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Article

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

by
Katarína Čekovská
1,
Radek Šanda
2,
Eva Ašenbrenerová
2,
Abderrahmane Kassar
3,
Dimitris Zogaris
4,
Anna Maria Pappalardo
5,
Ali Serhan Tarkan
6,7,
Ekaterina Vasil’eva
8,
David Santos
1,† and
Jasna Vukić
1,*
1
Department of Ecology, Faculty of Science, Charles University, Viničná 7, CZ-128 44 Prague, Czech Republic
2
Department of Zoology, National Museum of the Czech Republic, Václavské Nám. 68, CZ-115 79 Prague, Czech Republic
3
LCVRM, 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
4
Department of Ichthyology and Aquatic Environment, University of Thessaly, GR-38446 Volos, Greece
5
Department of Biological, Geological and Environmental Sciences, University of Catania, Via Androne 81, 95124 Catania, Italy
6
Department of Basic Sciences, Faculty of Fisheries, Muğla Sıtkı Koçman University, Muğla 48000, Turkey
7
Department of Ecology and Vertebrate Zoology, Faculty of Biology and Environmental Protection, University of Lodz, PL-90136 Lodz, Poland
8
Zoological Museum, Moscow State University, Moscow 121069, Russia
*
Author to whom correspondence should be addressed.
†
Current address: Marine and Environmental Sciences Centre (MARE), Faculty of Sciences, University of Lisbon, PT-2750-374 Cascais, Portugal.
J. Mar. Sci. Eng. 2023, 11(12), 2289; https://doi.org/10.3390/jmse11122289
Submission received: 30 October 2023 / Revised: 22 November 2023 / Accepted: 28 November 2023 / Published: 2 December 2023
(This article belongs to the Section Marine Ecology)
Browse Figures
Versions Notes

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.

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., [4,5,6,7,8,9]. Usually, new species were first discovered in a small geographic area, but subsequently, they were found in even geographically considerably distant localities [10,11,12,13,14,15], 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 Kovačić & 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 [18,20,21,22,23,24,25,26,27], 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., [4,5,6,7,8,9,11,12,14,15,18,28,30,31,32]. Several studies deal with phylogenetic relationships, e.g., [33,34,35,36], and also the population genetic structure and phylogeography were investigated for several species, e.g., [37,38,39,40,41,42,43].
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 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.

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 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., [31,34,35,36], 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 analyses only the long sequences were used. The dataset was further extended by available published sequences of cyt b of both species (FJ526784 [34] and KR811029-KR811059 [18]) (Table 1). Sequences of both species published by Kovačić & Šanda [18] were taken as a reference, 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 Kovačić 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].
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 Kovačić 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 SubareaLocalityCountryCoordinatesNo. in MapNew
Sequences		Published
Sequences
Gobius incognitusNE AtlanticGalePortugal37.076972, −8.31072217
Gobius incognitusNE AtlanticSesmarias, Praia de EvaristoPortugal37.074006, −8.30357526
Gobius incognitusAfrican W MedBou IsmailAlgeria36.650767, 2.69115334
Gobius incognitusAfrican W MedTipazaAlgeria36.594200, 2.44913145
Gobius incognitusAfrican W MedAlgiers, Sidi FredjAlgeria36.764291, 2.848401517
Gobius incognitusAfrican W MedAlgiers, Ain Benian Algeria36.802050, 2.898339619
Gobius incognitusSpanish W MedBenidormSpain38.533982, −0.12939574
Gobius incognitusGulf of LionBanyuls-sur-MerFrance42.481817, 3.1360328106 A
Gobius incognitusGulf of LionBanyuls-sur-Mer, Paulliles beachFrance42.505759, 3.123662973 A
Gobius incognitusGulf of LionPort VendresFrance42.523300, 3.109700101
Gobius incognitusCorsican shelfCorsica, LumioFrance42.574444, 8.8052781125
Gobius incognitusMaltese shelfGozo Island, RamlaMalta36.061869, 14.284042122
Gobius incognitusIonian SeaSicily, Aci CastelloItaly37.555419, 15.149383139
Gobius incognitusIonian SeaSicily, Aci TrezzaItaly37.562758, 15.163661149
Gobius incognitusIonian SeaSicily, Capo MuliniItaly37.574014, 15.173408158
Gobius bucchichiAdriatic SeaKraljevicaCroatia45.267660, 14.5622561611 A
Gobius bucchichiAdriatic SeaKrk Island, OmišaljCroatia45.218911, 14.55090817 2 A
Gobius bucchichiAdriatic SeaKrk Island, GlavotokCroatia45.094134, 14.43669418 1 B
Gobius bucchichiAdriatic SeaSelceCroatia45.152293, 14.7189281961 A
Gobius incognitusAdriatic SeaKrk Island, Sveti MarakCroatia45.105944, 14.668500202
Gobius bucchichi + Gobius incognitusAdriatic SeaBrač Island, SumartinCroatia43.285934, 16.8788292110 (5xG. inc., 5xG. buc.)6 A(G. inc.)
Gobius incognitusAdriatic SeaHvar Island, Smočiguzica Croatia43.233889, 16.574722221
Gobius incognitusAdriatic SeaHvar Island, Sveta NedjeljaCroatia43.134433, 16.5913812314
Gobius bucchichiAdriatic SeaBoka Kotorska, Kostanjica Montenegro42.485403, 18.669678246
Gobius bucchichiAdriatic SeaBoka Kotorska, StrpMontenegro42.503989, 18.669453254
Gobius incognitusAdriatic SeaBudva, Jaz beachMontenegro42.283836, 18.8090342612 A
Gobius incognitusAdriatic SeaKamenovoMontenegro42.273917, 18.887667275
Gobius incognitusAdriatic SeaSveti StefanMontenegro42.257180, 18.89211828 5 A
Gobius bucchichiIonian Seaoutflow of Butrint lagoonAlbania39.743614, 20.0186542915 A
Gobius incognitusIonian SeaSivotaGreece39.399800, 20.234664302
Gobius incognitusIonian SeaRomanos, PeloponnesosGreece36.985361, 21.651634311
Gobius incognitusIonian SeaPetalidi, PeloponnesosGreece36.959142, 21.934967321
Gobius incognitusAegean SeaEuboia Island, Petalii IslandsGreece38.014764, 24.280875333
Gobius incognitusAegean SeaEuboia Island, MarmariGreece38.048808, 24.318328344
Gobius incognitusAegean SeaEuboia Island, Kalamitsi beachGreece37.971372, 24.365764352
Gobius incognitusAegean SeaEuboia Island, Mnima penninsulaGreece37.978775, 24.399581364
Gobius incognitusAegean SeaEuboia Island, Agia Pelagia IsletGreece37.997008, 24.398186371
Gobius incognitusAegean SeaEuboia Island, KarystosGreece38.009667, 24.430804383
Gobius incognitusAegean SeaEuboia Island, Kastri BeachGreece37.973022, 24.537658391
Gobius incognitusAegean SeaEuboia Island, Mirmigia rocksGreece37.972336, 24.548042401
Gobius incognitusAegean SeaRachesGreece38.876144, 22.785700413
Gobius incognitusAegean SeaGavradiaGreece40.440278, 23.8397224218
Gobius incognitusAegean Seabetween Geyikli and Dalyan *Turkey39.789722, 26.1561114322
Gobius incognitusLevantine SeaAkamas, FontanaCyprus35.090108, 32.303844443
Gobius incognitusLevantine SeaAkamas, George IslandCyprus35.074958, 32.335222458
Gobius incognitusLevantine SeaCavo Greco, Tunnel—caveCyprus34.963592, 34.073078467
Gobius incognitusLevantine SeaCavo Greco, chappelCyprus34.976053, 34.076781472
Gobius incognitusLevantine SeaCavo Greco, Cyclops caveCyprus34.985631, 34.076928481
Gobius bucchichiBlack SeaSevastopolUkraine44.494094, 33.533650491
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.

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%.

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 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 between 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 between 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 divergence 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 syntopically, 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 Mediterranean 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 Kovačić & 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 [4,18,20,21,22,23,24,25,26,27,28], while other works, even those recently published, continue to report only G. bucchichi [60,61,62], probably being unaware of the description of G. incognitus.
Kovačić 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 Kovačić & 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 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 Kovačić 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]. Kovačić 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 polymorphism, 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.Č., E.A., J.V. and R.Š.; software, K.Č., E.A., J.V. and R.Š.; validation, J.V. and R.Š.; formal analysis, J.V. and R.Š.; investigation, K.Č., 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.Č.; writing—review and editing, A.K., D.Z., A.M.P., A.S.T., E.V. and D.S.; visualization, K.Č., 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.

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 Kovačić (PMR, Rijeka, Croatia), Mihurić 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. IΠ2921ΙΠ/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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Jmse 11 02289 g001
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.
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.
Jmse 11 02289 g001
Jmse 11 02289 g002
Figure 2. Map showing the localities from which material was analysed (circles) and distribution range of G. incognitus and G. bucchichi based on Kovačić et al. [20]. Locality numbers correspond to those in Table 1.
Figure 2. Map showing the localities from which material was analysed (circles) and distribution range of G. incognitus and G. bucchichi based on Kovačić et al. [20]. Locality numbers correspond to those in Table 1.
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Figure 3. The 50% majority-rule consensus Bayesian phylogenetic reconstructions based on mitochondrial cytochrome b sequence data. Numbers on branches represent posterior probabilities.
Figure 3. The 50% majority-rule consensus Bayesian phylogenetic reconstructions based on mitochondrial cytochrome b sequence data. Numbers on branches represent posterior probabilities.
Jmse 11 02289 g003
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.
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. incognitusG. bucchichiG. couchiG. falaxG. niger
G. incognitus (258)0.63 (±0.086)0.80.91.11.1
G. bucchichi (30)130.68 (±0.11)11.11.2
G. couchi (1)16.714.9N/A1.11.1
G. falax (1)16.615.314.3N/A1.2
G. niger (1)19.41818.919.7N/A
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Čekovská, K.; Šanda, R.; Ašenbrenerová, E.; Kassar, A.; Zogaris, D.; Pappalardo, A.M.; Tarkan, A.S.; Vasil’eva, E.; Santos, D.; Vukić, 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

AMA Style

Čekovská K, Šanda R, Ašenbrenerová E, Kassar A, Zogaris D, Pappalardo AM, Tarkan AS, Vasil’eva E, Santos D, Vukić J. Cytochrome b Sequencing as a Tool for Identification of Morphologically Similar Mediterranean Gobies Gobius incognitus and Gobius bucchichi (Actinopterygii: Gobiidae). Journal of Marine Science and Engineering. 2023; 11(12):2289. https://doi.org/10.3390/jmse11122289

Chicago/Turabian Style

Čekovská, Katarína, Radek Šanda, Eva Ašenbrenerová, Abderrahmane Kassar, Dimitris Zogaris, Anna Maria Pappalardo, Ali Serhan Tarkan, Ekaterina Vasil’eva, David Santos, and Jasna Vukić. 2023. "Cytochrome b Sequencing as a Tool for Identification of Morphologically Similar Mediterranean Gobies Gobius incognitus and Gobius bucchichi (Actinopterygii: Gobiidae)" Journal of Marine Science and Engineering 11, no. 12: 2289. https://doi.org/10.3390/jmse11122289

APA Style

Čekovská, K., Šanda, R., Ašenbrenerová, E., Kassar, A., Zogaris, D., Pappalardo, A. M., Tarkan, A. S., Vasil’eva, E., Santos, D., & Vukić, J. (2023). Cytochrome b Sequencing as a Tool for Identification of Morphologically Similar Mediterranean Gobies Gobius incognitus and Gobius bucchichi (Actinopterygii: Gobiidae). Journal of Marine Science and Engineering, 11(12), 2289. https://doi.org/10.3390/jmse11122289

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