Layout 1 INTRODUCTION Italian inland waters are known to be heavily affected by the occurrence of non-indigenous species (NIS) (Gher- ardi et al., 2007). These biological invaders are one of the major threats to native freshwater fauna, altering the habi- tat structure and assemblage composition and ultimately leading to a significant loss of native biodiversity (Naselli- Flores and Marrone, 2019 and references therein). Italy is characterized by a rich fauna of Cladocera, which includes 17 Daphnia O.F. Müller, 1785 species be- longing to the subgenera Daphnia s.s. and Ctenodaphnia, plus several subspecies or hybrid taxa of dubious taxo- nomical value (Ruffo and Stoch, 2005; Marrone et al., 2007). Within the species belonging to the genus Daphnia s.s. occurring in Italy, two are allochthonous taxa of Nearctic origin, i.e., D. ambigua Scourfield, 1947 and D. parvula Fordyce, 1901 (Margaritora, 1985; Riccardi et al., 2004). Furthermore, the presence of a non-native lineage of American origin within D. pulex Leydig, 1860 was recorded in Sardinia and Piedmont (Fadda et al., 2011; Marková et al., 2013). To date, the only non-native cladocerans positively known to occur in Sicily are Daphnia parvula and D. am- bigua (Marrone et al., 2006; Marrone and Naselli-Flores, 2015), although also D. galeata G.O. Sars, 1864 and D. cucullata G.O. Sars, 1862 have a dubious status in the re- gion (Marrone and Vecchioni, 2020). Moreover, no mo- lecular data are to date available on the Sicilian populations of D. pulex, so that no information about the native or non-native status of Sicilian D. pulex is avail- able. Furthermore, it must be taken into account that the taxonomy of the Daphnia pulex complex is rather difficult due to the absence of clear morphological features distin- guishing among taxa listed under Daphnia pulex (Conde- Porcuna et al., 2020 and reference therein), a binomen which is misleadingly used for distinct different evolu- tionary lineages belonging to different biogeographical regions (Mergeay et al., 2008; Crease et al., 2012; Ma et al., 2019). Among them, the Nearctic lineage has been in- troduced in several countries, attaining an almost world- wide distribution with the sole exception of Antarctica (Crease et al., 2012; Conde-Porcuna et al., 2020). In the light of the evidence of the presence of the al- lochthonous North American lineage of Daphnia pulex in Italian mainland and Sardinia (Fadda et al., 2011; Marková et al., 2013, 2017), the aim of this work was to identify the Daphnia lineages occurring in the Sicilian populations using a mitochondrial molecular marker, and thus verifying whether the evolutionary lineages occur- ring in Sicily belong to the allochthonous Nearctic or the autochthonous European groups. Accordingly, we carried out samplings in a selection of the known sites of occur- rence of the species, and in an unpublished site where the species was found to occur. METHODS Based on the data available in the literature, Daphnia pulex is currently known for 19 sites in Sicily, which are limited to medium-high altitudes of the Nebrodi area, Madonie area, Etna and Bosco della Ficuzza (Marrone and Vecchioni, 2020). The populations from which sam- ples of Daphnia pulex to be molecularly characterized ARTICLE Molecular data attest to the occurrence of autochthonous Daphnia pulex (Crustacea, Branchiopoda) populations in Sicily, Italy Luca Vecchioni,* Marco Arculeo, Federico Marrone Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, via Archirafi 18, 90123 Palermo, Italy ABSTRACT Biological invasions are known to be among the most important threats to the long-term conservation of native biota, and their effects might be even more difficult to contrast when they are cryptic, i.e., when the non-native invaders cannot be easily recognised based on morphology, and can thus be confused with native taxa. Such cryptic invasions are known to widely occur in the cladoceran genus Daphnia O.F. Müller, 1785, so that the actual distribution and status of most species and lineages need to be checked with a genetic approach. In the frame of this work, we investigated if the Sicilian populations of D. (Daphnia) pulex Leydig, 1860 be- longed to the allochthonous North American lineage, which is known to occur in several regions of the Palearctic and Afrotropical biogeographical regions, or rather to the autochthonous European lineage of the species. The molecular results obtained, based on a fragment of the mitochondrial gene encoding for NADH subunit dehydrogenase 5 (ND5), allowed us to rule out the allochthonous status of the species, confirming the presence of autochthonous relictual lineages of D. pulex in Sicily. The native status of these populations is in agreement with their local distribution, limited to natural and poorly-impacted water bodies mostly located in wooded areas at medium and high altitudes. The current local distribution of D. pulex in Sicily is possibly linked to the end of the last glacial maximum and the onset of warmer climatic conditions in the early Holocene, which led the species to take refuge in colder microthermal refugia located at high altitudes, determining their current relictual distribution. No n- co mm er cia l u se on ly L. Vecchioni et al.30 were collected were chosen based on the known occur- rence sites, with the aim of including in the analyses at least a population from each of the four major distribution subareas of the species on the island. In addition, one un- published site of occurrence was found in the context of this sampling campaign (“ME076” – 37.94158 N, 14.683676 E – 1559 m a.s.l.). A map of the known and sampled occurrence Sicilian sites of the species was made using QGIS software v. 3.18 (http://www.qgis.org). Microcrustaceans were collected both in open waters and in the littoral areas of each water body through the use of hand- and\or towing-plankton nets with a mesh size of 125-200 μm, depending on size, depth and vegetation cover of the sampled sites. The collected samples were fixed in situ with 96% ethanol and microcrustaceans were sorted out in laboratory under a stereomicroscope. Clado- cera were morphologically identified according to Mar- garitora (1985), Alonso (1996), and Benzie (2005). All the studied samples are currently stored in FM’s branchiopod collection at the University of Palermo, Italy, and are available for loan on request. A single D. pulex individual from each population was carefully cleaned of any impurities and soaked in double- distilled water for 5-10 minutes in order to eliminate the residual ethanol, and then processed for DNA extraction using the BIORON GmbH “Ron’s Tissue DNA Mini Kit”, following the manufacturer’s instructions. The selective amplification of a fragment from the gene encoding NADH subunit dehydrogenase 5 (ND5), was carried out by polymerase chain reaction (PCR) using the primers ND5newF (5’-AAA CCT CTA AAB TTC YKA RCT- 3 ‘) and ND5newR (5’-CAT RTT YAT RTC RGG GGT TGT- 3’), described by Dufresne et al. (2011). The PCR mix consisted of 18.9 μl of distilled water, 2.5 μl of Buffer 10X which includes 15 mM of MgCl2, 0.4 μl of dNTPs (10 mM for each), 0.4 μl of each of the primers (10 μM), 0.4 μl of Taq polymerase (5 U / μl) and 2 μl of template DNA, for a total volume of 25 μl. The thermal cycle consisted of 35 cycles of denaturation (94°C for 1 min), annealing (50°C for 1 min) and extension (72°C for 1 min), followed by five minutes at 72°C for the final extension step. After PCR, 5 μl of each PCR product were used to perform electrophoresis on 2% agarose gel at 90 V for 20 min and then visualized with a UV transilluminator. When PCR products showed a clear single band, of the expected length, they were purified using the Exo-SAP-IT® kit (Affymetrix USB, Santa Clara, CA, USA). Sequencing was performed by Macrogen Inc. (Madrid, Spain; https://dna.macrogen.com/eng/) using an ABI 3130xL (Applied Biosystems, Waltham, MA, USA) sequencer. The same primers used previously for PCR were subse- quently used for direct sequencing of the PCR products. The quality of the obtained chromatograms was checked through the measurement of their “Phred score” (Richterich, 1998). Only those sequences that showed continuous high quality base readings (QV > 20) were used. Chromatograms were analysed and manually proof- read using the software Chromas software v. 2.6.2 (Tech- nelysium, Pty. Ltd., South Brisbane, Australia). Overall, six novel ND5 sequences of Daphnia pulex were pro- duced. Moreover, in order to compare the new sequences with those publicly available, ten Daphnia pulex se- quences, eight D. pulicaria Forbes, 1893 sequences, four D. tenebrosa Sars, 1898 sequences and one D. magna Straus, 1820 sequence (used as an outgroup) were down- loaded from GenBank and included in the analyses (see Tab. 1 for their Accession Number, A.N.). All sequences were aligned with MEGAX software (Kumar et al., 2018) using the ClustalW method (Thomp- son et al., 1997). MrBayes software v. 3.2.6 (Ronquist et al., 2012) and PhyML v. 3 (Guindon and Gascuel, 2003) were used for inferring the molecular identification and phylogenetic re- lationships between taxa, using Bayesian Inference (BI) and Maximum Likelihood (ML) analyses. As support measures for the nodes, bootstrap values (Felsenstein, 1985) were calculated with 1000 replicates in ML trees, while the posterior probability values were reported in the BI tree. PartitionFinder v. 1.0.1 (Lanfear et al., 2012) was used to choose the best evolutionary model following the “Akaike Information Criterion” (AIC; Akaike, 1974). A Hasegawa – Kishino – Yano evolutionary sequence model with a proportion of gamma and invariant sites (HKY+I+Γ; nst = 2) was used in the BI and ML analyses. In the BI analysis, two independent Markov Chain Monte Carlo analyses were performed with 1 million generations (temp.: 0.2; default priors). The trees and parameter val- ues were sampled every 100 generations, resulting in 10,000 trees for each analysis. The convergence in the analysis was reached (Effective Sample Size (ESS) greater than 533.13 in all the analyses performed). The initial 25% of trees were discarded as “burn-in”. RESULTS In addition to five of the published sites from the Madonie, Nebrodi and Bosco della Ficuzza areas, the species was collected in an novel site on the Nebrodi mountains (ME076), located close to the site “ME004”, where the species was already known to occur (see Tab. 1 in Marrone and Vecchioni, 2020). In all the sampled populations, parthenogenetic females coexisted with males and ephippial females. Unfortunately, logistic con- straints made not possible to collect fresh samples in the only known site for the species within the Etna area (CT011, see Tab. 1 in Marrone and Vecchioni, 2020), which was thus not included in the analyses. No n- co mm er cia l u se on ly Molecular characterization of Daphnia pulex in Sicily 31 Overall, six new sequences belonging to Daphnia pulex were produced from six different Sicilian water bodies (Fig. 1, Tab. 1). After having trimmed out the se- quences, a properly aligned fragment of 624 bp long of the ND5 mtDNA gene was obtained. Novel sequences were deposited in GenBank (A.N., MZ489122- MZ489127). The BI and ML trees based on the mitochondrial ND5 mtDNA fragment and rooted on D. magna showed a con- gruent and well supported topology highlighting how the analysed sequences of Daphnia pulex s.l. create two pa- raphyletic clades: the first including North American D. pulex populations and the allochthonous North American D. pulex populations occurring in Europe (“NAPX”); the second one includes the autochthonous European D. pulex populations (“EPX”) including the analysed Sicilian pop- ulations (i.e., PA074, PA079, PA081, ME004, ME013, ME076; Tab. 1). All the Sicilian sequences clustered to- gether in a monophyletic clade showing only two slightly different haplotypes (i.e., there is only a single base of dif- ference between the sequence that belongs to PA079 and the remaining ones). Similarly to what was observed in D. pulex, also D. pulicaria shows a marked paraphyly (Fig. 2) that sepa- rates the North American populations (“NAPC”) from the European ones (“EPC”). DISCUSSION Based on the molecular evidence here reported, the Si- cilian populations of Daphnia pulex unequivocally belong to the native European lineage. Taking into account the distribution of the species in Sicily, this result is not surprising. In fact, Sicilian D. pulex populations seem to be linked to natural, poorly- Tab. 1. Origin and GenBank accession numbers (A.N.) for the analysed Daphnia specimens. Geographic coordinates are expressed as decimal degrees (Map Datum: WGS84). Taxon Country Location Latitude (N) Longitude (E) A.N. Clade Reference D. pulicaria Albania Ohrid 40.95 20.71 KC536551 EPC Marková et al., 2013 Switzerland Alps 46.67 8.04 KC536536 EPC Marková et al., 2013 Czech Republic Bohdaneč 49.78 15.23 KC536543 EPC Marková et al., 2013 Italy Dolomites 46.48 11.66 KC536525 EPC Marková et al., 2013 Greenland Nuuk 64.15 -51.31 KC536586 EPC Marková et al., 2013 Canada Winnipeg, Manitoba 52.12 -97.25 KC536611 NAPC Marková et al., 2013 USA Indiana 39.9 -85.43 KC536600 NAPC Marková et al., 2013 Iceland Reykjavik 64.13 -21.94 KC536621 NAPC Marková et al., 2013 D. pulex Czech Republic Blatná 49.42 13.78 KC536544 EPX Marková et al., 2013 Germany Regensburg 49 12.15 KC536593 EPX Marková et al., 2013 UK Streetly End 52.14 0.35 KC536555 EPX Marková et al., 2013 Lithuania Vilnius 54.75 25.29 KC536560 EPX Marková et al., 2013 Canada Listowel pond, Ontario 43.73 -80.95 KC536602 NAPX Marková et al., 2013 Canada Disputed Road, Ontario 42.22 -83.03 HQ434640 NAPX Vergilino et al., 2011 Canada Res. Duchesnier, Quebec 48.13 -68.63 KC536603 NAPX Marková et al., 2013 Italy PA079 37.889944 13.394575 MZ489122 EPX Present work Italy PA074 37.823410 14.127241 MZ489123 EPX Present work Italy PA081 37.901131 13.408438 MZ489124 EPX Present work Italy ME013 37.951944 14.698331 MZ489125 EPX Present work Italy ME076 37.941580 14.683670 MZ489126 EPX Present work Italy ME004 37.939456 14.682634 MZ489127 EPX Present work Italy Avigliana 45.070000 7.390000 KC536565a NAPX Marková et al., 2013 Italy Sardinia 40.560000 9.320000 KC536565b NAPX Marková et al., 2013 Italy Bodrio del Pastore III 45.001389 10.323889 KR233296 EPX Marková et al., 2017 Spain Lake Borreguil, Sierra Nevada 37.052 -03.30 MW883468 NAPX Conde-Porcuna et al., 2021 Sweden - - - HQ434644 EPX Vergilino et al., 2011 Kenya Lake Naivasha -0.771667 36.361667 DQ235240 NAPX Mergeay et al., 2006 South Africa Cape Flats -33.98 18.656667 DQ235233 NAPX Mergeay et al., 2006 Kenya Lake Baringo 0.535764 36.063759 DQ235242 NAPX Mergeay et al., 2006 Japan Kosugi, Toyama 36.68 137.09 JX532913 NAPX Crease et al., 2012 Japan Higashi Hiroshima, Hiroshima 34.35 132.8 JX532907 NAPX Crease et al., 2012 China Lake Wusutu 40.86 111.55 MH632088 NAPX Ma et al., 2019 D. tenebrosa Canada Churchill, Manitoba 58.77 -94.17 KC536605 - Marková et al., 2013 Russia Petchora Delta 68.06 53.58 KC536564 - Marková et al., 2013 Svalbard Storvatnet, Ny-Alesund 78.92 11.88 KC536580 - Marková et al., 2013 Russia Tsvetkov cape, Taimyr 74.92 112.62 KC536608 - Marková et al., 2013 D. magna - - - - MT199637 - Lee (Unpublished) No n- co mm er cia l u se on ly L. Vecchioni et al.32 mineralized ponds located within wooded areas at medium and high altitudes (Marrone and Vecchioni, 2020), where phenomena of biological invasions are less frequent than in disturbed water bodies located in highly- anthropized areas. In fact, in Italy, the non-native D. pulex lineage was to date found in artificial or highly-an- thropized water bodies both in Sardinia and Piedmont (Fadda et al., 2011; Marková et al., 2017). In light of these results, the hypothesis by Marrone et al. (2009) that Si- cilian D. pulex populations are relictual elements that colonised Sicily during the late Pleistocene glacial events, coming from the Balkan peninsula or northern Italy, and which later found a “cool” refuge at higher altitude with the onset of warmer and low-moisture climatic conditions in the early Holocene (Curry et al., 2016), seems to be supported. Interestingly, based on the currently available data, it seems that native European D. pulex populations prefer- entially inhabit small, natural water bodies, whereas the populations of the non-native American lineage are mostly occurring in artificial or highly-disturbed habitats (Schwenk et al., 2000; Fadda et al., 2011; Vergilino et al., 2011) at medium and low altitude with a single high-alti- tude exception reported for a Spanish lake located in the Sierra Nevada (Conde-Porcuna et al., 2020). Native and non-native D. pulex lineages are known to coexist sym- patrically, but not syntopically, in the Po River basin, with the native populations occurring in the “bodri” (i.e., tem- porary natural ponds) (Marková et al., 2017), and the non- native one found in the highly-anthropized Avigliana lakes (Virgilino et al., 2011; Marková et al., 2013). Con- versely, for Sardinian populations molecular data are available only from a single site, i.e., an artificial reservoir built in Sos Canales (Fadda et al., 2011). However, D. pulex is also occurring in smaller, natural astatic and tem- porary ponds located in the southern part of the island (Margaritora et al., 2021; Marrone and Stoch, unpublished data), and it should be checked whether the D. pulex pop- ulations inhabiting these marginal, natural habitats belong to the alien or the native lineage. Fig. 1. Geographic location of Daphnia pulex sampling sites for which molecular data are available. Red circles indicate sites where the North American lineages, “NAPX”, of Daphnia pulex occur. Green circles and diamonds indicate where the European lineages, “EPX”, of the species occur. Green diamonds indicate the novel sampled sites. Due to the scale of the map some of the novel sites overlap and thus are not displayed. See Tab. 1 for the coordinates of the sampling sites and for more information on the collected species. No n- co mm er cia l u se on ly Molecular characterization of Daphnia pulex in Sicily 33 Fig. 2. Bayesian phylogram of Daphnia spp. based on the 624 bp fragment of the mtDNA ND5. D. magna was used as outgroup. Node statistical support is reported as nodal posterior probabilities (Bayesian Inference of phylogeny, BI)/bootstrap values (Maximum Like- lihood, ML). *, Nodal statistical supports <0.50. Square brackets group the samples according to the current taxonomy of the genus. Novel sequences are reported in bold. NAPX, North American Pulex; NAPC, North American Pulicaria; EPX, European Pulex; EPC, European Pulicaria. The analysed specimens are reported using the codes listed in Tab. 1. No n- co mm er cia l u se on ly L. Vecchioni et al.34 CONCLUSIONS This study provides the first molecular data about the Sicilian populations of the water flea Daphnia pulex. All the sampled populations proved to belong to the au- tochthonous European lineage of the species, in accor- dance with their previously hypothesised native and relictual status (Marrone et al., 2009; Marrone and Vec- chioni, 2020). Sicilian D. pulex populations are thus im- portant management units, to be attentively managed and preserved. Considering the threats to native taxa linked with the occurrence and spreading of invasive, alien species (Marrone and Naselli-Flores, 2015), and the im- portance of timely acting to have a chance to effectively manage the biological invasions, the molecular character- ization of the D. pulex populations occurring in southern Sardinia, peninsular Italy, and Malta (Margaritora et al., 2021) is urgent and desirable. ACKNOWLEDGMENTS Sara La Franca is acknowledged for the help she pro- vided during the preparation of this manuscript. Two anonymous reviewers are acknowledged for their com- ments to a first draft of the manuscript. CONFLICT OF INTEREST The authors declare no competing interests. Corresponding author: luca.vecchioni@unipa.it Keywords: Cladocera; Anomopoda; biological invasions; cryptic invasions. Received: 1 July 2021. Accepted: 14 September 2021. This work is licensed under a Creative Commons Attribution Non- Commercial 4.0 License (CC BY-NC 4.0). ©Copyright: the Author(s), 2021 Licensee PAGEPress, Italy Advances in Oceanography and Limnology, 2021; 12:9947 DOI: 10.4081/aiol.2021.9947 REFERENCES Akaike H, 1974. A New look at the statistical model identifica- tion. IEEE Trans. Automat. Contr. 19:716–723. Alonso M, 1996. Crustacea, Branchiopoda. Fauna Iberica 7. Museo Nacional de Ciencias Naturales, CSIC, Madrid, 486 pp. Benzie JAH, 2005. Cladocera: the genus Daphnia (including Daphniopsis). Guides to the identification of the Microin- vertebrates of the Continental Waters of the World. 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