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Article

Molecular Data Confirm the Occurrence of the Allochthonous Gambusia holbrooki (Pisces: Poeciliidae) in Sicily and the Maltese Archipelago

by
Luca Vecchioni
1,*,
Mirko Liuzzo
2,
Arnold Sciberras
3,
Jeffrey Sciberras
3,
Justin Formosa
3,
Alan Deidun
4,
Gabriele Giacalone
1,
Vincenzo Arizza
1,
Marco Arculeo
1,
Federico Marrone
1 and
Francesco Paolo Faraone
1
1
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 18, 90123 Palermo, Italy
2
Department of Environmental Sciences, Informatics and Statistics (DAIS), Ca’ Foscari University of Venice, 30170 Venice, Italy
3
The Exterminator, Service Hub, Triq San Gorg, 5 In-Naxxar, NXR 2541 Naxxar, Malta
4
Oceanography Malta Research Group, Department of Geosciences, Faculty of Science, University of Malta, MSD 2080 Msida, Malta
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(1), 48; https://doi.org/10.3390/d17010048
Submission received: 20 November 2024 / Revised: 8 January 2025 / Accepted: 8 January 2025 / Published: 13 January 2025
(This article belongs to the Special Issue Evaluating the Influence of Environmental Variables on Fish Ecology and Diversity)
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Versions Notes

Abstract

:
A major threat to biodiversity is represented by Invasive Alien Species (IAS), particularly on freshwater ecosystems, which are already heavily altered by human activities. Two of the most pernicious IAS are the eastern and western mosquitofish, i.e., Gambusia holbrooki and G. affinis. These two poeciliids are morphologically very close to each other, and soon after their formal description, G. holbrooki was considered a subspecies of G. affinis. In the following years, several studies proved that these two entities belonged to two different species; nevertheless, it was only at the end of the 1990s that their separate taxonomic status was re-established. In the 1920s and 1930s, both G. holbrooki and G. affinis were asynchronously introduced from the United States into Europe and subsequently translocated globally as biocontrol agents of the malaria vector (i.e., the larvae of the Anopheles mosquitoes), with dramatic consequences for the inland water native fauna. However, due to taxonomic uncertainties and nomenclatural instability, for years, there were doubts about which Gambusia species had been introduced in different regions. The first available molecular studies confirmed the occurrence of G. holbrooki in Europe, but no evidence confirming the occurrence of G. affinis was found. Despite this, some records report the occurrence of western mosquitofish in Italy and Malta. Considering the negative effects that the mosquitofish has on the native biota, it is of paramount importance to know the precise biological diversity of the native and non-native species to better implement environmental management strategies to properly preserve the already-fragile waterbodies. Therefore, to check for the possible occurrence of G. affinis in Italy and Malta, we conducted extensive sampling in Sicily (Italy) and in the Maltese archipelago, aiming to verify the identity of Gambusia populations occurring in the study area. Based on sequences of the mitochondrial cytochrome b gene, we consistently observed the occurrence of only G. holbrooki in the investigated area, finding, almost exclusively, the most common haplotype known for the species in the whole invaded range (i.e., “HOL1”).

1. Introduction

Biological invasions by non-native species are among the most significant causes of biodiversity loss and ecosystem alteration [1,2,3,4,5]. In fact, the introduction of invasive alien species (hereafter IAS) through commercial trade represents a key factor in transforming biocenoses and natural ecosystems ([6] and references therein). Over centuries, humans have frequently introduced, deliberately or unwarily, non-native species worldwide for various purposes such as sport fishing, ornamental reasons and biocontrol attempts (e.g., [1,7]). In this context, inland waters are by far the most sensitive ecosystems to the introduction and spread of IAS, mainly due to the overexploitation of these habitats linked to human activities as well as the high dispersal ability of non-native species, the eradication of which, once established, is often difficult or impossible [8,9]. Furthermore, the occurrence of non-native species involves multi-faceted impacts, since they can predate on or outcompete native species (e.g., [10,11]) and lead to phenomena of niche displacement [12] or parasite spill-over [13], thus significantly compromising the stability of the native communities that inhabit the invaded waterbodies.
Native to the United States, the eastern and western mosquitofishes (also known as “plague minnows”) of the genus Gambusia Poey, 1854, i.e., G. holbrooki Girard, 1859 (Figure 1) and G. affinis (Baird & Girard, 1853) are small, viviparous poeciliid fishes inhabiting both fresh and brackish water. Gambusia usually reaches sexual maturity in 3–8 weeks, with females that can have multiple broods with an average clutch size of 5 to 100 over a single breeding season ([14] and references therein), thus having great population expansion potential. At the beginning of the 20th century, they were introduced worldwide as biocontrol agents (e.g., [14,15,16,17,18,19,20]) in an attempt to control the mosquitoes of the genus Anopheles Meigen, 1818, i.e., the dispersal vectors of malaria. Employing mosquitofishes was one of the best solutions found by Howard [21] and other investigators (see [16] and reference therein) to control and limit the spread of mosquito populations, i.e., the vectors of the disease. Therefore, from the beginning of the 20th century onwards, Gambusia spp. were imported from the USA and introduced to Europe [14,22], leading to one of the worst global invasions occurring in inland waters (e.g., [9,18,23,24,25,26,27,28,29,30]).
Eastern and western mosquitofishes are two phylogenetically and morphologically closely related poeciliid species. After their description, due to their alleged resemblance, Gambusia holbrooki was considered a subspecies of G. affinis, the latter also including another subspecies, namely G. affinis patruelis, later synonymised with G. affinis affinis (see [14] and references therein). Several years later, the distinct species status of the two taxa was re-established [31,32]. However, due to taxonomic uncertainties and nomenclatural instability, for years, there were doubts about which mosquitofish species were translocated throughout the world and where exactly they had been introduced. Although some studies correctly identified the species since its first introduction in Europe, it was only after the molecular study of Vidal et al. [33] (but see also [34,35,36]) that it was ascertained that G. holbrooki, the eastern mosquitofish, was the Gambusia species most frequently introduced in Europe.
In Italy, both Gambusia holbrooki and G. affinis were acclimated and introduced (cf. [37,38,39,40]). Despite the increasing amount of evidence attesting the impact of Gambusia spp. on native biocenoses (e.g., [41,42,43,44,45,46,47,48]), the reputation of the mosquitofish as an effective predator of mosquito larvae remained undiminished, thus leading to its official or informal active introduction into waterbodies throughout the country (e.g., [49]).
Nowadays, due to their known impact on native biocenoses, both Gambusia affinis and G. holbrooki are considered invasive alien species of Union concern according to the EU Regulations [50] and [51]; accordingly, their breeding, trade, introduction in the wild and translocation are prohibited across the entire European Union. However, unidentified mosquitofishes of unknown origin can still often be found for sale in pet shops and street markets as well as on the internet; accordingly, it is possible that multiple, independent introduction events posterior to those of the first half of the 20th century occurred and still occur to this very day.
In light of the likely occurrence of Gambusia affinis in Italy and possibly in Slovenia ([39,52,53,54,55,56], but see also [57]), and of the possibility that multiple introductions from different source areas took place in the past and in recent times in our study area, we aimed to establish, through an extensive sampling effort, which Gambusia species actually occur in the Sicilian and Maltese inland waters. Considering the fact that the mosquitofish negatively affects the inland water’s biota (e.g., [45,58]), the accurate and precise knowledge of the biological diversity of both the native and non-native species of both islands is in fact of paramount importance for the future environmental management of the heavily endangered aquatic ecosystems [59]. Furthermore, a reliable taxonomic assessment is also crucial from a legislative and management point of view, especially when dealing with groups of cryptic species or those that are difficult to identify on a solely morphological basis.
Diversity 17 00048 g001
Figure 1. Illustrations of female and male specimens of Gambusia holbrooki (modified from: Comes, [60]).
Figure 1. Illustrations of female and male specimens of Gambusia holbrooki (modified from: Comes, [60]).
Diversity 17 00048 g001

2. Materials and Methods

Prior to sampling in the Sicilian and Maltese freshwater waterbodies, an in-depth bibliographic research work through the use of the databases of SCOPUS (https://www.scopus.com, last accessed on 13 November 2024), Google Scholar (https://scholar.google.it/, last accessed on 13 November 2024) and ResearchGate (https://www.researchgate.net/, last accessed on 13 November 2024), was conducted to assess the current knowledge on the presence and distribution of Gambusia on both investigated areas, thus allowing us to correctly plan the subsequent sampling sessions. The query structure used to gather information was based on the following keywords: “mosquitofish”, “Gambusia”, “Gambusia holbrooki”, “Gambusia affinis”, “Sicil*”, “Malt*” “Italy”.
Mosquitofish samples were collected throughout mainland Sicily (Italy) and Malta (Figure 2). In the frame of this work, permanent natural and artificial ponds, dams, reservoirs and mouth rivers were sampled using a 250 µm mesh-sized hand net. In addition to the samples collected in Sicily, Gambusia samples from “Lago di Canterno” (province of Frosinone, Latium) and “Passo Campalto” (province of Venice, Veneto) were also investigated as comparative materials (see Table 1 for further details). Collected Gambusia individuals were preserved in situ in 96% ethanol for subsequent identification in the laboratory. Collected samples are now deposited at the Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Italy, under the responsibility of LV.
A map showing the geographic locations of the sampled sites was produced using QGIS software v. 3.30.2 (https://www.qgis.org/, last accessed on 13 November 2024).
A single individual from each site was processed for molecular analyses. Total genomic mosquitofish DNA was obtained through direct DNA extraction, starting with 40–50 mg of muscular tissue from each collected specimen, using the BIORON GmbH “Ron’s Tissue DNA Mini Kit”, following the manufacturer’s instructions. Afterwards, the extracted DNA was amplified by Polymerase Chain Reaction (PCR). The primers “CytBF1” and “CytBR1” [33] were used to amplify a fragment of the mitochondrial cytochrome b gene (Cytb), following the procedure described by Vidal et al. [33]. To check for the correct amplification of the Cytb fragment, 4 μL of each PCR product w used to perform electrophoresis on 1% agarose gel at 90 V for 30 min and then visualised with a UV transilluminator. Before sequencing, PCR products were purified using the Exo-SAP-IT® kit (Affymetrix USB, USA). Sequencing was performed by Macrogen Europe (Milan, Italy) using an ABI 3130xL sequencer (Applied Biosystems), using the same Cytb primers used previously for the PCRs. Obtained chromatograms were analysed and manually proofread using MEGA11 software [61]. The novel Cytb sequences of Gambusia were deposited in GenBank (see Table 1 for their Accession Numbers, A.N.’s). Furthermore, aiming to compare the 37-novel produced Cytb sequences with a selection of those publicly available, 86 Gambusia spp. Cytb sequences (including at least one sequence for each known haplotype of G. holbrooki and G. affinis) and a sequence of Belonesox belizanus Kner, 1860 (used as an outgroup) were downloaded from GenBank and included in the analyses (see Figure 3 and Figure S1 for their A.N.’s). All Cytb sequences were aligned with MEGA11 software through the ClustalW method. Novel Cytb sequences were translated into amino acids to check for any possible presence of frameshifts or stop codons, eventually highlighting the presence of sequencing errors or pseudogenes.
The molecular identification of the studied specimens and reconstruction of the phylogenetic relationships among taxa were performed based on the Bayesian Inference (BI) and Maximum Likelihood (ML) analyses using the MrBayes v. 3.2.7 [63] and PhyML v. 3.0 [64] software packages, respectively. The best evolutionary model was chosen using PartitionFinder v. 1.0.1 [65]. Both the BI and ML analyses were performed under a General Time Reversible sequence model of evolution for molecular data with a proportion of gamma and invariant sites (GTR+I+Γ; nst = 6). Two independent Markov Chain Monte Carlo (MCMC) analyses were run with 1.000.000 generations (temp.: 0.2; default priors). Twenty-five percent of the initial trees were conservatively discarded as “burn in”. Effective Sample Size values above 200 were obtained for all parameters analysed.
The haplotype network based on novel and already-published Cytb Gambusia holbrooki sequences, including one sequence for each known haplotype (sensu [33]) in each country available, was built using the software PopART v. 1.7 ([66]—https://popart.maths.otago.ac.nz/, accessed on 13 November 2024), implementing the Median-joining network algorithm [67].

3. Results

The bibliographic review showed that, at the beginning of the 20th century, mosquitofishes were introduced to Sicily and the rest of Italy, as well as neighbouring countries (49and reference therein), as an agent against the malaria-bearing mosquitoes of the genus Anopheles [68]. Sicily was among the first Italian regions where the mosquitofish were translocated: the first batch of individuals arrived in Catania in 1927 [68,69]; these belonged to a stock originally collected in Edenton (NC, USA), i.e., within the known distribution range of the eastern mosquitofish, Gambusia holbrooki. During those years, dozens of fish farms were instituted, and these started mass-producing mosquitofish to be translocated to more than 100 Sicilian waterbodies (see pag. 84 in [68]). To date, mosquitofish have been reported in several localities across the Sicilian mainland (e.g., [48,60,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86]), but their local distribution is still severely underestimated. Vidal et al. [33], based on mtDNA sequences, attested the occurrence of G. holbrooki in the only Sicilian locality they investigated (i.e., Catania). Presumably, during those years, a similar introduction pathway occurred in Malta as in most European countries; unfortunately, no evidence of this pattern was found in the frame of the conducted bibliographic review work. In fact, no information is available about the historical introduction of mosquitofish to Malta. However, the Maltese islands have also experienced malaria (e.g., [87,88]), and there is evidence of the presence of mosquitoes of the genus Anopheles on the islands until at least 1943, when the genus probably became locally extinct [89]. It is thus likely that mosquitofishes were introduced to the Maltese archipelago in the same period as in Sicily and the other circum-Mediterranean areas. Nowadays, mosquitofishes are widespread in the Maltese archipelago (e.g., [90,91,92]), most likely due to multiple, independent introduction events originating from unknown source populations.
Due to some uncertainties in Gambusia taxonomy and identification, it is not clear which species were actually introduced and which ones established self-sustaining populations in the study area. As a result, although the available data suggests a general prevalence of G. holbrooki across most of Europe (e.g., [33,93]), some evidence also points to the occurrence of sparse populations of G. affinis as well (e.g., [53]). Therefore, the identity of Gambusia populations inhabiting currently uninvestigated areas is still to be ascertained (cf. [33,54,57]).
The sampling activities led to the collection of Gambusia individuals from 37 sites, specifically 25 sites in Sicily and 10 in Malta; two further sampling sites are related to the comparative samples collected in Veneto and Latium (peninsular Italy). See also Table 1 and Figure 2.
Overall, 37 Cytb Gambusia sequences were obtained and included in the analyses (see Table 1 and Figure 3 and Figure S1). The length of the Cytb PCR product ranged from 345 to 356 bp. After having trimmed out the tails of the sequences, a properly aligned Cytb alignment of 305 bp was obtained (the alignment is available from the corresponding author on request). We used the BLAST tool (through the web interface available at https://blast.ncbi.nlm.nih.gov/, accessed on 13 November 2024) to preliminarily identify the species. The queries led us to 98 to 100% identity matches of the sequences with published sequences ascribed to G. holbrooki. The phylogenetic trees obtained based on BI and ML analyses and rooted on Belonesox belizanus showed a congruent topology in accordance with the current taxonomy of the analysed species (Figure 3A and Figure S1). All novel Sicilian and Maltese Gambusia sequences clustered together with the already-published Cytb G. holbrooki sequences.
In addition, from the molecular analyses conducted, we noticed that one of the sequences available on GenBank (i.e., A.N. KF013226), labelled as Gambusia affinis, is actually G. holbrooki (see Figure S1), thus highlighting a misidentification of the specimen by the authors of the sequence (i.e., [94]).
The Median-joining haplotype network based solely on the analysed Cytb Gambusia holbrooki sequences revealed the occurrence of three already-known haplotypes in our Sicilian and Maltese specimens (Figure 3B). All the Maltese and 23 of the Sicilian sequences matched with the haplotype “HOL1” detected by Vidal et al. [33], whereas the other three Sicilian G. holbrooki sequences matched the haplotypes “HOL5” and “HOL6” (see Figure 3B and Table 1 for further information).

4. Discussion

Based on the novel molecular data obtained within this work, the only mosquitofish species occurring in Sicily and Malta proved to be the eastern mosquitofish, Gambusia holbrooki. In fact, in all 37 sites sampled within the study area, only this species was found; this finding is in accordance with the available historical information about the origin of the Sicilian populations of the species, whereas it contrasts with the report of G. affinis in Malta [91], which thus needs to be revised.
The mitochondrial marker used in the present study (i.e., Cytb) highlights a low genetic diversity among samples, allowing us to detect the presence of three haplotypes, corresponding to “HOL1” (for 34/37 of the specimens examined), “HOL5” and “HOL6” (sensu [33], see also Figure 3). It is not surprising that the most frequent haplotype found is the one designated “HOL1”, since this is the most common haplotype found throughout the invaded range of the species, as already reported in the literature [24,27,30,33,95,96]. Moreover, Vidal et al. [33] reported that the occurrence of the “HOL1” haplotype is related to the first introduction event of the mosquitofish from Edenton (North Carolina, United States) to Spain, from where it was then translocated to Italy and to the rest of the world (see also [95]). As for the other two haplotypes found (i.e., “HOL5” and “HOL6”), these can be linked to subsequent, independent introduction events that occurred in France and Portugal, respectively, then followed by the likely translocation of individuals to Sicily [24,27,30,33].
Although the introduction of mosquitofishes into Europe at the beginning of the 20th century was aimed at protecting public health in an attempt to stem the spread of the malaria vector (i.e., anopheline mosquitoes), it led to dramatic environmental impacts that still afflict our fragile aquatic ecosystems. In fact, differing from what was initially thought (e.g., [21]), the efficiency of mosquitofish in limiting mosquitoes’ proliferation is not particularly high. Moreover, in natural ecosystems with complex and well-structured biocenoses, the introduction of mosquitofishes might even have the opposite effect, since it may indirectly increase the survival rate of mosquitoes through the elimination of efficient native competitors (e.g., Aphanius fasciatus Valenciennes, 1821) [78] or even invertebrate predators of mosquito larvae [97].
Nowadays, malaria has been mostly eradicated from most of the circum-Mediterranean countries, due in part to wetland reclamations and the use of chemical insecticides (e.g., “Paris green”, see [39,68]). Despite this, mosquitofish have long been sold in Europe, and they are still often available in local shops or through the web, under the belief that these poeciliid species are effective solutions for controlling and/or eradicating mosquitoes from waterbodies.
The trade and informal translocation of Gambusia spp. have further contributed to the uncontrolled spread of the species in inland waters, dramatically altering the conditions of these ecosystems and damaging their native invertebrate and vertebrate biota. Several studies highlighted how the occurrence of mosquitofish actively alters the delicate equilibria of the invaded waterbodies, for example, by competing with native species such as fish or amphibians (e.g., [45,58]).
In the study area, only a few native fish species inhabit inland waterbodies. These are Aphanius fasciatus, Salariopsis fluviatilis (Asso, 1801), Salmo trutta s.l. Linnaeus, 1758 and Syngnathus abaster Risso, 1827 [98,99,100,101]. Conversely, a rich and diversified non-native fish fauna is widespread in both Sicilian and Maltese inland waters [82,92,102]. Among the native fish species that are most negatively affected by the competition with the eastern mosquitofish, there is the “Mediterranean toothcarp” A. fasciatus, which is an endemic cyprinodontid to the Mediterranean basin, listed in Annex II of the Berne Convention (the Convention on the Conservation of European Wildlife and Natural Habitats) and in Annex II of the EU Habitats Directive (Council Directive 92/43/EEC). Accurate and updated data about the distribution of Gambusia holbrooki and A. fasciatus in Sicily and the Maltese Archipelago are currently missing, but the two species currently co-exist syntopically, at least in the wetlands and river mouths of eastern and south-eastern Sicily (e.g., [76]; pers. obs.). However, in accordance with what was reported by Monti et al. [46] for a site in Central Italy, in most Sicilian and Maltese localities, G. holbrooki seems to have displaced A. fasciatus to saline or even hyperaline waterbodies. The Mediterranean toothcarp is particularly sensitive not only to the presence of G. holbrooki but also to habitat degradation of coastal wetlands. For these reasons, the species is experiencing a dramatic decline of its populations throughout its known range [46,76,78].
In conclusion, the new molecular data pointed out the sole occurrence of Gambusia holbrooki in both Sicily and the Maltese archipelago. Furthermore, although the commercialisation of the species is prohibited in Europe, the sale of the species has still been observed locally. Consequently, in order to protect the autochthonous species of the Sicilian and Maltese inland waters, more attention should be paid by local authorities to prevent the commercialisation, breeding and translocation of mosquitofish. In addition, specific monitoring activities should be implemented to produce a comprehensive distribution map of this invasive alien species, aiming to protect the endangered native biota. Moreover, further actions should be taken to verify the impact of this species in Sicily. Understanding trophic interactions is particularly important in zoology [103,104] especially for the evaluation of the effect of non-native species [105,106], this is one of the unknown aspects of the impact of Sicilian populations of G. holbrooki that deserves to be further investigated.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17010048/s1: Figure S1: Bayesian phylogram of Gambusia spp. based on a 305 bp long Cytb fragment. Belonesox belizanus was used as outgroup to root the tree. Node supports are reported as posterior probabilities (BI)/bootstrap values (ML). Asterisks indicate support values lower than 50. Square brackets assort the samples according to their current taxonomy. Three-letter countries are reported according to the [62]. SIC: Sicily. Novel G. holbrooki sequences are reported in bold.

Author Contributions

Conceptualisation, M.A., A.D. and F.M.; methodology, F.P.F., M.L., A.S., J.S., J.F., G.G., F.M. and L.V.; validation, F.P.F., F.M. and L.V.; formal analysis, L.V.; writing—original draft preparation, L.V., F.P.F. and F.M.; writing—review and editing, L.V., M.L., A.D., A.S., M.A., V.A., G.G. and F.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the fund “NextGenerationEU” of the European Union (D.M. 737/2021—CUP B79J21038330001).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/, accessed on 13 November 2024 (A.N.’s: PQ737554-PQ737590).

Acknowledgments

We wish to thank Luca Altavilla, Simone Costa, Gina Curmi, Francesco Lillo, Angelica Rallo and Fabio Stoch for the help they provided us with the collection of some samples.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 2. Distribution of Gambusia spp. samples collected in Peninsular Italy, Sicily and Malta. Haplotype nomenclature of G. holbrooki follows [33]. See Table 1 for the coordinates and codes of the sampled sites. Photo of G. holbrooki by Robert Aguilar, Smithsonian Environmental Research Center (https://www.flickr.com/photos/serc_biodiversity/12597857535/, last accessed on 13 November 2024). Map created using the Free and Open Source QGIS software v. 3.30.2.
Figure 2. Distribution of Gambusia spp. samples collected in Peninsular Italy, Sicily and Malta. Haplotype nomenclature of G. holbrooki follows [33]. See Table 1 for the coordinates and codes of the sampled sites. Photo of G. holbrooki by Robert Aguilar, Smithsonian Environmental Research Center (https://www.flickr.com/photos/serc_biodiversity/12597857535/, last accessed on 13 November 2024). Map created using the Free and Open Source QGIS software v. 3.30.2.
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Figure 3. (A): Bayesian phylogram of Gambusia spp. haplotypes based on a 305 bp long Cytb fragment. Belonesox belizanus was used as an outgroup to root the tree. Node supports are reported as posterior probabilities (BI)/bootstrap values (ML). Asterisks indicate support values lower than 50. Square brackets assort the samples according to their current taxonomy. Gambusia holbrooki haplotypes found in our novel samples are reported in bold. Haplotype nomenclature of G. holbrooki follows [33]. (B): Median-joining haplotype network based on Cytb G. holbrooki sequences. Dashes indicate substitution steps. Each circle represents a haplotype, and its size is proportional to its frequency. Haplotype nomenclature of G. holbrooki follows [33]. Three-letter countries are reported according to the [62]. SIC: Sicily.
Figure 3. (A): Bayesian phylogram of Gambusia spp. haplotypes based on a 305 bp long Cytb fragment. Belonesox belizanus was used as an outgroup to root the tree. Node supports are reported as posterior probabilities (BI)/bootstrap values (ML). Asterisks indicate support values lower than 50. Square brackets assort the samples according to their current taxonomy. Gambusia holbrooki haplotypes found in our novel samples are reported in bold. Haplotype nomenclature of G. holbrooki follows [33]. (B): Median-joining haplotype network based on Cytb G. holbrooki sequences. Dashes indicate substitution steps. Each circle represents a haplotype, and its size is proportional to its frequency. Haplotype nomenclature of G. holbrooki follows [33]. Three-letter countries are reported according to the [62]. SIC: Sicily.
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Table 1. Geographic origins of the collected Gambusia holbrooki samples and GenBank accession numbers (A.N.’s). Geographical decimal coordinates are reported according to the WGS84 datum. Haplotype nomenclature of G. holbrooki follows [33].
Table 1. Geographic origins of the collected Gambusia holbrooki samples and GenBank accession numbers (A.N.’s). Geographical decimal coordinates are reported according to the WGS84 datum. Haplotype nomenclature of G. holbrooki follows [33].
Geographic OriginVoucher CodeLatitude NLongitude ECytb A.N.Holotype
Lago di Canterno, Frosinone, Latium, ItalyLAT0141.7571513.25021PQ737554Hol1
Affluente diga Trinità, Trapani, Sicily, ItalySIC0137.7238112.76003PQ737565Hol1
Augusta, Siracusa, Sicily, ItalySIC0237.2408215.16481PQ737566Hol1
Bevaio Accitella Cinisi, Palermo, Sicily, ItalySIC0338.1209613.13615PQ737567Hol6
Bevaio Piano Margi, Palermo, Sicily, ItalySIC0438.1211313.13779PQ737568Hol1
Biviere di Cesarò, Messina, Sicily, ItalySIC0537.9552414.71244PQ737569Hol1
Blufi, Palermo, Sicily, ItalySIC0637.7484114.05694PQ737570Hol5
Diga Nicoletti, Enna, Sicily, ItalySIC0737.6061114.34488PQ737571Hol1
Diga Santa Rosalia, Ragusa, Sicily, ItalySIC0836.9729914.76598PQ737572Hol1
Diga Villarosa, Enna, Sicily, ItalySIC0937.5888814.20618PQ737573Hol1
Foce fiume Modione, Trapani, Sicily, ItalySIC1037.5828812.82062PQ737574Hol1
Foce fiume Platani, Agrigento, Sicily, ItalySIC1137.4036413.28470PQ737575Hol1
Foce fiume Verdura, Agrigento, Sicily, ItalySIC1237.4756013.20380PQ737576Hol1
Lago di Piana degli Albanesi, Palermo, Sicily, ItalySIC1337.9783513.30099PQ737577Hol1
Licata, Agrigento, Sicily, ItalySIC1437.1307013.87576PQ737578Hol1
Longi, Messina, Sicily, ItalySIC1538.0086514.75579PQ737579Hol1
Foce Marina di Modica, Ragusa, Sicily, ItalySIC1636.7096814.78165PQ737580Hol1
Botanical Garder, Palermo, Sicily, ItalySIC1738.1120613.37431PQ737581Hol1
Private fish tank, Palermo, Sicily, ItalySIC1838.1567013.31826PQ737582Hol1
Pantano Baronello, Siracusa, Sicily, ItalySIC1936.6775915.05427PQ737583Hol1
Pantano Gariffi, Ragusa, Sicily, ItalySIC2036.7341414.93803PQ737584Hol1
R.N.O. “Fonte Ciane”, Siracusa, Sicily, ItalySIC2137.0419915.23482PQ737585Hol1
Rio Favara, Ragusa, Sicily, ItalySIC2236.7302214.89691PQ737586Hol1
Salinelle del fiume Simeto, Catania, Sicily, ItalySIC2337.5685714.86396PQ737587Hol5
Stagno di Balze Sottane, Bronte, Sicily, ItalySIC2437.8526514.83128PQ737588Hol5
Torrente Morello, Enna, Sicily, ItalySIC2537.5677514.20349PQ737589Hol1
Passo Campalto, Venezia, Veneto, ItalyVEN0145.4808512.29839PQ737590Hol1
Chadwick Lakes, Rabat village, Malta islandMLT0135.8841514.38234PQ737555Hol1
Pond, Ghaj il-Papri, Għajnsielem village, Gozo island, Malta islandsMLT0236.0227614.28793PQ737556Hol1
Pond, Ghajn Abdul, Gozo island, Malta islandsMLT0336.0487814.20874PQ737557Hol1
Pond, Għajnsielem village, Comino island, Malta islandsMLT0436.0160914.33691PQ737558Hol1
Vegetative pond, Għajnsielem village, Comino island, Malta islandsMLT0536.0097714.33558PQ737559Hol1
Reservoir, Għajnsielem village, Comino island, Malta islandsMLT0636.0125614.33750PQ737560Hol1
Reservoir, Nadur village, Gozo island, Malta islandsMLT0736.0501714.28233PQ737561Hol1
Pond, San Anton Palace, Attard village, Malta islandsMLT0835.8963214.44913PQ737562Hol1
Pond, Ta’ Qali, Attard village, Malta islandMLT0935.8927414.42058PQ737563Hol1
Sarraflu pond, Kerċem village, Gozo island, Malta islandsMLT1036.0366214.19906PQ737564Hol1
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MDPI and ACS Style

Vecchioni, L.; Liuzzo, M.; Sciberras, A.; Sciberras, J.; Formosa, J.; Deidun, A.; Giacalone, G.; Arizza, V.; Arculeo, M.; Marrone, F.; et al. Molecular Data Confirm the Occurrence of the Allochthonous Gambusia holbrooki (Pisces: Poeciliidae) in Sicily and the Maltese Archipelago. Diversity 2025, 17, 48. https://doi.org/10.3390/d17010048

AMA Style

Vecchioni L, Liuzzo M, Sciberras A, Sciberras J, Formosa J, Deidun A, Giacalone G, Arizza V, Arculeo M, Marrone F, et al. Molecular Data Confirm the Occurrence of the Allochthonous Gambusia holbrooki (Pisces: Poeciliidae) in Sicily and the Maltese Archipelago. Diversity. 2025; 17(1):48. https://doi.org/10.3390/d17010048

Chicago/Turabian Style

Vecchioni, Luca, Mirko Liuzzo, Arnold Sciberras, Jeffrey Sciberras, Justin Formosa, Alan Deidun, Gabriele Giacalone, Vincenzo Arizza, Marco Arculeo, Federico Marrone, and et al. 2025. "Molecular Data Confirm the Occurrence of the Allochthonous Gambusia holbrooki (Pisces: Poeciliidae) in Sicily and the Maltese Archipelago" Diversity 17, no. 1: 48. https://doi.org/10.3390/d17010048

APA Style

Vecchioni, L., Liuzzo, M., Sciberras, A., Sciberras, J., Formosa, J., Deidun, A., Giacalone, G., Arizza, V., Arculeo, M., Marrone, F., & Faraone, F. P. (2025). Molecular Data Confirm the Occurrence of the Allochthonous Gambusia holbrooki (Pisces: Poeciliidae) in Sicily and the Maltese Archipelago. Diversity, 17(1), 48. https://doi.org/10.3390/d17010048

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