A first attempt to track genetic signature of the colonization of the Mediterranean basin by the pigmy white-toothed shrew, Suncus etruscus (Eulipotyphla, Soricidae)

A first attempt to track genetic signature of the colonization of the Mediterranean basin by the pigmy white-toothed shrew, Suncus etruscus (Eulipotyphla, Soricidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report A first attempt to track genetic signature of the colonization of the Mediterranean basin by the pigmy white-toothed shrew, Suncus etruscus (Eulipotyphla, Soricidae) Riccardo Castiglia, Chiara Rotondi, Gaetano Aloise, Giovanni Amori, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2807604/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Mammal Research → Version 1 posted 4 You are reading this latest preprint version Abstract The pigmy white-toothed shrew Suncus etruscus is a widespread species whose distribution patterns are unclear. Paleontological data suggested an east-west pattern of dispersion in the Mediterranean basin during late Holocene but some doubts are still present especially considering the absence of fossil remains from key areas, as mainland Italy. Here we propose, a preliminary screening of the phylogeographic relationships among Italian pigmy white-toothed shrews and other Mediterranean areas. The Italian haplotypes were all very similar without an evident geographic structure, however we evidenced that the haplotype from Israel, the putative source area for the Mediterranean basin, is almost identical to the most common Italian haplotype. This excludes an ancient event of vicariance between the two areas and we can assume that these haplotypes arrived in the central Mediterranean through the eastward wave of colonization, in agreement with the relatively recent arrival of the species in the area. Mediterranean basin Mitochondrial DNA Phylogeography Soricidae Suncus etruscus Figures Figure 1 Figure 2 Full Text The pigmy white-toothed shrew Suncus etruscus (Savi, 1822) (Eulipotyphla, Soricidae) is one of the smallest living mammals (Fig. 1, Burgin and He, 2018). It is a widespread species, present from south-western Europe and northern Africa to the Middle East and Southeast Asia (Fig. 2, Aulagnier et al. 2017). In the Mediterranean area, it is restricted to the Mediterranean climate zone including large and small islands (Burgin and He 2018, Loy et al. 2019, Sciandra et al. 2022). It is also present in Socotra, Madagascar and in the island of Tenerife (Canary Island, Spain), where it has been recently introduced. The patterns and times of colonization of the species across the Mediterranean have recently been investigated by a complete review of the fossil data (Garcia et al. 2020), the oldest records come mainly from Middle and Late Pleistocene sites of Israel (280.000 – 33.000 ya) and this area is considered the origin for the colonization of all the Mediterranean basin. The records seem to support a scenario in which S. etruscus gradually spread through the Mediterranean from East to West, starting approximately 4.000 ya (late Holocene) and reaching the Iberian Peninsula (Estrecho Cave, Spain) around 2.000 ya (Garcia et al. 2020). In this scenario, however, most of the aspects of colonization of the central area of Mediterranean remains unclear. Some Middle Holocene records from central Mediterranean islands (Sicily and Sardinia, Lopez García et al. 2013, Sans-Coma et al. 1985) seem to suggest an earlier introduction (3.360-6.000 ya) in central Mediterranean area respect to the Eastern islands. However, this apparent anomaly is probably due to the dating of these records, which is not fully reliable (Garcia et al. 2020). Therefore, although an east-west dispersion pattern seems absolutely the most plausible, some doubts are still present especially considering the absence of fossils from key areas, as mainland Italy (Dobson 1998). Unlike other species that have had similar histories of colonization (Solano et al. 2013, Colangelo et al. 2015), the molecular phylogeography of S. etruscus has never been thoroughly investigated. This is due to the extreme difficulty of obtaining samples through the classic trapping methods used for small mammals (cf. Vogel 2012). Previous researches highlight the presence of two major mitochondrial clades: the eastern clade in South Asia (India and Sri-Lanka) and the western clade in the Near-East (Iran and Israel) and in the central Europe (France, Italy and Spain) (Adamson et al. 2011, Pinya et al. 2018, Shpirer et al. 2020). Therefore, not much can be said about the colonization of the central Mediterranean area aside from the fact that the colonization took place by the western clade. In this note, we want to contribute, with new data, to the phylogeography of S. etruscus in the Mediterranean, analyzing specimens from peninsular and insular Italy and investigating the genetic signature of the expansion in the Mediterranean basin from literature, emerged from fossil evidence. Twenty-five shrews from twelve localities were analyzed (Tab. 1). All the individuals were found dead (i.e. most likely captured by domestic cats or other predators) and are kept at the Museum of Comparative Anatomy “Giovanni Battista Grassi” (“La Sapienza” University, Rome). In addition, we analyzed two specimens from Catalonia provided by IRBio (University of Barcelona). These two specimens, already analyzed by Pinya et al. (2018) were re-sequenced to obtain a gene fragment that could be aligned with our sequences. Procedures for DNA extraction, amplification and sequencing followed Castiglia et al. (2007) and Colangelo et al. (2010). The new sequences have been aligned by MEGA X software (Kumar et al. 2018) to 13 sequences available in GenBank, obtaining an alignment of 411 bp. Some of the GenBank available haplotypes of S. etruscus have been eliminated because they were too short or to avoid gaps in the alignment. The final alignment included 39 specimens from 23 localities (Figure 2). Localities, vouchers and GenBank accession numbers of all animals are reported in Table 1S of supplementary information. Relationships among haplotypes were estimated by Network Analysis, considered the best approach when the number of SNPs is low (e.g., Silva et al. 2009, Nerva er al. 2021). We implemented three different reconstruction methods with POPART (Leigh and Bryant 2015) in default settings: the TCS (statistical parsimony), the median joining network (MJNs) and the integer neighbor joining network (IntNJ, Leigh and Bryant 2015). The first two provide the best estimate of the true genealogy when internal node haplotypes are not sampled (Cassens et al. 2005). Eleven total haplotypes (SUNC_1-SUNC_11) were identified (GenBank accession numbers will be provided upon acceptance) and of these, five are new haplotypes for S. etruscus . The alignment also detected 23 SNPs (17 parsimony informative and 6 singletons). The SUNC_1 haplotype is the most represented, present in 22 of the 39 total specimens. This haplotype has also a wide distribution being present in Israel, in Central and Southern Italy, in France and in Spain. The haplotype from Mallorca (Pinya et al. 2018) not used in the alignment because their short size, turned out to be an additional different haplotype close to SUNC_1 (differs by a single mutation over 236bp). The MJNs, the TCS (Fig. 2) and the IntNJ show overlapping topologies, with minimal differences in IntNJ that proposed an alternative connection of the SUNC_5 - SUNC_1 haplotypes. The networks clearly identified the two groups of haplotypes, attributable to the eastern and western clades, separated by a minimum of 12 SNPs (2.9% p-distance). The SUNC_1 has the highest number of connections with other haplotypes, four or five depending on the reconstruction method used. The wide diffusion of SUNC_1 haplotype and in particular its presence in Israel, considered the origin area for S. etruscus in the Mediterranean, may indicate that it represents an ancestral haplotype arrived in the central Mediterranean through the eastward wave of colonization. This scenario agrees with the paleontological findings. Moreover, the presence of this haplotype in both the study area and Israel excludes an ancient event of vicariance, in agreement with the relatively recent arrival of the species in the Mediterranean (Garcia et al. 2020). It can also be evidenced how several Italian haplotypes derive with star-like relationships from SUNC_1, differing for only 1 or 2 mutations. A similar condition has been found in the black rat from the same area and this have been interpreted as a colonization by a single matriarchal line followed by an in-situ diversification (Colangelo et al. 2015). The current state of knowledge is full of geographical gaps and does not allow to capture the genetic signatures of the eastward wave of colonization. Therefore, is difficult to say whether the pygmy shrew arrived firstly in Italy or in the eastern Mediterranean area (Garcia et al. 2020). More extensively, this work can encourage further comparative studies on Mediterranean colonization routes of S. etruscus and new sampling efforts in key areas, such as Turkey, Balkan Peninsula and North Africa, to get an extensive knowledge on colonization dynamics. Declarations Acknowledgements Authors would like to thank Mattia Menchetti and Francesc Munoz-Munoz for help in data collection. Thanks are extended to two anonymous reviewers for valuable advices on the first version of the manuscript. Conflict of Interest: The authors have no competing interests to declare that are relevant to the content of this article. Funding: RC received financial support from Sapienza University research project. Ethical Conduct: not applicable References Adamson EAS, Bhassu S, Hashim R, Ruedi M (2011) Phylogenetic relationships of Malayan and Malagasy pygmy shrews of the genus Suncus (Soricomorpha: Soricidae) inferred from mitochondrial cytochrome-b sequences. Raffles Bull Zool 59: 237-243. Aulagnier S, Hutterer R, Jenkins P, Bukhnikashvili A, Kryštufek B, Kock D (2017) Suncus etruscus . The IUCN Red List of Threatened Species 2017: e.T90389138A22288134. https://dx.doi.org/10.2305/IUCN.UK.2017-2.RLTS.T90389138A22288134.en. Accessed on 22 January 2023. Burgin CE, He K (2018). Family Soricidae (shrews). In: WilsonDE, Mittermeier RA (Eds) Handbook of the Mammals of the World, Insectivores, Sloths and Colugos. Volume 8. Lynx Edicions, Barcelona (Spain), pp. 332-352. Cassens I, Mardulyn P, Milinkovitch MC (2005) Evaluating intraspecific “network” construction methods using simulated sequence data: do existing algorithms outperform the global maximum parsimony approach? System Biol 54 : 363-372. https://doi.org/10.1080/10635150590945377 Castiglia R, Annesi F, Aloise G, Amori G (2007) Mitochondrial DNA reveals different phylogeographic structures in the water shrews Neomys anomalus and N. fodiens (Insectivora: Soricidae) in Europe. J Zool Syst Evol Res 45: 255-262. https://doi.org/10.1111/j.1439-0469.2006.00391.x Colangelo P, Bannikova AA, Kryštufek B, Lebedev VS, Annesi F, Capanna E, Loy A (2010) Molecular systematics and evolutionary biogeography of the genus Talpa (Soricomorpha: Talpidae). Mol Phyl Evol 55: 372-380. https://doi.org/10.1016/j.ympev.2010.01.038 Colangelo P, Abiadh A, Aloise G, Amori G, Capizzi D, Vasa E, Annesi F, Castiglia C (2015) Mitochondrial phylogeography of the black rat supports a single invasion of the western Mediterranean basin. Biol Invasions 17: 1859-1868. https://doi.org/10.1007/s10530-015-0842-2 Dobson M (1998) Mammal distributions in the western Mediterranean: the role of human intervention. Mammal Rev 28: 77-88. https://doi.org/10.1046/j.1365-2907.1998.00027.x García ÁCD, Laplana C, Sevilla P (2020) Early reliable evidence of the Etruscan shrew ( Suncus etruscus ) in southwestern Europe during ancient times. Reconstructing its dispersal process along the Mediterranean Basin. Quaternary Science Reviews 250: 106690. https://doi.org/10.1016/j.quascirev.2020.106690 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547. https://doi.org/10.1093/molbev/msy096 Leigh JW, Bryant D (2015) POPART: full-feature software for haplotype network construction. Meth Ecol Evol 6: 1110-1116. https://doi.org/10.1111/2041-210X.12410 Lopez-Garcia JM, Blain HA, Pagano E, Olle A, Vergès JM, Forgia V (2013) The small mammals (insectivores, bats and rodents) from the Holocene archaeological site of Vallone Inferno (Scillato, Lower Imera Valley, Northwestern Sicily). Rivista Italiana di Paleontologia e Stratigrafia 119: 229-244. https://doi.org/10.13130/2039-4942/6037 Loy A, Aloise G, Ancillotto L, Angelici FM, Bertolino S, Capizzi D, Castiglia R, Colangelo P, Contoli L, Cozzi B, Fontaneto D, Lapini L, Maio N, Monaco A, Mori E, Nappi A, Podestà M, Russo D, Sarà M. Scandura M, Amori G (2019) Mammals of Italy: an annotated checklist. Hystrix 30: 87-106. https://doi.org/10.4404/hystrix-00196-2019 Nerva L, Iannucci A, Menchetti M, Andreoni A, Chitarra W, Martini M, Mueller N, Peters TMJ, Pesenti E, Verbeylen G, Zozzoli R, Mori E (2021) Where do Chip and Dale come from? Origins of invasive populations of the Siberian chipmunk in Europe. Mammal Res 66: 525-529. https://doi.org/10.1007/s13364-021-00569-4 Pinya S, Bover P, Jurado-Rivera JA, Trenado S, Parpal L, Férriz I, Talavera A, Hinckley A, Pons J, Lopez-Fuster MJ, Alcover JA (2018) Recent island colonization by an introduced shrew in the western Mediterranean. Hystrix 29: 232-235. https://doi.org/10.4404/hystrix-00075-2018 Sans-Coma V, Alcover JA, Lopez-Fuster J (1985) Morphometrischer Vergleich rezenter und subfossiler Etruskerspitzmäuse Suncus etruscus (Savi, 1822) von der Insel Sardinien. Säugetierkd Mitt 32: 151-158. Shpirer E, Haddas-Sasson M, Spivak-Glater M, Feldstein T, Meiri S, Huchon D (2021) Molecular relationships of the Israeli shrews (Eulipotyphla: Soricidae) based on cytochrome b sequences. Mammalia 85: 79-89. https://doi.org/10.1515/mammalia-2019-0143 Sciandra C, Mori E, Solano E, Mazza G, Viviano A, Scarfò M, Bona F, Annesi F, Castiglia R (2022) Mice on the borders: genetic identification of rat and house mouse species in Lampedusa and Pantelleria islands (Southern Italy). Biogeographia 37: a013. https://doi.org/10.21426/B637155716 Silva P, Guan X, Ho‐Shing O, Jones J, Xu J, Hui D, Notter D, Smith E (2009) Mitochondrial DNA‐based analysis of genetic variation and relatedness among Sri Lankan indigenous chickens and the Ceylon junglefowl ( Gallus lafayetti ). Animal Genetics 40: 1-9. https://doi.org/10.1111/j.1365-2052.2008.01783.x Solano E, Franchini P, Colangelo P, Capanna E, & Castiglia R (2013) Multiple origins of the western European house mouse in the Aeolian Archipelago: clues from mtDNA and chromosomes. Biol Invasions 15: 729-739. Vogel P (2012) New trapping method to survey for presence of the Etruscan shrew Suncus etruscus , the smallest mammal. Mammal Review 42: 314-318. https://doi.org/10.1111/j.1365-2907.2012.00215.x Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2023 Read the published version in Mammal Research → Version 1 posted Reviewers agreed at journal 20 Apr, 2023 Reviewers invited by journal 18 Apr, 2023 Editor assigned by journal 17 Apr, 2023 First submitted to journal 11 Apr, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2807604","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":192841525,"identity":"58816194-9936-4464-8798-ad7645946e8c","order_by":0,"name":"Riccardo Castiglia","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-9011-302X","institution":"Sapienza Università di Roma: Universita degli Studi di Roma La Sapienza","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Castiglia","suffix":""},{"id":192841526,"identity":"066942c6-c8e8-418a-aac2-0f842e5ac2c6","order_by":1,"name":"Chiara Rotondi","email":"","orcid":"","institution":"Universita degli Studi di Roma La Sapienza Dipartimento di Biologia e Biotecnologie Charles Darwin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Rotondi","suffix":""},{"id":192841527,"identity":"dd915a6b-a7ab-44e3-afc7-803a573aa3e9","order_by":2,"name":"Gaetano Aloise","email":"","orcid":"","institution":"Università della Calabria: Universita della Calabria","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gaetano","middleName":"","lastName":"Aloise","suffix":""},{"id":192841528,"identity":"735c6f64-dd39-4abe-a218-e2b89e807ef1","order_by":3,"name":"Giovanni Amori","email":"","orcid":"","institution":"IRET CNR: Istituto di Ricerca sugli Ecosistemi Terrestri Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"Amori","suffix":""},{"id":192841529,"identity":"a0c1d0e2-075c-45eb-9339-ed1113be937f","order_by":4,"name":"Flavia Annesi","email":"","orcid":"","institution":"UNIROMA1 BBCD: Universita degli Studi di Roma La Sapienza Dipartimento di Biologia e Biotecnologie Charles Darwin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Flavia","middleName":"","lastName":"Annesi","suffix":""},{"id":192841530,"identity":"3fe8af6f-ae9d-44bb-8642-812d26037e68","order_by":5,"name":"Emanuela Solano","email":"","orcid":"","institution":"IRET CNR: Istituto di Ricerca sugli Ecosistemi Terrestri Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emanuela","middleName":"","lastName":"Solano","suffix":""},{"id":192841531,"identity":"048a5558-240c-4627-ba14-8c3366ad6c18","order_by":6,"name":"Emiliano Mori","email":"","orcid":"","institution":"IRET CNR: Istituto di Ricerca sugli Ecosistemi Terrestri Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emiliano","middleName":"","lastName":"Mori","suffix":""}],"badges":[],"createdAt":"2023-04-12 15:45:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2807604/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2807604/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13364-023-00706-1","type":"published","date":"2023-07-28T21:47:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36063848,"identity":"b910a94b-8bbe-4fde-b312-b961aee30e23","added_by":"auto","created_at":"2023-04-20 13:27:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":444074,"visible":true,"origin":"","legend":"\u003cp\u003eA pigmy white-toothed shrew (Photo by Riccardo Castiglia).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2807604/v1/707e94a1ebc4897ff1709754.jpeg"},{"id":36063849,"identity":"73b226ea-b2cf-4d1a-9cc6-ca3a364cd194","added_by":"auto","created_at":"2023-04-20 13:27:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324826,"visible":true,"origin":"","legend":"\u003cp\u003ea) Sampling localities of \u003cem\u003eSuncus etruscus\u003c/em\u003e. Colors and numbers refer to haplotypes of\u003c/p\u003e\n\u003cp\u003efigure 2b. Localities of the Eastern Clade belonging to Madagascar, India and Sri-Lanka are not shown in the map. b) Median-Joining haplotype network topology obtained for the cytb sequences\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2807604/v1/f519a365e2cc3ed59a853273.jpeg"},{"id":44736519,"identity":"9546aeee-940f-4b4d-99d4-cd4208a18c10","added_by":"auto","created_at":"2023-10-16 22:30:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":524881,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2807604/v1/6acd5378-2099-45a9-8c10-89760c63e6d4.pdf"},{"id":36063850,"identity":"cc277931-2597-44ae-944b-44e0111185f0","added_by":"auto","created_at":"2023-04-20 13:27:28","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16814,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2807604/v1/e874533ca22ffb7791a547a3.docx"}],"financialInterests":"","formattedTitle":"A first attempt to track genetic signature of the colonization of the Mediterranean basin by the pigmy white-toothed shrew, Suncus etruscus (Eulipotyphla, Soricidae)","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThe pigmy white-toothed shrew \u003cem\u003eSuncus etruscus\u003c/em\u003e (Savi, 1822) (Eulipotyphla, Soricidae) is one of the smallest living mammals (Fig. 1, Burgin and He, 2018). It is a widespread species, present from south-western Europe and northern Africa to the Middle East and Southeast Asia (Fig. 2, Aulagnier et al. 2017). In the Mediterranean area, it is restricted to the Mediterranean climate zone including large and small islands (Burgin and He 2018, Loy et al. 2019, Sciandra et al. 2022). \u0026nbsp;It is also present in Socotra, Madagascar and in the island of Tenerife (Canary Island, Spain), where it has been recently introduced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe patterns and times of colonization of the species across the Mediterranean have recently been investigated by a complete review of the fossil data (Garcia et al. 2020), the oldest records come mainly from Middle and Late Pleistocene sites of Israel (280.000 \u0026ndash; 33.000 ya) and this area is considered the origin for the colonization of all the Mediterranean basin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe records seem to support a scenario in which \u003cem\u003eS. etruscus\u003c/em\u003e gradually spread through the Mediterranean from East to West, starting approximately 4.000 ya (late Holocene) and reaching the Iberian Peninsula (Estrecho Cave, Spain) around 2.000 ya (Garcia et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this scenario, however, most of the aspects of colonization of the central area of Mediterranean remains unclear.\u0026nbsp;Some Middle Holocene records from central Mediterranean islands (Sicily and Sardinia, Lopez Garc\u0026iacute;a et al. 2013, Sans-Coma et al. 1985) seem to suggest an earlier introduction (3.360-6.000 ya) in central Mediterranean area respect to the Eastern islands. However, this apparent anomaly\u0026nbsp;is probably due to the\u0026nbsp;dating of these records, which is not fully reliable (Garcia et al. 2020). Therefore, although an east-west dispersion pattern seems absolutely the most plausible, some doubts are still present especially considering the absence of fossils from key areas, as mainland Italy (Dobson 1998).\u003c/p\u003e\n\u003cp\u003eUnlike other species that have had similar histories of colonization (Solano et al. 2013, Colangelo et al. 2015), the molecular phylogeography of \u003cem\u003eS. etruscus\u003c/em\u003e has never been thoroughly investigated. This is due to the extreme difficulty of obtaining samples through the classic trapping methods used for small mammals (cf. Vogel 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious researches highlight the presence of two major mitochondrial clades: the eastern clade in South Asia (India and Sri-Lanka) and the western clade in the Near-East (Iran and Israel) and in the central Europe (France, Italy and Spain) (Adamson et al. 2011,\u0026nbsp;Pinya et al. 2018, Shpirer et al. 2020).\u0026nbsp;Therefore, not much can be said about the colonization of the central Mediterranean area aside from the fact that the colonization took place by the western clade.\u003c/p\u003e\n\u003cp\u003eIn this note, we want to contribute, with new data, to the phylogeography of \u003cem\u003eS. etruscus\u003c/em\u003e in the Mediterranean, analyzing specimens from peninsular and insular Italy and investigating the genetic signature of the expansion in the Mediterranean basin from literature, emerged from fossil evidence.\u003c/p\u003e\n\u003cp\u003eTwenty-five shrews from twelve localities were analyzed (Tab. 1). All the individuals were found dead (i.e. most likely captured by domestic cats or other predators) and are kept at the Museum of Comparative Anatomy \u0026ldquo;Giovanni Battista Grassi\u0026rdquo; (\u0026ldquo;La Sapienza\u0026rdquo; University, Rome). In addition, we analyzed two specimens from Catalonia provided by IRBio (University of Barcelona). These two specimens, already analyzed by Pinya et al. (2018) were re-sequenced to obtain a gene fragment that could be aligned with our sequences. Procedures for DNA extraction, amplification and sequencing followed Castiglia et al. (2007) and Colangelo et al. (2010). The new sequences have been aligned by MEGA X software (Kumar et al. 2018) to 13 sequences available in GenBank, obtaining an alignment of 411 bp. Some of the GenBank available haplotypes of \u003cem\u003eS. etruscus\u003c/em\u003e have been eliminated because they were too short or to avoid gaps in the alignment. The final alignment included 39 specimens from 23 localities (Figure 2). Localities, vouchers and GenBank accession numbers of all animals are reported in Table 1S of supplementary information.\u003c/p\u003e\n\u003cp\u003eRelationships among haplotypes were estimated by Network Analysis, considered the best approach\u0026nbsp;when the number of SNPs is low\u0026nbsp;(e.g., Silva et al. 2009, Nerva er al. 2021). We implemented three different reconstruction methods with POPART (Leigh and Bryant 2015) in default settings: the TCS (statistical parsimony), the median joining network (MJNs) and the integer neighbor joining network\u0026nbsp;(IntNJ,\u0026nbsp;Leigh and Bryant 2015).\u0026nbsp;The first two provide the best estimate of the true genealogy when internal node haplotypes are not sampled (Cassens\u0026nbsp;et al. 2005).\u003c/p\u003e\n\u003cp\u003eEleven total haplotypes (SUNC_1-SUNC_11) were identified (GenBank accession numbers will be provided upon acceptance) and of these, five are new haplotypes for \u003cem\u003eS. etruscus\u003c/em\u003e.\u0026nbsp;The alignment also detected 23 SNPs (17 parsimony informative and 6 singletons).\u0026nbsp;The SUNC_1 haplotype is the most represented, present in 22 of the 39 total specimens.\u0026nbsp;This haplotype has also a wide distribution being present in Israel, in Central and Southern Italy, in France and in Spain. The haplotype from Mallorca\u0026nbsp;(Pinya et al. 2018)\u0026nbsp;not used in the alignment because their short size, turned out to be an additional different haplotype close to SUNC_1 (differs by a single mutation over 236bp). The\u0026nbsp;MJNs, the TCS (Fig. 2) and the\u0026nbsp;IntNJ\u0026nbsp;show overlapping topologies, with minimal differences in\u0026nbsp;IntNJ\u0026nbsp;that proposed an alternative connection of the SUNC_5 - SUNC_1 haplotypes. The networks clearly identified the two groups of haplotypes, attributable to the eastern and western clades, separated by a minimum of 12 SNPs (2.9% p-distance).\u0026nbsp;The SUNC_1\u0026nbsp;has the highest number of connections with other haplotypes, four or five depending on the reconstruction method used.\u003c/p\u003e\n\u003cp\u003eThe wide diffusion of SUNC_1 haplotype and in particular its presence in Israel, considered the origin area for \u003cem\u003eS. etruscus\u0026nbsp;\u003c/em\u003ein the Mediterranean, may indicate that it represents an ancestral haplotype arrived in the central Mediterranean through the eastward wave of colonization. This scenario agrees with the paleontological findings. Moreover, the presence of this haplotype in both the study area and Israel excludes an ancient event of vicariance, in agreement with the relatively recent arrival of the species in the Mediterranean (Garcia et al. 2020). It can also be evidenced how several Italian haplotypes derive with star-like relationships from SUNC_1, differing for only 1 or 2 mutations. A similar condition has been found in the black rat from the same area and this have been interpreted as a colonization by a single matriarchal line followed by an \u003cem\u003ein-situ\u003c/em\u003e diversification (Colangelo et al. 2015). The current state of knowledge is full of geographical gaps and does not allow to capture the genetic signatures of the eastward wave of colonization. Therefore, is difficult to say whether the pygmy shrew arrived firstly in Italy or in the eastern Mediterranean area (Garcia et al. 2020). More extensively, this work can encourage further comparative studies on Mediterranean colonization routes of \u003cem\u003eS. etruscus\u003c/em\u003e and new sampling efforts in key areas, such as Turkey, Balkan Peninsula and North Africa, to get an extensive knowledge on colonization dynamics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to thank Mattia Menchetti and Francesc Munoz-Munoz for help in data collection. Thanks are extended to two anonymous reviewers for valuable advices on the first version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eRC received\u0026nbsp;financial support from Sapienza University research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Conduct:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdamson EAS, Bhassu S, Hashim R, Ruedi M (2011) Phylogenetic relationships of Malayan and Malagasy pygmy shrews of the genus \u003cem\u003eSuncus\u003c/em\u003e (Soricomorpha: Soricidae) inferred from mitochondrial cytochrome-b sequences. 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Mammal Review 42: 314-318. https://doi.org/10.1111/j.1365-2907.2012.00215.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mammal-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acth","sideBox":"Learn more about [Mammal Research](http://link.springer.com/journal/13364)","snPcode":"13364","submissionUrl":"https://www.editorialmanager.com/acth/default2.aspx","title":"Mammal Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mediterranean basin, Mitochondrial DNA, Phylogeography, Soricidae, Suncus etruscus","lastPublishedDoi":"10.21203/rs.3.rs-2807604/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2807604/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pigmy white-toothed shrew \u003cem\u003eSuncus etruscus\u003c/em\u003e is a widespread species whose distribution patterns are unclear. Paleontological data suggested an east-west pattern of dispersion in the Mediterranean basin during late Holocene but some doubts are still present especially considering the absence of fossil remains from key areas, as mainland Italy. Here we propose, a preliminary screening of the phylogeographic relationships among Italian pigmy white-toothed shrews and other Mediterranean areas. The Italian haplotypes were all very similar without an evident geographic structure, however we evidenced that the haplotype from Israel, the putative source area for the Mediterranean basin, is almost identical to the most common Italian haplotype. This excludes an ancient event of vicariance between the two areas and we can assume that these haplotypes arrived in the central Mediterranean through the eastward wave of colonization, in agreement with the relatively recent arrival of the species in the area.\u003c/p\u003e","manuscriptTitle":"A first attempt to track genetic signature of the colonization of the Mediterranean basin by the pigmy white-toothed shrew, Suncus etruscus (Eulipotyphla, Soricidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-20 13:27:23","doi":"10.21203/rs.3.rs-2807604/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-04-20T16:44:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-18T07:56:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-17T07:08:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mammal Research","date":"2023-04-12T03:32:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mammal-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acth","sideBox":"Learn more about [Mammal Research](http://link.springer.com/journal/13364)","snPcode":"13364","submissionUrl":"https://www.editorialmanager.com/acth/default2.aspx","title":"Mammal Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"56079eae-5bb7-42a3-a86f-d96dba275db3","owner":[],"postedDate":"April 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:22:18+00:00","versionOfRecord":{"articleIdentity":"rs-2807604","link":"https://doi.org/10.1007/s13364-023-00706-1","journal":{"identity":"mammal-research","isVorOnly":false,"title":"Mammal Research"},"publishedOn":"2023-07-28 21:47:25","publishedOnDateReadable":"July 28th, 2023"},"versionCreatedAt":"2023-04-20 13:27:23","video":"","vorDoi":"10.1007/s13364-023-00706-1","vorDoiUrl":"https://doi.org/10.1007/s13364-023-00706-1","workflowStages":[]},"version":"v1","identity":"rs-2807604","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2807604","identity":"rs-2807604","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}