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Article

First Record of the Alien Species Procambarus virginalis Lyko, 2017 in Fresh Waters of Sardinia and Insight into Its Genetic Variability

by
Daria Sanna
1,*,†,
Ilenia Azzena
1,2,†,
Fabio Scarpa
2,
Piero Cossu
2,
Angela Pira
3,
Flavio Gagliardi
3 and
Marco Casu
2
1
Dipartimento di Scienze Biomediche, Università di Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
2
Dipartimento di Medicina Veterinaria, Università di Sassari, Via Vienna 2, 07100 Sassari, Italy
3
Acquario di Cala Gonone, Via La Favorita, 08020 Cala Gonone, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Life 2021, 11(7), 606; https://doi.org/10.3390/life11070606
Submission received: 24 May 2021 / Revised: 11 June 2021 / Accepted: 22 June 2021 / Published: 24 June 2021
(This article belongs to the Special Issue Biological Invasions and Biodiversity)
Browse Figures
Versions Notes

Abstract

:
In the fresh waters of Sardinia (Italy), the non-indigenous crayfish species Procambarus clarkii has been reported from 2005, but, starting from 2019, there have been several reports of a new non-indigenous crayfish in southern and central areas of this Mediterranean island, and its morphology suggests that this species may be the marbled crayfish Procambarus virginalis. Forty-seven individuals of this putative species were analyzed, using the mitochondrial gene Cytochrome c Oxidase subunit I as molecular marker to identify this crayfish and investigate the level of genetic variability within the recently established population. Phylogenetic and phylogeographic analyses were carried out on a dataset including sequences from the Sardinian individuals and from all congenerics available in GenBank. Results showed that the new Sardinian crayfish belong to the species P. virginalis. All the sequences belonging to P. virginalis from European countries are identical, with only few exceptions found among Sardinian individuals. In conclusion, this paper highlights the occurrence of a new further alien species in the Sardinian fresh waters, which are already characterized by the high presence of non-indigenous species.

1. Introduction

The so-called freshwater crayfish (Malacostraca, Decapoda) are a monophyletic group of crustaceans with challenging taxonomy and phylogeny [1,2,3], which are present in each continent, except for Antarctica and mainland Africa [4,5], and occur in almost every type of freshwater habitat, both lentic and lotic [5,6,7].
As they are among the largest invertebrate predators in their habitats, freshwater crayfish are an important component in the structuring of the aquatic fauna [8,9]. Indeed, many studies showed a clear impact as a keystone species, due to their feeding activity, which mainly involves vegetation, invertebrates, and vertebrates such as fish. Furthermore, as ecosystem engineers, they can create major impacts on their habitat, affecting the sediment transport as a consequence of their constant search for new refugia, from riffles to pools and vice versa [7,8].
Over the years, multiple crayfish species were translocated, either deliberately or accidentally [10,11,12]. For this reason, at least one non-indigenous crayfish species (hereafter NICS, following Holdich [13]) has been reported in most European countries, and the general number of NICS is rapidly growing [14,15,16].
The presence of NICS can have serious consequences on native ecosystems [17], not only due to interspecific competition with indigenous species or habitat modification, but also by carrying diseases, such as the “crayfish plague”, i.e., Aphanomyces astaci [17]. This water mould is mainly transmitted by the largely resistant introduced North American crayfish species, and can lead to high levels of mortality, causing declines of indigenous fauna [18,19,20]. Furthermore, as this oomycete can survive on fish skin and use these animals as vectors, another way of transmission is represented by fish moving away from areas where the crayfish plague is present, which can thus spread the infection among drainage basins [21].
In Europe, there are few native species of crayfish [22,23,24], and the first documented introduction of a NICS dates back to 1890 in Barnowko village (Poland), where the spiny-cheek crayfish, Faxonius limosus (Rafinesque, 1817), was introduced for commercial purposes [25] from Pennsylvania (USA). It was followed by other NICS introductions, mainly Procambarus clarkii Girard, 1852 (firstly introduced in Spain in 1973), which is considered as a very harmful problematic NICS due to the plastic life cycle, capability of naturalization, and rapid potential for dispersal [26,27].
Later, in the 90s, in a German pet trade, a new species was identified for the first time, it was the marbled crayfish, Procambarus virginalis Lyko, 2017 [28].Preliminary studies hypothesized that P. virginalis might represent the product of hybridization between the sexually reproducing Procambarus fallax (Hagen, 1870) (from FL, USA) and its congeneric P. clarkii (from LA, USA) [29]. However, recent studies have highlighted the autopolyploid nature of the marbled crayfish, which is a triploid organism that differentiated from its mother species P. fallax [30,31]. All marbled crayfish show common phenotypic, genetic, and epigenetic characteristics, despite their broad geographical distribution [31] and reproduce by parthenogenesis, being able to quickly create “wild” populations in the temperate zones of the planet [32,33].
Considering the high interest in the marbled crayfish from aquarists, their presence in European ecosystems is probably due to the voluntary release into the wild carried out by some owners and traders [32,33,34,35,36,37]. To date, stable populations of P. virginalis in Europe are known from: Germany and Netherlands [10], Croatia [38], Romania [39], Slovakia [40], Estonia [41], Czech Republic [42], Hungary [43], Ukraine [44], and Sweden [45]. The spread of this species is not limited to Europe, in fact its presence has been also reported in Japan [46] and Madagascar [29,47]. Furthermore, although not yet mentioned in scientific publications, several individuals of marbled crayfish have been discovered in different freshwater areas of Poland, Taiwan, and Macau, based on recent local reports. In Italy, (where four autochthonous crayfish species occur [48]), the first report of P. virginalis dates back to 2008 [11], but, at present, stable populations are not reported.
In 2019, some individuals morphologically attributable to the marbled crayfish were found in freshwater habitats of southern Sardinia (Italy). Just one year later, individuals that can be identified as marbled crayfish have been found in many other areas of Sardinia (pers. obs.). In this Mediterranean island, two other species of crayfish are known so far: Austropotamobius pallipes Lereboullet, 1858, which is indigenous to Italy, and is rarely found in Sardinian freshwaters, where, likely, its occurrence is not natural but due to translocation events [49]; and the NICS P. clarkii, which was recorded for the first time in 2005 [50] and it is now widely distributed across the island.
This paper aimed to: (1) identify the new freshwater crayfish found in Sardinia, applying a species delimitation approach; (2) investigate the levels of the species’ genetic variability in Sardinia; and (3) perform phylogenetic and phylogeographic analyses on the newly established population (the only known in Italy), comparing sequences of Sardinian individuals with those already available in the literature, in order to place the data obtained in a wider geographic background.

2. Materials and Methods

2.1. Sample Collection

Forty-seven individuals were analysed (Table 1), whose morphology was consistent with marbled crayfish or the closely related slough crayfish, Procambarus fallax following Hobbs [51].
Forty-one adult individuals, which were tentatively identified as the marbled crayfish (Procambarus virginalis), were collected from May to December 2019 in the central and southern areas of Sardinia which are the alleged center of the first dispersion of this crayfish in the island (see Figure 1 and Table 1 for details). Six eggs (specimens PFMO8–PFMO13 in Table 1), taken from one of the adult individuals, were added to our molecular analyses. In particular, we performed a non-standardized survey in the three areas of the island where the possible occurrence of this species was reported by local communities (see Figure 1 and Table 1 for details on sampling stations). The waterbodies of these sampling areas are characterized by slow flowing, irregular streams and temporary natural or artificial ponds, mainly with muddy and poorly vegetated riverbeds. Natural streams are often connected one another by drainage canals, which greatly facilitate crayfish spreading. Live crayfish, collected using fish traps, were delivered to the laboratory to be sacrificed by an anaesthetic overdose, and then stored at −20 °C until the DNA extraction.

2.2. Diagnostic Molecular Analysis

Total genomic DNA was isolated from a portion of muscle tissue using the Macherey-Nagel Nucleo Spin Tissue Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) following the supplier’s instructions. DNA solutions were quantified using the Nanodrop™ Lite Spectrophotometer (by Thermo Scientific; Waltham, MA, USA), which showed an average yield of approximately 30 ng/μL.
A fragment of the subunit I of the mitochondrial Cytochrome c Oxidase gene (COI) was amplified by standard PCR using universal primers [52]. Reactions were carried out in a total volume of 25 μL. On average, 10 ng of total genomic DNA were combined with 0.6 μM of each primer and one pellet of PuReTaq Ready-To-Go PCR beads (GE Healthcare, Wauwatosa, WI, USA) containing stabilizers, bovine serum albumin (BSA), deoxynucleotide triphosphates, 2.5 units of PuReTaq DNA polymerase, and reaction buffer. When a bead was reconstituted to a 25 μL final volume, the concentration of each dNTP and MgCl2 was set at 200 μM and 1.5 mM, respectively. PCRs were performed in a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, Waltham, MA, USA), programmed as follows: 1 cycle of 4 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 48 °C, and 30 s at 72 °C. At the end, a post-treatment of 10 min at 72 °C and a final cooling at 4 °C were carried out. Both positive (high-quality DNA samples from the congeneric P. clarkii) and negative controls were used to test the effectiveness of the PCR protocols, and the absence of possible contaminations. Electrophoresis was carried out on 2% agarose gels, prepared using 1× TAE buffer (Tris-Acetate-EDTA, pH 8.3) and stained with Gel Red Nucleic Acid Stain (Biotium Inc., Fremont, CA, USA). PCR products were purified by ExoSAP-IT (USB Corporation, Cleveland, OH, USA) and sequenced for forward and reverse strands (by means of the same primers used for PCR), using an external sequencing core service (Macrogen, Europe, Amsterdam, The Netherlands).

2.3. Phylogenetic and Phylogeographic Analyses

All the sequences obtained from crayfish adults and eggs were identified as belonging to the species Procambarus virginalis through BLAST analysis implemented in the GenBank nucleotide database (www.ncbi.nlm.nih.gov (accessed on 21 July 2020)) that showed a 100% identity.
Sequences were aligned using the package Clustal Omega [53] (available at https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 4 May 2021)) after a manual checking and editing by means of Unipro UGENE v.35 (by the Unipro Center for Information Technologies, Novosibirsk, Russia) [54].
COI sequences of the marbled crayfish collected in Sardinia were aligned with the sequences belonging to P. virginalis and P. fallax from other localities (Belgium, Czech Republic, Germany, Italy, Sweden, Japan, and Florida—USA) so far available on GenBank (last update 5 April 2021) (see Table 1 for details). As outgroups, three sequences belonging to the species P. clarkii from two different localities were chosen: two from Sardinia (obtained in the present study) (Genbank accession numbers: MZ099652, MZ099653), and one from Alabama (USA) (Genbank accession number: KX417114).
Levels of genetic variation among sequences were assessed estimating the number of polymorphic sites (S), number of haplotypes (H), nucleotide diversity (π), and haplotype diversity (h), using the software package DnaSP 6.12.03 (by Universitat de Barcelona, Barcelona, Spain) [55].
To assess the taxonomic status of crayfish collected in Sardinia, the nucleotide divergence threshold (NDT) method of species delimitation [56] was also performed. The NDT method is based on genetic distances and does not consider the phylogenetic relationships within the dataset. It works on sequences to rank taxa into taxonomic entities applying the fixed threshold of 2% given by Hebert et al. [56] for DNA barcodes, using the pairwise Kimura (1980) two-parameter model (K2P) to compute the matrix of genetic distances [57]. The analysis was performed by means of a script [58] written in the R statistical environment (available at https://cran.r-project.org/ (accessed on 5 May 2021)).
The probabilistic model of sequence evolution that better fit the sequence data was detected using the software JmodelTest 2.1.7 [59]. Based on the best-fitting model, a Bayesian phylogenetic species tree was obtained using the software MrBayes 3.2.7 [60] setting as model parameters: NST = 6, rates = invgamma, ngammacat = 4. Two independent runs, each consisting of four Metropolis-Coupled MCMC chains (one cold and three heated chains), were run simultaneously for 5,000,000 generations, sampling trees every 1000 generations. The first 25% of the 10,000 sampled trees was discarded as burn-in. To assess the convergence of chains, we checked that the Average Standard Deviation of Split Frequencies (ASDSF) approached 0 [60], and the Potential Scale Reduction Factor (PSRF) was around 1 [61], following Scarpa et al. [62]. The phylogenetic tree was visualized and edited using FigTree 1.4.0 (available at http://tree.bio.ed.ac.uk/software/figtree/).
To distinguish genetic clusters, a Principal Coordinate Analysis (PCoA) was also performed on a K2P pairwise genetic distance matrix using GenAlEX 6.5 [63]. Thevariation rate among sites was modelled with a gamma distribution and all ambiguous positions were removed for each sequence pair.
A median-joining network [64] was constructed using the software package Network 10.0.0.0 (www.fluxus-engineering.com) to infer the genetic relationships among haplotypes and to detect the occurrence (if any) of discrete genetic clusters. The transitions and transversions were equally weighted. Due to the lack of knowledge on the possible occurrence of retromutation events, the same weight (10) was assigned to each observed polymorphism.

3. Results

In the present study, 47 COI sequences of 617 bp length were obtained for Procambarus virginalis and deposited in GenBank (see Table 1 for accession numbers). Among the sequences belonging to Sardinian P. virginalis, a very low level of genetic divergence was found, with three polymorphic sites resulting in four haplotypes (see Table 2 for details on genetic divergence estimates). A high percentage (93.62%) of Sardinian P. virginalis share the same haplotype, while 6.38% of the Sardinian individuals showed a private lineage.
The genetic analysis performed on the dataset including all the Sardinian COI sequences obtained in the present study, and those belonging to P. virginalis and P. fallax deposited in GenBank to date, evidenced 14 polymorphic sites and 9 haplotypes resulting in a low level of genetic variability (see Table 2 for details on genetic divergence estimates) for P. virginalis and P. fallax. In particular, three haplotypes were found in Sardinian individuals. The NDT species delimitation method ranked all the sequences belonging to P. virginalis (also including the Sardinian sequences) and P. fallax, into a unique taxonomic entity.
Accordingly, in the phylogenetic tree (Figure 2), all the sequences belonging to P. virginalis and P. fallax from Sardinia, other European countries, and Japan, were included in a well-supported monophyletic clade, with an internal sub-cluster that included a sequence of P. fallax from Florida (USA) and two sequences of P. fallax isolated in Germany. The other sequences belonging to P. fallax from Florida were set outside the main clade.
The PCoA plot (Figure 3) showed the occurrence of four groups of sequences (groups 1–4). A genetic similarity was found along the x-axis between the group 2—including all the Sardinian sequences and almost all the sequences of P. virginalis and P. fallax from European countries and Japan—and the groups 1 and 3 which included sequences of P. fallax isolated in Florida (see Appendix A Table A1 for details). A further divergent group (group 4) of three sequences, already evidenced in the phylogenetic tree (Figure 2), was separated along the x-axis, and included two sequences of P. fallax isolated in Germany and one sequence of P. fallax isolated in Florida.
The network analysis (Figure 4) showed a typical star-like shape, with the occurrence of a common haplotype shared by 93.75% and 25.00% of individuals belonging to P. virginalis and P. fallax (all collected in Germany and Sweden), respectively. Four sequences of P. virginalis diverged from the common haplotype by one- or two-point mutations; three of them were isolated in Sardinia (two from Arborea and one from Sanluri) and one in Japan. Further sequences belonging to P. fallax collected in Florida and Germany diverged by 3- to 4-point mutations.

4. Discussion

This paper reports the first record of Procambarus virginalis in Sardinia, supported by molecular identification. This study also represents the first insight into the genetic variability of the first Italian population of P. virginalis, as only one individual from Tuscany [10] and three from Veneto [65] are known from Italy to date. New inferences are also provided on the global genetic variation of this species.
Overall, the Sardinian population of the marble crayfish is characterized by a very low level of genetic variability, with a high percentage of specimens sharing the same haplotype, apart from three individuals which show new, slightly divergent haplotypes. Two possible scenarios may be invoked to explain this remarkable finding: (1) the parthenogenetic marble crayfish was introduced in Sardinia with a highly variable group of individuals, and further studies, involving a greater number of specimens from areas outside Sardinia, are needed to check for the occurrence of these never reported haplotypes in the mainland; or (2) based on the current knowledge, we cannot rule out that the three private haplotypes found in Sardinia might stem as the result of strong selective pressures acting on the population of this recently introduced invasive species.
Indeed, all the sequences belonging to P. virginalis from European countries are identical, with only few exceptions. The most common haplotype of P. virginalis also occurs in P. fallax from Germany and Sweden, which may represent individuals directly imported from the USA. Interestingly, no one of the COI sequences isolated in P. fallax from the USA correspond to the most common lineage found in P. virginalis from Europe and Japan. The lack of the most common, worldwide spread haplotype of P. virginalis among the sequences of P. fallax from the USA might reflect their low number so far available on GenBank. However, this pattern may also depend on the genetic drift resulting from the small number of founders selected in the USA to be imported in Europe for the pet trade. In this latter case, only a small and poorly representative portion of the source population genetic variability might have been transferred to the European populations, thus resulting in the lack of shared haplotypes between the two continents.
Furthermore, when adaptive evolution takes place, the molecular lineages of introduced populations can be rare, or not expressed in the source population but become common and distinctive among individuals in the new colonized habitats as a consequence of founder events and natural selection [66]. Although representing an uncommon trend, selective pressure on mtDNA promoted by new environmental conditions was already reported for the highly invasive Lessepsian migrant Fistularia commersonii [67], whose recently established Mediterranean population does not include any mitochondrial lineages present among individuals of the source population. For this reason, in the case of P. virginalis, mitochondrial variants, arrived in Europe from the USA, may have undergone a selective sweep which might have favoured the spread in European populations of rare alleles that likely allow a better adaptation of the species to new environmental conditions.

5. Conclusions

Although there is no evidence of the presence of native crayfish in Sardinia, the autochthonous nature of Austrapotamobius pallipes, included in Annexes II and V of the European Union Habitats Directive 2000 (92/43/EEC), is still controversial. Nonetheless, there are endemic species of amphibians that may suffer due to the presence of this new invasive alien species in their fresh waters. For instance, Souty-Grosset et al. [68] and Oficialdegui et al. [69] showed that crayfish eat amphibian eggs and tadpoles. Additionally, competition for food could also indirectly reduce amphibian populations, as the grazing activity that crayfish exert on periphyton may lead periphyton-associated invertebrates to disappear [70]. Lastly, there is evidence that crayfish could carry the chytrid Batrachochytrium dendrobatidis (see Battisti and Scalici [26] and references therein), which may cause diseases in populations of Sardinian endemic amphibians.
Therefore, future studies that investigate the stomach content of these crayfish would be useful to provide a clear idea of their impact on Sardinian native fauna. At the same time, further studies are needed to investigate the presence in the island of the oomycete Aphanomyces astaci, which is generally associated to crayfish. Indeed, the presence of A. astaci in other crustaceans, such as Eriocheir sinensis [71,72], suggests the occurrence of spill over events.

Author Contributions

Conceptualization, D.S., F.S. and M.C.; methodology, D.S. and I.A.; software, F.S.; validation, D.S., F.S. and M.C.; formal analysis, D.S., I.A. and F.S.; investigation, D.S., I.A. and F.S.; resources, D.S. and M.C.; data curation, D.S., I.A. and F.S.; writing—original draft preparation, D.S., I.A. and M.C.; writing—review and editing, D.S., I.A., F.S., P.C. and M.C.; visualization, D.S., I.A., F.S., P.C., I.A., A.P., F.G. and M.C.; supervision, D.S. and M.C.; project administration, D.S. and M.C.; and funding acquisition, D.S. and M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by FONDO DI ATENEO PER LA RICERCA 2019 (to Daria Sanna and Marco Casu) of the University of Sassari (Italy), grant numbers FAR2019SANNAD and FAR2019CASUM.

Institutional Review Board Statement

Scientific research and collecting of specimens of P. fallax f. virginalis in Sardinia were authorised by Regione Autonoma della Sardegna during the activities for management of invasive alien species (Regulation (EU) no. 1143/2014 and Legislative Decree 230 of 15 December 2017): Procambarus fallax f. virginalis—presentation of regional guidelines for rapid eradication. Project protocol n. 18164—17 September 2020.

Informed Consent Statement

Not applicable.

Data Availability Statement

Sequences obtained in the present study for the mitochondrial Cytochrome c Oxidase subunit I gene isolated in Sardinian crayfish were deposited in the GenBank database under the accession numbers MZ097549-95, MZ099652-53.

Acknowledgments

We are truly indebted with the veterinary technical officer Davide Brugnone, of the Assessorato della Difesa dell’Ambiente of the Regione Autonoma della Sardegna for his precious and invaluable support during the preliminary phases of this study. Furthermore, we would like to thank Commissioner Maria Tiziana Pinna and Senior Inspector Giuseppe Santucciu of the Servizio Ispettorato Ripartimentale di Oristano—Stazione Forestale and of the Vigilanza Ambientale di Marrubiu; Paolo Briguglio of the Duemari veterinary clinic of Oristano; and Angelo Ruiu of the Istituto Zooprofilattivo Sperimentale G. Pegreffi di Oristano, for their help in the sampling collection.

Conflicts of Interest

The authors and the founders declare no conflicts of interest.

Appendix A

Table
Table A1. Principal Coordinate Analysis (PCoA) results. The table shows the specific composition of the four groups of sequences.
Table A1. Principal Coordinate Analysis (PCoA) results. The table shows the specific composition of the four groups of sequences.
Group 1
NORTH AMERICA
Sample IDCountrySpecies
PFFL1Florida—USAProcambarus fallax
PFFL2Florida—USAProcambarus fallax
PFFL3Florida—USAProcambarus fallax
PFFL7Florida—USAProcambarus fallax
Group 2
EUROPE
Sample IDCountrySpecies
PFGE2GermanyProcambarus fallax
PFGE3GermanyProcambarus fallax
PFGE4GermanyProcambarus virginalis
PFGE5GermanyProcambarus virginalis
PFGE6GermanyProcambarus virginalis
PFGE7GermanyProcambarus virginalis
PFCR1Czech RepublicProcambarus virginalis
PFSW1SwedenProcambarus virginalis
PFBE1BelgiumProcambarus virginalis
PFBE2BelgiumProcambarus virginalis
PFBE3BelgiumProcambarus virginalis
PFBE4BelgiumProcambarus virginalis
PFBE5BelgiumProcambarus virginalis
PFBE6BelgiumProcambarus virginalis
PFBE7BelgiumProcambarus virginalis
PFBE8BelgiumProcambarus virginalis
PFBE9BelgiumProcambarus virginalis
PFBE10BelgiumProcambarus virginalis
PFIT1Peninsular Italy—VenetoProcambarus virginalis
PFMO1Insular Italy—SardiniaProcambarus virginalis
PFMO2Insular Italy—SardiniaProcambarus virginalis
PFMO3Insular Italy—SardiniaProcambarus virginalis
PFMO4Insular Italy—SardiniaProcambarus virginalis
PFMO5Insular Italy—SardiniaProcambarus virginalis
PFMO6Insular Italy—SardiniaProcambarus virginalis
PFMO7Insular Italy—SardiniaProcambarus virginalis
PFMO8Insular Italy—SardiniaProcambarus virginalis
PFMO9Insular Italy—SardiniaProcambarus virginalis
PFMO10Insular Italy—SardiniaProcambarus virginalis
PFMO11Insular Italy—SardiniaProcambarus virginalis
PFMO12Insular Italy—SardiniaProcambarus virginalis
PFMO13Insular Italy—SardiniaProcambarus virginalis
PFOR1Insular Italy—SardiniaProcambarus virginalis
PFOR2Insular Italy—SardiniaProcambarus virginalis
PFOR5Insular Italy—SardiniaProcambarus virginalis
PFOR6Insular Italy—SardiniaProcambarus virginalis
PFOR7Insular Italy—SardiniaProcambarus virginalis
PFOR8Insular Italy—SardiniaProcambarus virginalis
PFOR9Insular Italy—SardiniaProcambarus virginalis
PFOR10Insular Italy—SardiniaProcambarus virginalis
PFOR11Insular Italy—SardiniaProcambarus virginalis
PFOR12Insular Italy—SardiniaProcambarus virginalis
PFOR13Insular Italy—SardiniaProcambarus virginalis
PFOR14Insular Italy—SardiniaProcambarus virginalis
PFOR15Insular Italy—SardiniaProcambarus virginalis
PFOR16Insular Italy—SardiniaProcambarus virginalis
PFOR17Insular Italy—SardiniaProcambarus virginalis
PFOR18Insular Italy—SardiniaProcambarus virginalis
PFOR19Insular Italy—SardiniaProcambarus virginalis
PFOR20Insular Italy—SardiniaProcambarus virginalis
PFOR21Insular Italy—SardiniaProcambarus virginalis
PFOR22Insular Italy—SardiniaProcambarus virginalis
PFOR23Insular Italy—SardiniaProcambarus virginalis
PFOR24Insular Italy—SardiniaProcambarus virginalis
PFOR25Insular Italy—SardiniaProcambarus virginalis
PFSL1Insular Italy—SardiniaProcambarus virginalis
PFSL2Insular Italy—SardiniaProcambarus virginalis
PFSL3Insular Italy—SardiniaProcambarus virginalis
PFSL4Insular Italy—SardiniaProcambarus virginalis
PFSL5Insular Italy—SardiniaProcambarus virginalis
PFSL6Insular Italy—SardiniaProcambarus virginalis
PFSL7Insular Italy—SardiniaProcambarus virginalis
PFSL8Insular Italy—SardiniaProcambarus virginalis
PFSL9Insular Italy—SardiniaProcambarus virginalis
PFSL10Insular Italy—SardiniaProcambarus virginalis
PFSL11Insular Italy—SardiniaProcambarus virginalis
ASIA
Sample IDCountrySpecies
PFJA1JapanProcambarus virginalis
Group 3
NORTH AMERICA
Sample IDCountrySpecies
PFFL5Florida—USAProcambarus fallax
PFFL6Florida—USAProcambarus fallax
Group 4
NORTH AMERICA
Sample IDCountrySpecies
PFFL4Florida—USAProcambarus fallax
EUROPE
Sample IDCountrySpecies
PFGE1GermanyProcambarus fallax
PFGE8GermanyProcambarus fallax

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Figure 1. The three geographic areas of the sites where samples were collected are indicated in the map of the Mediterranean island of Sardinia.
Figure 1. The three geographic areas of the sites where samples were collected are indicated in the map of the Mediterranean island of Sardinia.
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Figure 2. Bayesian phylogenetic tree showing the relationships among Procambarus virginalis from Sardinia (indicated with a red font) and marble crayfish from all over the world. Values of support at each node are expressed in Posterior Probabilities. The samples codes are as reported in Table 1.
Figure 2. Bayesian phylogenetic tree showing the relationships among Procambarus virginalis from Sardinia (indicated with a red font) and marble crayfish from all over the world. Values of support at each node are expressed in Posterior Probabilities. The samples codes are as reported in Table 1.
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Figure 3. The plot of the Principal Coordinate Analysis (PCoA) evidences the genetic relationships among sequences (and sample sites) based on a matrix of genetic distances. The codes of sequences indicated on the right are as reported in Table 1.
Figure 3. The plot of the Principal Coordinate Analysis (PCoA) evidences the genetic relationships among sequences (and sample sites) based on a matrix of genetic distances. The codes of sequences indicated on the right are as reported in Table 1.
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Figure 4. The median-joining network shows the relationships among COI gene haplotypes. The small red spots on the nodes show median vectors representing the hypothetical connecting sequences that were calculated using the maximum parsimony method. The numbers of mutations between sequences that are greater than 1 are reported on network branches.
Figure 4. The median-joining network shows the relationships among COI gene haplotypes. The small red spots on the nodes show median vectors representing the hypothetical connecting sequences that were calculated using the maximum parsimony method. The numbers of mutations between sequences that are greater than 1 are reported on network branches.
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Table
Table 1. The table reports data on the sampling collection. Sampling sites for each geographic area are indicated for the individuals of Procambarus virginalis collected in Sardinia during the present study. Details are also provided for the GenBank sequences of P. virginalis and P. fallax from all over the world used for the analyses.
Table 1. The table reports data on the sampling collection. Sampling sites for each geographic area are indicated for the individuals of Procambarus virginalis collected in Sardinia during the present study. Details are also provided for the GenBank sequences of P. virginalis and P. fallax from all over the world used for the analyses.
Sample IDSampling SiteSpeciesGB #CoordinatesHabitatSampling Date
LongLat
PFMO1Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975498,6854028639,6536863CreekMay 2019
PFMO2Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975508,6854028639,6536863CreekMay 2019
PFMO3Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975518,6854028639,6536863CreekMay 2019
PFMO7Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975558,6854028639,6536863CreekMay 2019
PFOR1Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975598,57123324439,72729907CreekMay 2019
PFMO4Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975528,6854028639,6536863CreekJune 2019
PFMO5Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975538,6854028639,6536863CreekJune 2019
PFMO6Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975548,6854028639,6536863CreekJune 2019
PFMO8Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975568,6854028639,6536863CreekJune 2019
PFMO9Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975578,6854028639,6536863CreekJune 2019
PFMO10Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975588,6854028639,6536863CreekJune 2019
PFMO11Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975608,6854028639,6536863CreekJune 2019
PFMO12Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975618,6854028639,6536863CreekJune 2019
PFMO13Italy (Sardinia)—MorimentaProcambarus virginalisMZ0975628,6854028639,6536863CreekJune 2019
PFOR2Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975638,57123324439,72729907CreekNovember 2019
PFOR5Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975648,57123324439,72729907CreekNovember 2019
PFOR6Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975658,57123324439,72729907CreekNovember 2019
PFOR7Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975668,57123324439,72729907CreekNovember 2019
PFOR8Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975678,57123324439,72729907CreekNovember 2019
PFOR9Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975688,57123324439,72729907CreekNovember 2019
PFOR10Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975698,57123324439,72729907CreekNovember 2019
PFOR11Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975708,62150848139,82035494CreekNovember 2019
PFOR12Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975718,62150848139,82035494CreekNovember 2019
PFOR13Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975728,62150848139,82035494CreekNovember 2019
PFOR14Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975738,62150848139,82035494CreekNovember 2019
PFOR15Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975748,62150848139,82035494CreekNovember 2019
PFOR16Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975758,57845711839,82507761CreekNovember 2019
PFOR17Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975768,57845711839,82507761CreekNovember 2019
PFOR18Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975778,54928494339,74396601CreekNovember 2019
PFOR19Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975788,6087350839,8203551CreekNovember 2019
PFOR20Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975798,6087350839,8203551CreekNovember 2019
PFOR21Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975808,6087350839,8203551CreekNovember 2019
PFOR22Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975818,6087350839,8203551CreekNovember 2019
PFOR23Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975828,6087350839,8203551CreekNovember 2019
PFOR24Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975838,6087350839,8203551CreekNovember 2019
PFOR25Italy (Sardinia)—ArboreaProcambarus virginalisMZ0975848,6087350839,8203551CreekNovember 2019
PFSL1Italy (Sardinia)—SanluriProcambarus virginalisMZ0975858,84262859239,51785049CreekDecember 2019
PFSL2Italy (Sardinia)—SanluriProcambarus virginalisMZ0975868,84262859239,51785049CreekDecember 2019
PFSL3Italy (Sardinia)—SanluriProcambarus virginalisMZ0975878,84262859239,51785049CreekDecember 2019
PFSL4Italy (Sardinia)—SanluriProcambarus virginalisMZ0975888,84262859239,51785049CreekDecember 2019
PFSL5Italy (Sardinia)—SanluriProcambarus virginalisMZ0975898,84262859239,51785049CreekDecember 2019
PFSL6Italy (Sardinia)—SanluriProcambarus virginalisMZ0975908,84429500539,51785048CreekDecember 2019
PFSL7Italy (Sardinia)—SanluriProcambarus virginalisMZ0975918,84429500539,51785048CreekDecember 2019
PFSL8Italy (Sardinia)—SanluriProcambarus virginalisMZ0975928,84429500539,51785048CreekDecember 2019
PFSL9Italy (Sardinia)—SanluriProcambarus virginalisMZ0975938,84429500539,51785048CreekDecember 2019
PFSL10Italy (Sardinia)—SanluriProcambarus virginalisMZ0975948,84429500539,51785048CreekDecember 2019
PFSL11Italy (Sardinia)—SanluriProcambarus virginalisMZ0975958,84429500539,51785048CreekDecember 2019
PFBE8BelgiumProcambarus virginalisLR884227--PondMay 2020
PFBE9BelgiumProcambarus virginalisLR884226--PondMay 2020
PFBE10BelgiumProcambarus virginalisLR884225--MoatMay 2020
PFBE5BelgiumProcambarus virginalisLR884230--PondJune 2020
PFBE6BelgiumProcambarus virginalisLR884229--PondJune 2020
PFBE7BelgiumProcambarus virginalisLR884228--BasinJune 2020
PFBE1BelgiumProcambarus virginalisLR884234--MoatJuly 2020
PFBE2BelgiumProcambarus virginalisLR884233--MoatJuly 2020
PFBE3BelgiumProcambarus virginalisLR884232--PondJuly 2020
PFBE4BelgiumProcambarus virginalisLR884231--PondJuly 2020
PFCR1Czech RepublicProcambarus virginalisMK439899----
PFIT1Italy (Veneto)Procambarus virginalisKJ690261--ChannelApril 2009
PFJA1JapanProcambarus virginalisLC228303---November 2016
PFGE2GermanyProcambarus virginalisHM358011--StreamOctober 2009
PFGE5GermanyProcambarus virginalisKC107813----
PFGE6GermanyProcambarus virginalisKT074364----
PFGE4GermanyProcambarus virginalisHM358010--StreamOctober 2009
PFGE1GermanyProcambarus fallaxHM358012---October 2009
PFGE3GermanyProcambarus fallaxJF438007--Lake-
PFGE8GermanyProcambarus fallaxKT074365----
PFGE7GermanyProcambarus fallaxNC_020021----
PFFL1Florida—USAProcambarus fallaxHQ171459----
PFFL2Florida—USAProcambarus fallaxHQ171458----
PFFL3Florida—USAProcambarus fallaxHQ171457----
PFFL4Florida—USAProcambarus fallaxHQ171456----
PFFL5Florida—USAProcambarus fallaxHQ171455----
PFFL6Florida—USAProcambarus fallaxHQ171453----
PFFL7Florida—USAProcambarus fallaxHQ171454----
PFSW1SwedenProcambarus fallaxKF033123--RiverDecember 2012
# stands for accession number.
Table
Table 2. Genetic divergence estimates for Procambarus virginalis and Procambarus fallax based on COI gene sequences.
Table 2. Genetic divergence estimates for Procambarus virginalis and Procambarus fallax based on COI gene sequences.
NbpSHhdπ
Sardinia47617340.125 ± 0.0650.00021
Whole dataset766171490.312 ± 0.0690.00178
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Sanna, D.; Azzena, I.; Scarpa, F.; Cossu, P.; Pira, A.; Gagliardi, F.; Casu, M. First Record of the Alien Species Procambarus virginalis Lyko, 2017 in Fresh Waters of Sardinia and Insight into Its Genetic Variability. Life 2021, 11, 606. https://doi.org/10.3390/life11070606

AMA Style

Sanna D, Azzena I, Scarpa F, Cossu P, Pira A, Gagliardi F, Casu M. First Record of the Alien Species Procambarus virginalis Lyko, 2017 in Fresh Waters of Sardinia and Insight into Its Genetic Variability. Life. 2021; 11(7):606. https://doi.org/10.3390/life11070606

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Sanna, Daria, Ilenia Azzena, Fabio Scarpa, Piero Cossu, Angela Pira, Flavio Gagliardi, and Marco Casu. 2021. "First Record of the Alien Species Procambarus virginalis Lyko, 2017 in Fresh Waters of Sardinia and Insight into Its Genetic Variability" Life 11, no. 7: 606. https://doi.org/10.3390/life11070606

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