The genus Muticaria Lindholm, 1925, is currently distributed either in Southeastern Sicily or in the Malteseislands and comprises the species M. syracusana (Philippi, 1836), M. neuteboomi Beckmann, 1990 and M.macrostoma (Cantraine, 1835). For the first time, we report a molecular study on the topotypicous populationsof M. syracusana and M. neuteboomi carried out on fragments of the ribosomal 16S rDNA subunit and thecytochrome oxydase I (COI) mitochondrial genes by Neighbour Joining, Maximum Likelihood, MaximumParsimony and Bayesian Inference algorithms. Our results revealed the existence of nucleotide-sequencedivergence (Dxy: 5% for 16S rDNA and 12% for COI sequences) between the two taxa

M. Stella Colomba

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INTRODUCTIONThe genus Muticaria Lindholm, 1925, curren-tly attributed to the subfamily Alopiinae A. J.Wagner, 1913, tribe Medorini H. Nordsieck,1997 (Nordsieck, 1997; Nordsieck, 2007; Bank,2010), is distributed in Southeastern Sicily andMaltese islands (except Filfla) and shows mor-phological affinities with the genera Leuco-stigma A. J. Wagner, 1919, widespread in theApennine Italy, and Lampedusa O. Boettger,1877 occurring in the Pelagie (Sicily) and Mal-tese islands. Thake (1985; Giusti et al., 1995)suggested that the group might have originatedfrom ancestral Alopiinae that entered ApennineItaly from the Balcans and, successively, duringthe Messinian salinity crisis (Upper Tertiary),colonized Sicily and the Maltese islands. After-wards, when the sea water re-invaded the Medi-terranean basin with the so-called “Zancleanflood” (Pliocene) these molluscs separated inmany populations which diverged and differen-tiated in the genera Muticaria, Leucostigma andLampedusa. Within Muticaria, further differen-tiations at both specific and subspecific levelsoccurred during glacial and interglacial periods(Pleistocene) when sensitive fluctuations of thesea level went into a cycle of emersions andimmersions of land bridges connecting the Sici-lian mainland with the Maltese region.At present, Muticaria comprises three species,M. syracusana (Philippi, 1836), M. neuteboomiBeckmann, 1990 and M. macrostoma (Cantraine,1835), this latter with a few subspecific taxa (Nor-dsieck, 2007; Bank, 2010): M. macrostoma macro-stoma reported for Gozo, Comino, Cominotto andMalta; M. macrostoma scalaris (L. Pfeiffer, 1850)inhabiting a very limited area on the northwesterncoast of Malta (Tal-Blata, Mistra Bay); M. macro-stoma oscitans (Charpentier, 1852) reported forGozo and Malta; and M. macrostoma mamotica(Gulia, 1861) occurring in a very limited area onthe Munxar side of Xlendi Valley in Gozo (Beck-mann & Gittenberger, 1987; Schembri, 2003; Nor-Molecular analysis of Muticaria syracusana and M. neuteboomi fromSoutheastern Sicily, Italy (Gastropoda, Pulmonata, Clausiliidae).M. Stella Colomba1*,Armando Gregorini1, Fabio Liberto2,Agatino Reitano3, Salvatore Giglio4 & Ignazio Sparacio51Università di Urbino, DiSTeVA, via Maggetti 22 (loc. Sasso), 61029 Urbino, Italy. 2Strada Provinciale Cefalù-Gibilmanna n° 93, 90015Cefalù, Italy. 3Via Gravina 77, 95030 Tremestieri Etneo, Italy. 4Contrada Settefrati, 90015 Cefalù, Italy. 5Via E. Notarbartolo 54 int. 13, 90145Palermo, Italy.  *Email. mariastella.colomba@uniurb.itBiodiversity Journal, 2010, 1(1-4): 7-14ABSTRACT The genus Muticaria Lindholm, 1925, is currently distributed either in Southeastern Sicily or in the Malteseislands and comprises the species M. syracusana (Philippi, 1836), M. neuteboomi Beckmann, 1990 and M.macrostoma (Cantraine, 1835). For the first time, we report a molecular study on the topotypicous populationsof M. syracusana and M. neuteboomi carried out on fragments of the ribosomal 16S rDNA subunit and thecytochrome oxydase I (COI) mitochondrial genes by Neighbour Joining, Maximum Likelihood, MaximumParsimony and Bayesian Inference algorithms. Our results revealed the existence of nucleotide-sequencedivergence (Dxy: 5% for 16S rDNA and 12% for COI sequences) between the two taxa.KEY WORDS Door snails; Muticaria; Clausiliidae; Molecular taxonomy; 16S rDNA; COI.Received  18.10.2010; accepted 06.12.2010; printed 30.12.2010dsieck, 2007; Bank, 2010). Moreover, interestinglythere are a few populations which show a shell rib-bing intermediate between macrostoma and osci-tans, suggesting that a possible hybridization of thetwo might have occurred (see Giusti et al., 1995).M. syracusana locus typicus Siracusa(Philippi, 1836), and M. neuteboomi describedfor Cava d’Ispica, (Modica, Ragusa province)(Beckmann, 1990) are quite widespread in Sou-theastern Sicily. Giusti et al. (1995) reported M.neuteboomi as a local variation of M. syracusana(foreseeing Badino and colleagues’ results –which remained unpublished – on allozymicpolymorphism within the genus Muticaria), but afew years later, M. neuteboomi was recognizedvalid species and since then this statement hasnot been denied (Nordsieck, 1997; 2007; Bank,2010). Recently, a preliminary molecular studyon 16S rDNA partial sequences (Gregorini et al.,2008) revealed that the two taxa show some dif-ferences at genetic level. In this study, continua-tion of previous work, we carried out a moredetailed molecular analysis on several M. syracu-sana (from Roman amphitheatre, Siracusa) andM. neuteboomi (from Cava d’Ispica, Ragusa)specimens by a comparative investigation of themitochondrial 16S rDNA and cytochrome oxi-dase I (COI) partial sequences, in order toimprove current taxonomic knowledge andassess the level of differentiation between thetwo species from a molecular point of view.MATERIALS AND METHODSSample collection, DNA extraction, Amplificationand SequencingA total of ten specimens (five M. syracusanaand five M. neuteboomi; collection sites: nearbythe Roman amphitheatre of Siracusa and Cavad’Ispica, respectively, SE Sicily) were analysed.Samples were stored separately at -20 °C in testtubes. For each individual, the entire animal wasused for total DNA extraction (by Wizard Geno-mic DNA Purification Kit, Promega). Additionalspecimens belonging to the same batch were sto-red as vouchers in the laboratory of Cytogeneticsand Molecular Biology (University of Urbino,via Maggetti 22). Present study was based on acomparative analysis of 16S rDNA and COI par-tial sequences, frequently used as genetic mar-kers in the investigation of evolutionary proces-ses at the specific level.Fragments of 16S rDNA sequences (297 bp)were amplified by a couple of primers (MED16F:5’-actgtgcaaaggtagcatcc-3; ’MED16R: 5’-ccaa-catcgaggtcacaa-3’) designed on alignments ofseveral homologous sequences of Clausiliidae spe-cies downloaded from GenBank database. Whe-reas for COI fragments (660 bp), the universal pri-mers LCO1490: 5’-ggtcaacaaatcataaagatattgg-3’and HCO2198: 5’-taaacttcagggtgaccaaaaaatca-3’were employed according to Folmer et al. (1994)with slight modifications. PCR thermal cycleswere as follows: 95 °C for 5 min; 95 °C for 1 min,55 °C for 1 min, 72 °C for 1 min (35 cycles); 72 °Cfor 5 min. To remove primers and unincorporatednucleotides, the amplified products were purifiedby the Wizard SV gel and PCR Clean-up kit (Pro-mega). Sequencing of the purified PCR productswas carried out using automated DNA sequencersat Eurofins MWG Operon (Germany). GenBankaccession numbers for sequences generated in thisstudy are HQ696866-HQ696869.Phylogenetic analysesSequences were visualized with BioEditSequence Alignment Editor 7 (Hall, 1999), ali-gned with the ClustalW option included in thissoftware and double checked by eye. Standardmeasures of nucleotide polymorphism werecomputed using MEGA 4 (Tamura et al., 2007).Phylogenetic analyses were conducted in MEGA4, PhyML 3.0 (Guindon & Gascuel, 2003) andMrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001),the latter two available at http://www.phylo-geny.fr/version2_cgi/one_task.cgi?task_type=phyml and http://www.phylogeny.fr/version2_cgi/one_task.cgi?task_type=mrbayes (see Dereeper etal., 2008). To address the phylogenetic relation-ships among taxa, many different analyticalmethods were used: Maximum Likelihood (ML),Neighbour Joining (NJ), Maximum Parsimony(MP) and Bayesian Inference (BI). For Maxi-mum Likelihood analyses, the most appropriatemodel of DNA substitution was selected by Fin-dmodel (available via http://www.hiv.lanl.gov/cgi-bin/findmodel/findmodel.cgi), a web imple-mentation of Modeltest by Posada & Crandall(1998). Models were as follows: 8 M.S. COLOMBA, A. GREGORINI, F. LIBERTO, A. REITANO, S. GIGLIO & I. SPARACIOFigure 1. Neighbour Joining bootstrap consensus tree showing a possible reconstruction of evolutionary relationships of the ana-lysed taxa based on mitochondrial 16S rDNA partial sequences. Branches corresponding to partitions reproduced in less than50% bootstrap replicates are collapsed; bootstrap values are reported near the branches. The rate variation among sites wasmodeled with a gamma distribution (shape parameter = 0.57). All positions containing gaps and missing data were eliminatedfrom the dataset (Complete deletion option). Albinaria caerulea has been employed as outgroup to root the trees (legend, NEU= M. neuteboomi SYR = M. syracusana).Figure 2. Neighbour Joining bootstrap consensus tree showing a possible reconstruction of evolutionary relationships of the ana-lysed taxa based on mitochondrial COI partial sequences. Branches corresponding to partitions reproduced in less than 50%bootstrap replicates are collapsed; bootstrap values are reported near the branches. The rate variation among sites was mode-led with a gamma distribution (shape parameter = 0.15). All positions containing gaps and missing data were eliminated from thedataset (Complete deletion option). Albinaria caerulea has been employed as outgroup to root the trees (legend, NEU = M. neu-teboomi SYR = M. syracusana).9Molecular analysis of Muticaria syracusana and M. neuteboomi from Southeastern Sicily, Italy- for 16S rDNA data: K81uf (Unequal-fre-quency Kimura 3-parameter) plus gamma distri-bution, with an alpha parameter of 0.57;- for COI data: TVM (Transversion Model)plus gamma distribution, with an alpha parame-ter of 0.15;Neighbour Joining trees were constructedtreating gaps as missing data. MP trees wereobtained using the Close-Neighbour-Inter-change (CNI) algorithm with search level 3 inwhich the initial trees were achieved with therandom addition of sequences (10 replicates).All positions containing gaps and missing datawere eliminated from the dataset (completedeletion option). Characters were assignedequal weights. MP trees were collapsed toobtain a 50% majority rule consensus tree.Support for the internodes was assessed bybootstrap percentages (BP) (1,000 resamplingsteps for NJ and MP; 100 replicates for ML).For Bayesian analyses, the number of substitu-tion types was fixed to 6, with a standard(4by4) model of nucleotide substitution and arate variation across sites fixed to gamma. FourMarkov Chain Monte Carlo (MCMC) chainswere run for 100,000 generations, samplingevery 10 generations; from the 10,000 treesfound the first 1,000 were discarded as “burn-in”. Finally, a 50% majority rule consensus treewas constructed. All phylogenetic trees wererooted using homologous nucleotide sequencesof Albinaria caerulea (Deshayes, 1835) (Pul-monata, Mollusca, Clausiliidae) (GenBankIDs: DQ665343, NC_001761).RESULTS AND DISCUSSIONIn spite of the algorithm (NJ, MP, ML and BI)employed, all phylogenetic reconstructionsrevealed that the two taxa were clearly separated.Moreover, evolutionary trees showed highly con-gruent topologies that agreed on clustering andbranching patterns supported by high bootstrapvalues. Since analyses produced almost identicalresults not changing any of the interpretation,only the phylogenetic NJ consensus trees aredisplayed as an example. As shown in figures 1and 2, one cluster comprises only M. syracusana,whereas the second one includes just M. neute-boomi specimens. These findings not only con-firmed data previously reported for 16S rDNA(Gregorini et al., 2008), but also corroborated thevalidity of the two species. In fact, nucleotidedistances (5% for 16S rDNA and 12% for COIsequences) suggest strong isolation and strongdivergence which may justify considering thetwo taxa under investigation as different (phylo-genetic) species.From a morphological point of view, M.neuteboomi differs from M. syracusana forshell morphology showing more roundedwhorls, more closely spaced ribs  (Figs. 3-4)and the clausilium shorter, narrower and shar-per (Figs. 5-6), and for relationship betweenprincipalis and upper palatalis plica (Beck-mann, 1990; Giusti et al., 1995; Reitano et al.,2009). In particular, M. syracusana has princi-pal plica very short and fused to upper palatalplica, while M. neuteboomi has principal plicaindependent of upper palatal plica (Figs. 7-10).According to Beckmann (1990), the relation-ship between principalis and upper plica repor-ted for M. neuteboomi might be considered ple-siomorphic with respect to that observed in M.syracusana.Gregorini et al. (2008) also provided a contri-bute to better define the distribution area of bothspecies revealing that M. syracusana occurs onlyalong the entire coast embracing Siracusa province,whereas M. neuteboomi inhabits a broader areaincluding Siracusa, Ragusa, Caltanissetta and Cata-nia provinces, and generally occurs at higher altitu-des than those reported for M. syracusana. By acomparison of morphological data, M. neuteboomipopulations, a part from the exception of the topo-typicous one (employed in this study) show a mar-ked variability of shell morphology which, at leastin some cases, is very similar to that observed in M.syracusana (Figs. 11-14).Hence, combining previously results with alldata described herein, we suggest, that M. syra-cusana (with limited distribution area and a quitehomogeneous shell morphology) might have ori-ginated more recently than M. neuteboomi (witha much wider distribution area and heterogenousshell morphology). Anyway, further and more detailed analyseson other Muticaria populations are still in pro-gress in order to better define molecular, mor-phological and ecological features of this highlyinteresting group.10 M.S. COLOMBA, A. GREGORINI, F. LIBERTO, A. REITANO, S. GIGLIO & I. SPARACIO11Molecular analysis of Muticaria syracusana and M. neuteboomi from Southeastern Sicily, ItalyFigure 3. Muticaria syracusana. Siracusa, Teatro Romano, 19 m, 37°04’28” N, 15°16’45” E, legit A. Reitano (coll. Liberto);h: 14.5 m; D: 4 mm. – Figure 4. Muticaria neuteboomi. Ragusa, Cava d’Ispica, 340 m, 36°51’11” N, 14°50’14” E, legit A. Reitano(coll. Liberto); h: 11.9 mm; D: 4.3 mm.12 M.S. COLOMBA, A. GREGORINI, F. LIBERTO, A. REITANO, S. GIGLIO & I. SPARACIOFigure 5. Clausilium of Muticaria syracusana, length 2.46 mm. – Figure 6. Clausilium of Muticaria neuteboomi, length 2.48 mm. –Figure 7. Parietum of Muticaria syracusana. Legend: CL = columellar lamella; L = lunella; PL = parietal lamella; PLL = parallellamella; PP = principal plica; SL = spiral lamella; SP = sutural plica; UPP = upper palatal plica;  – Figure 8. Parietum of Mutica-ria neuteboomi. – Figure 9. Palatum of Muticaria neuteboomi. – Figure 10. Palatum of Muticaria syracusana.13Molecular analysis of Muticaria syracusana and M. neuteboomi from Southeastern Sicily, ItalyFigures 11-14. Variability of the shells and palatum of M. neuteboomi from Siracusa, Sortino, Pantalica, 265 m, 37°08’28” N,15°01’54”E, legit A. Reitano (coll. Liberto).  Figure 11. h:11.5; D: 4.3; Figure 12. h: 13.7; D: 4.3 mm.REFERENCESBank R.A., 2010. Fauna Europea: Gastropoda, Clausiliidae.Fauna Europea version 1.1. http//www.faunaeur.org.Last access: October 20th 2010.Beckmann K.H. & Gittenberger E., 1987. The Clausiliidae(Gastropoda) of the Maltese islands, some additionaldata. Journal of Conchology, 32: 335-338.Beckmann K.H., 1990. Beitrage zur Kenntnis der Landmol-luskenfauna Sizilien mit der Beschreibung von Mutica-ria neuteboomi spec. nov. (Gastropoda Pulmonata:Clausiliidae). Basteria, 54: 75-85.Dereeper A., Guignon V., Blanc  G., Audic S., Buffet S.,Chevenet F., Dufayard J.F., Guindon S., Lefort V.,Lescot M., Claverie J.M. & Gascuel O., 2008. Phylo-geny.fr: robust phylogenetic analysis for the non-specia-list. Nucleic Acids Research, 136 (Web Server issue),W465-469.Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R.,1994. DNA primers for amplification of mitochondrialcytochrome c oxidase subunit I from diverse metazoaninvertebrates. Molecular Marine Biology and Biote-chnology, 3: 294-299.Giusti F., Manganelli G. & Schembri P.J., 1995. The non-marine molluscs of the Maltese Islands. MonografieMuseo Regionale Scienze Naturali, Torino, 15: 1-607.Gregorini A., Colomba M.S., Reitano A., Liberto F., Ger-manà A. & Sparacio I., 2008. Analisi molecolari sulgenere Muticaria Lindholm, 1925 (Gastropoda Pulmo-nata Clausilidae). 37° Congresso Nazionale Italiano diBiogeografia, Catania 7-10 ottobre 2008 (abstract).Guindon S. & Gascuel O., 2003. A simple, fast and accuratealgorithm to estimate large phylogenies by maximumlikelihood. Systematic Biology, 52: 696-704.Hall T.A., 1999. BioEdit: a user-friendly biologicalsequence alignment editor and analysis program forWindows 95/98/NT. Nucleic Acids Symposium Series,41: 95-98.Huelsenbeck J.P. & Ronquist F., 2001. MRBAYES: Baye-sian inference of phylogenetic trees. Bioinformatics, 17:754-755.Nordsieck H., 1997. Phylogeny of and within the Albinaria-Isabellaria group (Gastropoda: Pulmonata: Clausilii-dae), Heldia 4: 53–61.Nordsieck H., 2007. Worldwide Door Snails (Clausiliidae),recent and fossil. ConchBooks, Hackenheim, 214 pp.Posada D. & Crandall K.A., 1998. Modeltest: testing themodel of DNA substitution. Bioinformatics: Applica-tion Note, 114: 817-818.Reitano A., Liberto F., Sparacio I. & Giglio S., 2009. I mol-luschi terrestri della R.N.I. “Grotta Palombara” (Melilli,Sicilia sud-orientale) (Gastropoda Architaenioglossa,Neotaenioglossa, Stylommatophora). Il Naturalista sici-liano, 33: 177-205.Schembri P.J., 2003. Current state of knowledge of the Mal-tese non-marine fauna. In: Malta Environment and Plan-ning Authority Annual report and accounts 2003.Floriana, Malta, 33-65.Tamura  K., Dudley J., Nei M. & Kumar S., 2007. MEGA4:Molecular Evolutionary Genetics Analysis (MEGA)software version 4.0. Molecular Biology and Evolution,24: 1596-1599. (Publication PDF at http://www.kumar-lab.net/publications).Thake M.A., 1985. The biogeography of the Malteseislands, illustrated by the Clausiliidae. Journal of Bio-geography, 12: 269-287.14 M.S. COLOMBA, A. GREGORINI, F. LIBERTO, A. REITANO, S. GIGLIO & I. SPARACIO

Molecular analysis of Muticaria syracusana and M. neuteboomi from Southeastern Sicily, Italy (Gastropoda, Pulmonata, Clausiliidae)

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Molecular analysis of Muticaria syracusana and M. neuteboomi from Southeastern Sicily, Italy (Gastropoda, Pulmonata, Clausiliidae)

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