description of a number
of new species in the last years, e.g., [
4
–
9
]. Usually, new species were first discovered
in a small geographic area, but subsequently, they were found in even geographically
considerably distant localities [
10
–
15
], indicating that they are rather widespread, and
that the knowledge about their distribution could be improved by increased research
effort [
16
]. Furthermore, some of the new species were described from greater depths
or are typical cryptobenthic species that are difficult to sample by conventional fishing
methods, but also by SCUBA divers (e.g., by active sampling methods or by a photographic
evidence). Thus, it is more difficult to record them despite their often widespread presence.
However, one of the recently described species was discovered in a very shallow water,
where it is very common and abundant, and its late discovery is only due to its overall
superficial morphological similarity with another, well-known species, which used to
be considered one of the most common shallow-water gobies in the Mediterranean Sea
region [
17
]. This species pair is a long-time known Gobius bucchichi Steindachner, 1870
and a newly described Gobius incognitus Kovaˇci´c & Šanda, 2016. Although cursorily
very similar (Figure 1), both species are in fact readily distinguishable by morphological
character [
18
]; they can even be reliably identified from photographs [
19
]. Both species also
seem to be genetically considerably distinct [
4
,
18
]. Data on the distribution of both species
were recently comprehensively reviewed [
20
]. While the newly described G. incognitus
is widespread throughout the Mediterranean Sea, reaching the Atlantic Ocean in the
Gibraltar straight region [
20
], G. bucchichi was confirmed only in a few scattered areas
in the northern part of the Mediterranean Sea (in the Adriatic, Ionian, and Aegean Seas),
and the Marmara and Black Seas [
20
]. However, the studies differentiating both species
and providing reliable data about the exact distribution of either of these species were
based mostly on morphological evidence: either on morphological examination or on
photographic evidence [
18
,
20
–
27
], while the genetic methods were used only scarcely and
on very limited amounts of material [
4
,
18
,
28
]. Even though these two gobies have no
economic value, their biogeography is of particular interest, especially in the case of G.
bucchichi, which seems to have a unique distribution pattern among the Mediterranean
gobies [
20
]. Moreover, the conservation status of both species needs an urgent update. In
the international IUCN Red List of threatened species, only G. bucchichi is included, and is
evaluated as Least Concern [
29
], while G. incognitus has not yet been evaluated. However,
the conservation status of G. bucchichi [
29
] is based on data that include both species, and
thus is no longer valid.
Molecular methods were used in numerous studies on gobies from the Mediterranean
Sea and in general from Europe. Most of the studies focused on taxonomy or distribution,
e.g., [
4
–
9
,
11
,
12
,
14
,
15
,
18
,
28
,
30
–
32
]. Several studies deal with phylogenetic relationships,
e.g., [
33
–
36
], and also the population genetic structure and phylogeography were investi-
gated for several species, e.g., [37–43].
The aims of this work were to test the suitability of the genetic data (mitochondrial
marker cytochrome b) for the identification and distinguishing between the goby species
G. bucchichi and G. incognitus, and to assess the congruency between the distribution records
based on genetic data and those based on morphological identification, which are, for a
large part, based on citizen science [
20
]. Genetics has been proven to be a useful tool for the
identification of taxonomically complicated gobiid species complexes in the Mediterranean
region [
30
]. Moreover, if proved to be reliable, this type of identification can be applied also

J. Mar. Sci. Eng. 2023,11, 2289 3 of 12
in the case when voucher material, apart from a tissue, is absent, or on specimens that are
too damaged to be used for unambiguous morphological identification.
J. Mar. Sci. Eng. 2023,11, x FOR PEER REVIEW 4of 14
can be applied also in the case when voucher material, apart from a tissue, is absent, or
on specimens that are too damaged to be used for unambiguous morphological
identification.
Figure 1. Gobius incognitus from Hvar Island, Sveta Nedjelja, Croatia (loc. 23 in Table 1) (upper)
and Gobius bucchichi from Selce, Croatia (loc. 19 in Table 1) (lower) photographed in an aquarium.
Both specimens are deposited in the collection of the National Museum of the Czech Republic in
Prague and were morphologically determined. Photo: R. Šanda.
2. Materials and Methods
Tissue samples for molecular analyses (fin clips) were preserved in 96% ethanol.
Genomic DNA was extracted with the Geneaid Genomic DNA Mini Kit (Tissue)
(Geneaid Biotech, Taiwan). Mitochondrial gene cytochrome b (cyt b) was used for the
identification, considering the relatively high divergence between both species on this
marker observed by Kovačić & Šanda [18]. Cyt b was confirmed to be a useful marker for
species delimitation in gobies, e.g., [9,18,32], reconstruction of phylogenetic relationships
between species e.g., [31,34–36], as well as for studying phylogeography and population
genetics e.g., [37,38,40,42,43]. Cyt b was amplified by polymerase chain reaction (PCR)
using PPP Master Mix (Top-Bio, Czech Republic) following primers and protocols
described in Šanda et al. [44]. Sequencing was performed by Macrogen Inc using newly
designed primers (GbuchR1: 5’-TGGGGGAAAAGAGGGCAAGG-3’; GbuchF1:
5’-GGCTTCTCCGTTGACAATGC-3’). These primers were designed based on previously
published cyt b sequences of both species [18,34] using Geneious Prime 2021.1
(https://www.geneious.com, accessed on 20 January 2022).
In total, 304 sequences of G. bucchichi and G. incognitus originating from 49 localities
were analysed (Table 1, Figure 2). Thirty-nine localities covered only new material, seven
localities were based on published data supplemented by newly analysed specimens,
while three localities were based solely on the published data (Table 1). The newly
analysed material was morphologically identified following [2,18] if the voucher
specimens were available (Table 1).
All sequences were visually checked and manually edited using Chromas v.2.6.4,
and aligned in Bioedit v.7.2.6.1 [45]. New sequences were collapsed to haplotypes in
Fabox [46], and the haplotypes were deposited in GenBank under the accession numbers
OR834513–OR834734 . The dataset included three sequences, which were considerably
shorter than most of the sequences due to problematic sequencing. These sequences
were used only for the identification, which was unambiguous in each case, while for the
Figure 1.
Gobius incognitus from Hvar Island, Sveta Nedjelja, Croatia (loc. 23 in Table 1) (upper) and
Gobius bucchichi from Selce, Croatia (loc. 19 in Table 1) (lower) photographed in an aquarium. Both
specimens are deposited in the collection of the National Museum of the Czech Republic in Prague
and were morphologically determined. Photo: R. Šanda.
2. Materials and Methods
Tissue samples for molecular analyses (fin clips) were preserved in 96% ethanol.
Genomic DNA was extracted with the Geneaid Genomic DNA Mini Kit (Tissue) (Geneaid
Biotech, New Taipei City, Taiwan). Mitochondrial gene cytochrome b (cyt b) was used
for the identification, considering the relatively high divergence between both species
on this marker observed by Kovaˇci´c & Šanda [
18
]. Cyt b was confirmed to be a useful
marker for species delimitation in gobies, e.g., [
9
,
18
,
32
], reconstruction of phylogenetic
relationships between species e.g., [
31
,
34
–
36
], as well as for studying phylogeography
and population genetics e.g., [
37
,
38
,
40
,
42
,
43
]. Cyt b was amplified by polymerase chain
reaction (PCR) using PPP Master Mix (Top-Bio, Praha, Czech Republic) following primers
and protocols described in Šanda et al. [
44
]. Sequencing was performed by Macrogen
Inc using newly designed primers (GbuchR1: 5’-TGGGGGAAAAGAGGGCAAGG-3’;
GbuchF1: 5’-GGCTTCTCCGTTGACAATGC-3’). These primers were designed based on
previously published cyt b sequences of both species [
18
,
34
] using Geneious Prime 2021.1
(https://www.geneious.com, accessed on 20 January 2022).
In total, 304 sequences of G. bucchichi and G. incognitus originating from 49 localities
were analysed (Table 1, Figure 2). Thirty-nine localities covered only new material, seven
localities were based on published data supplemented by newly analysed specimens, while
three localities were based solely on the published data (Table 1). The newly analysed
material was morphologically identified following [
2
,
18
] if the voucher specimens were
available (Table 1).
All sequences were visually checked and manually edited using Chromas v.2.6.4,
and aligned in Bioedit v.7.2.6.1 [
45
]. New sequences were collapsed to haplotypes in
Fabox [
46
], and the haplotypes were deposited in GenBank under the accession numbers
OR834513–OR834734. The dataset included three sequences, which were considerably
shorter than most of the sequences due to problematic sequencing. These sequences were
used only for the identification, which was unambiguous in each case, while for the anal-
yses only the long sequences were used. The dataset was further extended by available

J. Mar. Sci. Eng. 2023,11, 2289 4 of 12
published sequences of cyt b of both species (FJ526784 [
34
] and KR811029-KR811059 [
18
])
(Table 1). Sequences of both species published by Kovaˇci´c & Šanda [
18
] were taken as a refer-
ence, because they include sequences of type material of G. incognitus and morphologically
confirmed specimens of G. bucchichi based on redescription of this species.
Table 1.
Overview of the analysed material of G. incognitus and G. bucchichi. For each included
locality, geographic coordinates and sea subarea are provided. Sea subareas names follow Kovaˇci´c
et al. [
20
]. Locality numbers correspond to the numbers of localities in Figure 2. Voucher specimens
were available for all localities, where new material was included, except for the locality marked with
* (loc. 43). NE—north-eastern, W Med—western Mediterranean. Published sequences originated
from A[18] and B[34].
Detected Species
on Locality Sea Subarea Locality Country Coordinates No. in Map New
Sequences Published
Sequences
Gobius incognitus NE Atlantic Gale Portugal 37.076972, −8.310722 1 7
Gobius incognitus NE Atlantic Sesmarias, Praia de
Evaristo Portugal 37.074006, −8.303575 2 6
Gobius incognitus African W Med Bou Ismail Algeria 36.650767, 2.691153 3 4
Gobius incognitus African W Med Tipaza Algeria 36.594200, 2.449131 4 5
Gobius incognitus African W Med Algiers, Sidi Fredj Algeria 36.764291, 2.848401 5 17
Gobius incognitus African W Med Algiers, Ain Benian Algeria 36.802050, 2.898339 6 19
Gobius incognitus Spanish W Med Benidorm Spain 38.533982, −0.129395 7 4
Gobius incognitus Gulf of Lion Banyuls-sur-Mer France 42.481817, 3.136032 8 10 6A
Gobius incognitus Gulf of Lion Banyuls-sur-Mer,
Paulliles beach France 42.505759, 3.123662 9 7 3A
Gobius incognitus Gulf of Lion Port Vendres France 42.523300, 3.109700 10 1
Gobius incognitus Corsican shelf Corsica, Lumio France 42.574444, 8.805278 11 25
Gobius incognitus Maltese shelf Gozo Island, Ramla Malta 36.061869, 14.284042 12 2
Gobius incognitus Ionian Sea Sicily, Aci Castello Italy 37.555419, 15.149383 13 9
Gobius incognitus Ionian Sea Sicily, Aci Trezza Italy 37.562758, 15.163661 14 9
Gobius incognitus Ionian Sea Sicily, Capo Mulini Italy 37.574014, 15.173408 15 8
Gobius bucchichi Adriatic Sea Kraljevica Croatia 45.267660, 14.562256 16 1 1A
Gobius bucchichi Adriatic Sea Krk Island, Omišalj Croatia 45.218911, 14.550908 17 2A
Gobius bucchichi Adriatic Sea Krk Island, Glavotok Croatia 45.094134, 14.436694 18 1B
Gobius bucchichi Adriatic Sea Selce Croatia 45.152293, 14.718928 19 6 1A
Gobius incognitus Adriatic Sea Krk Island, Sveti Marak Croatia 45.105944, 14.668500 20 2
Gobius bucchichi +
Gobius incognitus Adriatic Sea Braˇc Island, Sumartin Croatia 43.285934, 16.878829 21 10 (5xG. inc.,
5xG. buc.)6A(G. inc.)
Gobius incognitus Adriatic Sea Hvar Island, Smoˇciguzica Croatia 43.233889, 16.574722 22 1
Gobius incognitus Adriatic Sea Hvar Island,
Sveta Nedjelja Croatia 43.134433, 16.591381 23 14
Gobius bucchichi Adriatic Sea Boka Kotorska, Kostanjica Montenegro 42.485403, 18.669678 24 6
Gobius bucchichi Adriatic Sea Boka Kotorska, Strp Montenegro 42.503989, 18.669453 25 4
Gobius incognitus Adriatic Sea Budva, Jaz beach Montenegro 42.283836, 18.809034 26 1 2A
Gobius incognitus Adriatic Sea Kamenovo Montenegro 42.273917, 18.887667 27 5
Gobius incognitus Adriatic Sea Sveti Stefan Montenegro 42.257180, 18.892118 28 5A
Gobius bucchichi Ionian Sea outflow of Butrint lagoon Albania 39.743614, 20.018654 29 1 5A
Gobius incognitus Ionian Sea Sivota Greece 39.399800, 20.234664 30 2
Gobius incognitus Ionian Sea Romanos, Peloponnesos Greece 36.985361, 21.651634 31 1
Gobius incognitus Ionian Sea Petalidi, Peloponnesos Greece 36.959142, 21.934967 32 1
Gobius incognitus Aegean Sea Euboia Island,
Petalii Islands Greece 38.014764, 24.280875 33 3
Gobius incognitus Aegean Sea Euboia Island, Marmari Greece 38.048808, 24.318328 34 4
Gobius incognitus Aegean Sea Euboia Island,
Kalamitsi beach Greece 37.971372, 24.365764 35 2
Gobius incognitus Aegean Sea Euboia Island, Mnima
penninsula Greece 37.978775, 24.399581 36 4
Gobius incognitus Aegean Sea Euboia Island,
Agia Pelagia Islet Greece 37.997008, 24.398186 37 1
Gobius incognitus Aegean Sea Euboia Island, Karystos Greece 38.009667, 24.430804 38 3
Gobius incognitus Aegean Sea Euboia Island, Kastri
Beach Greece 37.973022, 24.537658 39 1
Gobius incognitus Aegean Sea Euboia Island, Mirmigia
rocks Greece 37.972336, 24.548042 40 1
Gobius incognitus Aegean Sea Raches Greece 38.876144, 22.785700 41 3
Gobius incognitus Aegean Sea Gavradia Greece 40.440278, 23.839722 42 18
Gobius incognitus Aegean Sea between Geyikli and
Dalyan * Turkey 39.789722, 26.156111 43 22
Gobius incognitus Levantine Sea Akamas, Fontana Cyprus 35.090108, 32.303844 44 3
Gobius incognitus Levantine Sea Akamas, George Island Cyprus 35.074958, 32.335222 45 8
Gobius incognitus Levantine Sea Cavo Greco, Tunnel—cave Cyprus 34.963592, 34.073078 46 7
Gobius incognitus Levantine Sea Cavo Greco, chappel Cyprus 34.976053, 34.076781 47 2
Gobius incognitus Levantine Sea Cavo Greco, Cyclops cave Cyprus 34.985631, 34.076928 48 1
Gobius bucchichi Black Sea Sevastopol Ukraine 44.494094, 33.533650 49 1

J. Mar. Sci. Eng. 2023,11, 2289 5 of 12
Reconstruction of the phylogenetic relationships of the analysed material was es-
timated by Bayesian Inference (BI). Only unique haplotypes collapsed in Fabox [
46
]
were used for the phylogenetic reconstruction. Sequences of Gobius niger Linnaeus, 1758
(FJ526782 [
34
]), Gobius fallax Sarato, 1889 (FJ526785 [
34
]), and Gobius couchi Miller & El-
Tawil, 1974 (FJ389196 [
47
]) were used for comparison as well, while Pomatoschistus minutus
(Risso, 1810) (FJ526776 [
34
]) and Economidichthys pygmeus (Holly, 1929) (KF415544 [
33
])
were used as outgroup. The appropriate model of nucleotide substitution was determined
in jModeltest v.2.1.9 [
48
] based on Bayesian information criterion (BIC). BI was assessed
in MrBayes v.3.2.2 [
49
] under the GTR+I+G model selected by JModeltest. Two indepen-
dent runs, each consisting of four Markov Chain Monte Carlo (MCMC) methods, were
simultaneously run for 5 million generations. Sampling of the trees was performed every
1000 generations. TRACER v.1.7.0 [
50
] was used to analyse the trace files generated by
MCMC runs and visualise the convergence of chains. The first 25% of trees were discarded
as burn-in and kept trees were used for the construction of a 50% majority-rule consensus
tree. The final tree was visualised in FigTree v.1.4.4 [
51
] and edited in Inkscape version
0.92.3 [
52
]. Program MEGA 10 [
53
] was used to calculate genetic distance (uncorrected
p-distances) between and within the species. Fabox [
46
] was used to visualise the variable
sites and assess fixed mutations between the two species.
J. Mar. Sci. Eng. 2023,11, x FOR PEER REVIEW 6of 14
Islet
Gobius incognitus
Aegean Sea
Euboia Island, Karystos
Greece
38.009667, 24.430804
38
3
Gobius incognitus
Aegean Sea
Euboia Island, Kastri Beach
Greece
37.973022, 24.537658
39
1
Gobius incognitus
Aegean Sea
Euboia Island, Mirmigia
rocks
Greece
37.972336, 24.548042
40
1
Gobius incognitus
Aegean Sea
Raches
Greece
38.876144, 22.785700
41
3
Gobius incognitus
Aegean Sea
Gavradia
Greece
40.440278, 23.839722
42
18
Gobius incognitus
Aegean Sea
between Geyikli and Dalyan
*
Turkey
39.789722, 26.156111
43
22
Gobius incognitus
Levantine Sea
Akamas, Fontana
Cyprus
35.090108, 32.303844
44
3
Gobius incognitus
Levantine Sea
Akamas, George Island
Cyprus
35.074958, 32.335222
45
8
Gobius incognitus
Levantine Sea
Cavo Greco, Tunnel—cave
Cyprus
34.963592, 34.073078
46
7
Gobius incognitus
Levantine Sea
Cavo Greco, chappel
Cyprus
34.976053, 34.076781
47
2
Gobius incognitus
Levantine Sea
Cavo Greco, Cyclops cave
Cyprus
34.985631, 34.076928
48
1
Gobius bucchichi
Black Sea
Sevastopol
Ukraine
44.494094, 33.533650
49
1
Reconstruction of the phylogenetic relationships of the analysed material was
estimated by Bayesian Inference (BI). Only unique haplotypes collapsed in Fabox [46]
were used for the phylogenetic reconstruction. Sequences of Gobius niger Linnaeus,1758
(FJ526782 [34]), Gobius fallax Sarato, 1889 (FJ526785 [34]), and Gobius couchi Miller &
El-Tawil, 1974 (FJ389196 [47]) were used for comparison as well, while Pomatoschistus
minutus (Risso, 1810) (FJ526776 [34]) and Economidichthys pygmeus (Holly, 1929)
(KF415544 [33]) were used as outgroup. The appropriate model of nucleotide
substitution was determined in jModeltest v.2.1.9 [48] based on Bayesian information
criterion (BIC). BI was assessed in MrBayes v.3.2.2 [49] under the GTR+I+G model
selected by JModeltest. Two independent runs, each consisting of four Markov Chain
Monte Carlo (MCMC) methods, were simultaneously run for 5 million generations.
Sampling of the trees was performed every 1000 generations. TRACER v.1.7.0 [50] was
used to analyse the trace files generated by MCMC runs and visualise the convergence
of chains. The first 25% of trees were discarded as burn-in and kept trees were used for
the construction of a 50% majority-rule consensus tree. The final tree was visualised in
FigTree v.1.4.4 [51] and edited in Inkscape version 0.92.3 [52]. Program MEGA 10 [53]
was used to calculate genetic distance (uncorrected p-distances) between and within the
species. Fabox [46] was used to visualise the variable sites and assess fixed mutations
between the two species.
Figure 2.
Map showing the localities from which material was analysed (circles) and distribution
range of G. incognitus and G. bucchichi based on Kovaˇci´c et al. [
20
]. Locality numbers correspond to
those in Table 1.
3. Results
3.1. Genetic Distinctiveness
In this work, we used altogether 304 sequences of cyt b, of which 272 were new and 32
were published. For G. incognitus, there were 270 sequences, of which 22 were published
and 248 were newly obtained. Two new sequences of G. incognitus were shorter and were
used only for identification. The remaining 258 sequences were 1077 base pairs long and
represented 208 different haplotypes. For G. bucchichi, there were 34 sequences, of which
10 were published and 24 were new. A single new sequence of G. bucchichi was shorter and
was used only for the identification. The remaining 33 sequences were 1077 base pairs long
and they represented 29 different haplotypes. Altogether, 79 sites with fixed mutations
discriminating between both species were detected.

J. Mar. Sci. Eng. 2023,11, 2289 6 of 12
The phylogenetic reconstruction analysis showed each of the included Gobius species
as very distinct (Figure 3). The sequence species identity matches the morphological
identification for all specimens, for which vouchers were available. All calculated ge-
netic divergences are summarized in Table 2. The mean uncorrected p-distance between
G. incognitus and G. bucchichi was 13% (
±
0.8% S.E.), while the mean intraspecific distances
were much lower (G. incognitus 0.63%
±
0.086 S.E.; G. bucchichi 0.68%
±
0.11% S.E.). The
maximum observed intraspecific genetic distance between G. incognitus sequences was
1.76%, while for G. bucchichi it was 1.21%.
J. Mar. Sci. Eng. 2023,11, x FOR PEER REVIEW 8of 14
3. Results
3.1. Genetic Distinctiveness
In this work, we used altogether 304 sequences of cyt b, of which 272 were new and
32 were published. For G. incognitus, there were 270 sequences, of which 22 were
published and 248 were newly obtained. Two new sequences of G. incognitus were
shorter and were used only for identification. The remaining 258 sequences were 1077
base pairs long and represented 208 different haplotypes. For G. bucchichi, there were 34
sequences, of which 10 were published and 24 were new. A single new sequence of G.
bucchichi was shorter and was used only for the identification. The remaining 33
sequences were 1077 base pairs long and they represented 29 different haplotypes.
Altogether, 79 sites with fixed mutations discriminating between both species were
detected.
The phylogenetic reconstruction analysis showed each of the included Gobius
species as very distinct (Figure 3). The sequence species identity matches the
morphological identification for all specimens, for which vouchers were available. All
calculated genetic divergences are summarized in Table 2. The mean uncorrected
p-distance between G. incognitus and G. bucchichi was 13% (±0.8% S.E.), while the mean
intraspecific distances were much lower (G. incognitus 0.63% ± 0.086 S.E.; G. bucchichi
0.68% ± 0.11% S.E.). The maximum observed intraspecific genetic distance between G.
incognitus sequences was 1.76%, while for G. bucchichi it was 1.21%.
Figure 3. The 50% majority-rule consensus Bayesian phylogenetic reconstructions based on
mitochondrial cytochrome b sequence data. Numbers on branches represent posterior probabilities.
Table 2. Genetic distance (uncorrected p-distances, in %) based on cytochrome b sequences for the
analyzed Gobius species. Mean genetic distances between species are below diagonal, S.E. above
diagonal. On the diagonal, mean intraspecific genetic divergence is shown, with S.E. in
parentheses. Numbers in parentheses after species name in the first column are numbers of
analyzed sequences for species. N/A – not applicable.
G. incognitus
G. bucchichi
G. couchi
G. falax
G. niger
G. incognitus (258)
0.63 (±0.086)
0.8
0.9
1.1
1.1
G. bucchichi (30)
13
0.68 (±0.11)
1
1.1
1.2
G. couchi (1)
16.7
14.9
N/A
1.1
1.1
G. falax (1)
16.6
15.3
14.3
N/A
1.2
G. niger (1)
19.4
18
18.9
19.7
N/A
Figure 3.
The 50% majority-rule consensus Bayesian phylogenetic reconstructions based on mito-
chondrial cytochrome b sequence data. Numbers on branches represent posterior probabilities.
Table 2.
Genetic distance (uncorrected p-distances, in %) based on cytochrome b sequences for the
analyzed Gobius species. Mean genetic distances between species are below diagonal, S.E. above
diagonal. On the diagonal, mean intraspecific genetic divergence is shown, with S.E. in parentheses.
Numbers in parentheses after species name in the first column are numbers of analyzed sequences
for species. N/A—not applicable.
G. incognitus G. bucchichi G. couchi G. falax G. niger
G. incognitus (258) 0.63 (±0.086) 0.8 0.9 1.1 1.1
G. bucchichi (30) 13 0.68 (±0.11) 1 1.1 1.2
G. couchi (1) 16.7 14.9 N/A 1.1 1.1
G. falax (1) 16.6 15.3 14.3 N/A 1.2
G. niger (1) 19.4 18 18.9 19.7 N/A
3.2. Distribution
We analyzed material of G. incognitus and G. bucchichi from 49 localities covering
considerable parts of the Mediterranean Sea, Black Sea, and the Atlantic Ocean near
Gibraltar (Figure 2, Table 1). Some localities are close to each other, within a distance of
only a few km, and the samples cover 19 geographically well-separated areas (Figure 2).
Gobius incognitus is widespread, occurring throughout the Mediterranean, and in the
Atlantic Ocean near Gibraltar (Figure 2, Table 1). However, the documented distribution of
G. bucchichi is much more restricted (Figure 2, Table 1). Genetic data confirmed its presence
from three areas in the northern coastal region of the Adriatic Sea, one in the north-western
part of the Ionian Sea, and from the Crimean region of the Black Sea. Both species were
recorded sympatrically in four areas (Figure 2), though mostly from different localities

J. Mar. Sci. Eng. 2023,11, 2289 7 of 12
within the area (Table 1, Figure 2). However, a syntopic presence (i.e., at the same locality)
of both species was revealed at a single locality (Table 1, Figure 2).
4. Discussion
The mitochondrial marker cyt b was confirmed to be a highly reliable tool for the
genetic identification of morphologically similar and often confused gobies G. incognitus
and G. bucchichi. Both species showed very high haplotype variability, relatively uncommon
in coastal species, whose dispersal capability is limited by local oceanographic patterns [
54
].
However, G. incognitus and G. bucchichi can be genetically easily unambiguously identified,
given that the average interspecific genetic divergence between them is almost 20 times
higher than the average intraspecific divergence, and the number of fixed mutations be-
tween species on cyt b sequence data is nearly 80. Even the highest observed value of
intraspecific distance (1.76% in G. incognitus) is six to seven times lower than the lowest
interspecific genetic distance between both studied species. In general, gobies of the genus
Gobius are easily identifiable by mtDNA markers, as proved by Iglésias et al. [
4
], where
many species were analyzed by cytochrome c oxidase, and minimum genetic distance be-
tween species was 8–9% (with the only exception of the Gobius auratus complex), while
the intraspecific distance was always several times lower. The overall high genetic di-
vergence between species of the genus Gobius could be a result of the considerably long
evolutionary history of this genus; the oldest paleontological record of Gobius based on
complete skeleton fossil is dated to 20 MY ago [
55
]. It is probable that the evolution of the
lineages leading to the extant species started in the very early diversification of the genus,
allowing many mutations to accumulate over time. Nevertheless, the intraspecific genetic
distance within the Mediterranean gobies on mtDNA markers can also be relatively high,
commonly reaching 1.5–2% [
15
,
18
,
37
], and rarely even 5% [
4
,
9
], so molecular identification
of genetically more similar species of gobies may be challenging. However, as this is not a
case of G. bucchichi and G. incognitus, even the advanced genetic methods for identification
of the species presence from environmental samples (eDNA approach) are applicable to
these species, but also actually to most of the gobies from the Mediterranean Sea. The data
from eDNA could potentially fill in the gaps in the knowledge on distribution of many
goby species. A limitation of the use of the mtDNA markers for the species identification
is that potential hybrids or mito–nuclear discordance cannot be detected, as mtDNA is
uniparentally inherited. Gobius incognitus and G. bucchichi occur sympatrically, even syn-
topically, this work, [
20
]); their natural hybridisation, although not reported, cannot be a
priori excluded. So far, the hybridisation of gobies from the European seas was reported
only in the genus Pomatoschistus [
56
,
57
]. The hybridisation between European marine
gobies can be assumed to be rare owing to their complex mating system, which includes
nest building and species-specific sound production [
58
,
59
], which can serve as important
prezygotic barrier through strong auditory-based assortative mating.
Gobius bucchichi was for a long time considered a widespread species in the Mediter-
ranean and Black seas, present also in the European Atlantic near the Gibraltar strait up
to Algarve coast of Portugal [
17
]. However, this assumption was actually based on the
distribution of two morphologically similar species. After the discovery and description
of G. incognitus by Kovaˇci´c & Šanda [
18
], the distribution of both species was questioned.
Almost a decade after the description of G. incognitus, we can state that only several
publications have provided unambiguous distribution data, accompanied by appropriate
identification [
4
,
18
,
20
–
28
], while other works, even those recently published, continue to
report only G. bucchichi [
60
–
62
], probably being unaware of the description of G. incognitus.
Kovaˇci´c et al. [
20
] have summarized the available information on the distribution of
both G. incognitus and G. bucchichi and confirmed that distribution range of G. incognitus
covers a considerable part of the Mediterranean Sea coastal region, and the European
Atlantic near the Gibraltar strait up to Algarve (Figure 2), as previously suggested by
Kovaˇci´c & Šanda [
18
]. In contrast, G. bucchichi was confirmed for a very restricted and
scattered area (Figure 2). It has been reported only from the northern part of the Adriatic

J. Mar. Sci. Eng. 2023,11, 2289 8 of 12
Sea, from Italy to Montenegro, from a few localities in the northern Ionian Sea in Albania
and Greece, from one locality in the south-western Aegean Sea in Greece, south-eastern
and south-western part of the Aegean Sea in Turkey, from the Sea of Marmara, and from
the two areas of the northern Black Sea in Ukraine and Russia [
20
]. The range of both
species in the Mediterranean Sea completely overlaps, while for the Black Sea and Marmara
Sea, only the presence of G. bucchichi was confirmed [
20
]. Our results for distribution
based on genetic data, often including a considerable number of analyzed specimens per
locality/geographic area, are completely congruent with the revision of Kovaˇci´c et al. [
20
].
In our data, G. bucchichi was recorded only from the northern side of the Adriatic Sea,
westernmost Ionian Sea, and from the Crimean coast in the northern Black Sea (Figure 2,
Table 1), while G. incognitus was detected throughout the whole Mediterranean Sea and the
southern Portuguese Atlantic Ocean coast (Figure 2, Table 1).
However, for the large areas of the previously reported range of G. bucchichi [
17
],
respectively for large portions of the Mediterranean coastal region in general, we are still
awaiting clarification on which of the two species is present (Figure 2). This applies to
almost all of African Mediterranean coastal region, as G. bucchichi was reported only from
Libya [
63
] and Egypt [
64
], but this was not supplemented by morphological or molecular
evidence; moreover, G. bucchichi had been previously reported from Morocco [
65
] and
was recently confirmed to be G. incognitus instead [
20
]. Data are missing also for part of
the Levantine coastal region of Turkey [
20
] and the coastal regions of Syria and Lebanon,
from where neither of the species was reported (Syria [
66
]), or only G. bucchichi was listed
(Lebanon [
60
,
62
]). The Black Sea is another area with unsatisfactory data on the presence of
G. incognitus and G. bucchichi.Gobius bucchichi is the only unambiguously confirmed species
from this region and, moreover, only from a small part of the northern coast [
20
] (our data).
Nevertheless, at least one of these species is apparently widespread around the Black Sea,
as there are reports on G. bucchichi from Turkey [
67
], Russia [
68
,
69
], Ukraine [
70
,
71
], as well
as Bulgaria [
72
]. Finally, the lack of distribution data for the Italian Adriatic coastal region
(Figure 2) is surprising, as it was considered a part of the distribution range of G. bucchichi
by Miller [
17
]. Kovaˇci´c et al. [
20
] assumed that this gap in distribution may be real, as the
area is well studied, and it could be a result of lack of suitable habitats in this region.
Further research should focus not only on the clarification of the distribution of both
species, but especially on their ecology, including microhabitat use of each species and their
niche separation, since nothing is known about how the species coexist/interact in areas of
sympatric and syntopic occurrence.
The results of this study suggest an alternative, easier, and reliable identification
method of morphologically similar fish species. A high divergence in cyt b between
G. incognitus
and G. bucchichi, including numerous sites with fixed mutations (79 in total),
enables clear distinctions between the two goby species at the mtDNA level. This relatively
fast and low-cost method complements an array of molecular methods, such as multiplex
PCR assays, melt curve analysis real-time PCR, or restriction fragment length polymor-
phism, which are successfully used for fish identification [
73
]. Importantly, molecular tools
could also aid potential conservation efforts of less common species, for example, in this
particular case, of G. bucchichi. At the same time, our results demonstrate how insufficient
our knowledge on the life in the Mediterranean Sea remains, and point to the great potential
of molecular methods to achieve even a partial improvement.
Author Contributions:
Conceptualization, R.Š. and J.V.; methodology, K. ˇ
C., E.A., J.V. and R.Š.;
software, K. ˇ
C., E.A., J.V. and R.Š.; validation, J.V. and R.Š.; formal analysis, J.V. and R.Š.; investigation,
K. ˇ
C., J.V. and R.Š.; resources, J.V., R.Š., A.K., D.Z., A.M.P., A.S.T., E.V. and D.S.; data curation, R.Š.
and J.V.; writing—original draft preparation, J.V., R.Š. and K. ˇ
C.; writing—review and editing, A.K.,
D.Z., A.M.P., A.S.T., E.V. and D.S.; visualization, K. ˇ
C., E.A., J.V. and R.Š.; supervision, J.V.; project
administration, R.Š. and J.V.; funding acquisition, R.Š. and J.V. All authors have read and agreed to
the published version of the manuscript.

J. Mar. Sci. Eng. 2023,11, 2289 9 of 12
Funding:
This research was funded by the Ministry of Culture of the Czech Republic (DKRVO
2019–2023/6.III.e National Museum, 00023272) and by institutional resources of the Ministry of
Education, Youth, and Sports of the Czech Republic. Scientific studies of EV were carried within a
State Project of Moscow State University №121032300105-0.
Institutional Review Board Statement: Not applicable. Work conducted on museum material.
Informed Consent Statement: Not applicable.
Data Availability Statement: Sequences are deposited in GenBank.
Acknowledgments:
We are grateful to Konstantinos Moustakas and Ioulianos Pantelides (DFMR,
Cyprus), Stamatis Zogaris (HCMR, Greece), the Dikelas Dive Center (Karystos, Greece), Marcelo
Kovaˇci´c (PMR, Rijeka, Croatia), Mihuri´c Dive Center (Selce, Croatia), and Iain Wilson (Albania)
for their support or providing material used in this study. Sampling in Greece and Cyprus was
conducted under permission of the Hellenic Ministry of Environment (through collecting licence to
HCMR, no. 220965/2583/22-8-2011) and the Cyprus Ministry of Agriculture, Rural Development and
Environment, through the Department of Fisheries and Marine Research (no. I
Π
2921I
Π
/10-11-2017)
respectively. Collection of part of the material was supported by the EU FP7 project ASSEMBLE
at CCMar/Centre of Marine Sciences of Algarve, Faro, Portugal, and Observatoire oceanologique
de Banyuls/Mer, Laboratoire Arago, Banyuls/Mer. Computational resources were provided by the
e-INFRA CZ project (MetaCentrum, ID:90254), supported by the Ministry of Education, Youth and
Sports of the Czech Republic.
Conflicts of Interest: The authors declare no conflict of interest.
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