Amphibia-Reptilia 40 (2019): 121-127 brill.com/amre

Short Note

Phylogeographic evidence for multiple long-distance introductions of the common wall lizard associated with human trade and transport

Joana L. Santos1, Anamarija Žagar1,2,3,∗, Katarina Drašler3, Catarina Rato1, César Ayres4, D. James Harris1, Miguel A. Carretero1, Daniele Salvi1,5,*

Abstract. The common wall lizard has been widely introduced across Europe and overseas. We investigated the origin of putatively introduced Podarcis muralis populations from two southern Europe localities: (i) Ljubljana (Slovenia), where uncommon phenotypes were observed near the railway tracks and (ii) the port of Vigo (Spain), where the species was recently found 150 km far from its previously known range. We compared cytochrome-b mtDNA sequences of lizards from these populations with published sequences across the native range. Our results support the allochthonous status and multiple, long-distance origins in both populations. In Ljubljana, results support two different origins, Serbia and Italy. In Vigo, at least two separate origins are inferred, from western and eastern France. Such results confirm that human-mediated transport is promoting biological invasion and lineage admixture in this species. Solid knowledge of the origin and invasion pathways, as well as population monitoring, is crucial for management strategies to be successful.

Keywords: biological invasions, human-mediated introduction, Podarcis muralis, population admixture, Slovenia, Spain.

Introduction al., 2013). Species are transported to new lo- cations by different means, the importance of Biological invasions are one of the main threats which varies taxonomically, geographically and to biodiversity and together with habitat de- temporally. Often such introductions pose a risk struction and fragmentation are considered the to native biota, via processes such as competi- main causes behind recent biodiversity loss tive exclusion of native populations of ecolog- (Lee, 2002; Schulte et al., 2012; Simberloff et ically similar species, predation of native taxa, or through the possibility of invaders carry- ing diseases (Simberloff et al., 2013; Sacks et 1 - CIBIO Research Centre in Biodiversity and Genetic al., 2011). Consequently, this can lead to a de- Resources, InBIO, Universidade do Porto, Campus de Vairão, Rua Padre Armando Quintas, N° 7, 4485-661 crease of population size and fitness, promot- Vairão, Vila do Conde, Portugal ing partial isolation and strong selective pres- 2 - National insitute of Biology, Vecnaˇ pot 111, 1000 sure, compromising the continuity and stability Ljubljana, Slovenia of native populations (e.g. Sacks et al., 2011; 3 - Herpetological society – Societas herpetological Schulte et al., 2012). One particular problem slovenica, Vecnaˇ pot 111, 1000 Ljubljana, Slovenia 4 - Asociación Herpetológica Española, Museo Nacional is when populations of closely related species de Ciencias Naturales, C/ José Gutiérrez Abascal 2, or evolutionary lineages are introduced in the 28006 Madrid, Spain range of another, since they can compete with 5 - Department of Health, Life and Environmental Sci- native populations or interfere with reproduc- ences, University of L’Aquila, Via Vetoio, 67100 tion, resulting in hybridization (Allendorf et Coppito, L’Aquila, Italy ∗Corresponding authors; al. 2001; Lee, 2002; Schulte et al., 2012). In e-mails: [email protected]; fact, admixture between multiple conspecific [email protected] lineages, introduced into the same locality, can

© Koninklijke Brill NV, Leiden, 2018. DOI:10.1163/15685381-20181040 122 J.L. Santos et al. increase genetic diversity and thus the invasive- This species is also widely introduced outside ness potential of a given species (Wagner et of its native range in Europe and North Amer- al., 2017). A remarkable number of cases of ica (Brown et al., 1995; Schulte et al., 2008; biological invasion in terrestrial habitats corre- Michaelides et al., 2013, 2015). While many spond to reptiles (with over 4500 introductions studies have assessed the origin and paths of reported since 1850; Kraus, 2009). The overall introductions in northern and central European frequency of species introductions is increasing, populations using molecular data (e.g. Schulte and lizards are not exceptions (e.g. Michaelides et al., 2015). Therefore, understanding the ori- et al., 2008, 2012; Michaelides et al., 2013, gin and pathways of introductions is essential 2015), to date little is known in the rest of the to corroborate the alien status of putative intro- species range duced populations, as well as to design effective We used mitochondrial cytochrome-b (cytb) management strategies to control or prevent in- DNA sequence data to assess the alien sta- vasions. tus and phylogeographic origin of two possi- The common wall lizard, Podarcis muralis bly introduced populations in urban and heavily (Laurenti, 1768) has the largest distribution humanized landscapes: (i) one from Ljubljana range among species of the genus Podarcis (Slovenia), where lizards showing uncommon (Wagler, 1830), including most of central Eu- phenotypes in terms of colouration were re- rope, Mediterranean peninsulas (Iberia, Italia, ported; and (ii) the second in Vigo (Spain), Balkans), and NW Anatolia (Sillero et al., 2014). Across this range, up to 23 evolution- which is outside the known species’ range. By ary lineages were identified, likely originat- comparing our new DNA sequences with those ing from multiple glacial refugia within the from native populations, we aimed to assess three Mediterranean peninsulas, but also in ex- their origin and, in case of introduction, to in- tra Mediterranean regions (Salvi et al., 2013). fer their possible pathway.

Figure 1. (a) Maps of sampling locations for the two study-cases and representation of the distribution range of Podarcis muralis. (b) Satellite images with sampling localities and photographs of the habitat type for the two study-cases: Ljubljana, Slovenia and (c) port of Vigo, Spain. New introduced populations of Podarcis muralis 123

Materials and methods a morphological pattern distinct from native P. bocagei,also present in the area. Namely, P. muralis lizards from Vigo Two independent samplings were conducted in this study. In showed dark flanks contrasting with the dorsum, and bluish July 2013, nine samples were collected in Ljubljana (Slove- ocelli surrounded by dark colour on the shoulder, dark spots nia), in two different places separated by 1.5 km, across arranged in rows on the submaxilar scales and reddish or transects of approximately 200 m, next to the railway tracks white belly. (Location A1: Lat. 46.059°N, Lon. 14.504°E; Location A2: DNA extraction was performed using a high-saline Lat. 46.049°N, Lon. 14.491°E; fig. 1a, b). Podarcis muralis method (Sambrook et al., 1989) from tail tip tissues. The mi- is relatively common and widespread in Ljubljana (Krofel tochondrial cytb gene was amplified by Polymerase Chain et al., 2009), with a typical mostly uniformly brown dor- Reaction using the primers CytbG and cb2R (Palumbi et al., sal colouration, and a red or white ventral colouration in 1991). The amplified cytb fragment overlaps with the frag- both males and females. However, the observed sampled in- ment sequenced by Salvi et al. (2013). Amplification con- dividuals had uncommonly interconnected dark patches and ditions were as in Mendes et al. (2017) and sequencing re- a light greenish colouration on the dorsal side, with an or- actions were conducted by an external company, Genewiz ange or white ventral colouration. (UK). In September 2017, four samples of P. muralis were col- Electropherograms were checked and consensus se- lected at the maritime port of Vigo (NW Spain), along the quences were aligned using MUSCLE implemented in Bouzas seafront near a carpark for cargo boats (Location Geneious v4.8.5 (Biomatters Ltd). The dataset includes nine B1: Lat. 42.232°N, Lon. −8.761W; fig. 1a, c). This popu- sequences of P. muralis from Ljubljana and four from Vigo, lation was found 150 km to the west of the closest previ- generated in this study, and 185 sequences downloaded ously known population, and outside of the range predicted from GenBank used in Salvi et al. (2013). Podarcis liolepis by ecological models (Cabana et al., 2016). A tissue sample (GenBank accession number KF372218) was designated as from the tail and photographs were collected for each indi- an outgroup. Phylogenetic relationships were inferred us- vidual. The sampled lizards were easily identified, showing ing the Maximum Likelihood (ML) method implemented

Figure 2. (a) Maximum likelihood phylogenetic tree based on 198 cytb mtDNA sequences of Podarcis muralis sampled for this study and across its native range. Bootstrap values over 1,000 replicates are reported in correspondence of the nodes; nodes with a support lower than 30% were collapsed. The mitochondrial clades are numbered from 1 to 23, according to Salvi et al. (2013). The clades including the 13 new samples from Ljubljana (highlighted in pink) and Vigo (highlighted in cyan) are expanded in the boxes with detailed information on geographic origin of sequenced individuals (SRB: Serbia; SL: Slovenia; KR: Croatia; ITA: Italy; AUS: Austria; FR: France; ES: Spain; Andorra: Andorra; GER: Germany). (b) Map with the putative distribution range of P. muralis lineages (modified from Salvi et al., 2013); grey shade: native range; dots: introduced populations known in Germany and Great Britain; arrows: lineages of origin of introduced populations in Vigo and Ljubljana. (c) Possible introduction routes based on the marine trade of PSA-Citröen factory, in Vigo and the railways tracks of Ljubljana. 124 J.L. Santos et al. in MEGA 7 (Tamura et al., 2017), applying the nucleotide transportation by train may be the potential vec- substitution model K2P+G (Kimura, 1980), selected under tor for introductions (fig. 2c). However, we can- the Akaike Information Criterion (Akaike, 1974) and sup- not exclude the hypotheses that the Serbian lin- port for nodes was evaluated with 1,000 bootstrap repli- eage may also be distributed in Slovenia, since cates. The obtained phylogenetic trees were visualised and the sample collected in North-eastern Croatia edited using FigTree v1.4.2 (Rambaut, 2014). New se- (DQ001025; Podnar et al., 2007), belongs to quences were submitted to GenBank (accession numbers the Serbian clade (Salvi et al., 2013), suggest- MH911656-MH911668). ing a widespread distribution of this lineage. Still, since the sampled Croatian location cited above is also close to the same railway line Results and discussion connecting Serbia, Croatia and Slovenia, it may be that along this line multiple introductions of For all individuals, we obtained cytb sequences several different populations of P. muralis have of 399 base pairs. The ML phylogenetic tree occurred. Indeed, this is not the first time that (fig. 2a, b) showed the same 23 clades previ- range expansion mediated by railways has been ously identified by Salvi et al. (2013). Remark- proposed (Gherghel et al., 2009; Urosevic et al., ably, the nine sequences from Ljubljana fell 2016). into three distinct clades: 4, 10 and 11, other- Inside Vigo’s harbour (fig. 2b), our genetic re- wise known from Serbia, Central Italy (Marche sults indicate that the origin of this introduction region) and Slovenia, respectively (fig. 2a, b). was not from the Iberian Peninsula, but from In particular, samples M20776 and M20781 two distant and distinct regions within the na- clustered with individuals from Serbia, sam- tive range, i.e. the western and eastern France ples M20772, M20777, M20783, M20787 and lineages. This then resembles the Ljubljana sce- M20874 clustered with individuals from Slove- nario. The proximity to a large car storage depot nia and, samples M20784 and M20865 clus- (of the PSA Peugeot-Citroën factory) beside a large loading dock may account for the intro- tered with individuals from Marche region. duction pathway of the lizards. This company Similarly, the four sequences from Vigo fell into uses the port of Vigo as a key node for the trade two distinct clades – 1 and 6 (fig. 2a, b). Partic- of cars produced in France – namely in Saint- ularly, samples M28241 and M28246 clustered Nazaire and Rennes, as well as in Sochaux with individuals from western France and sam- and Mulhouse – towards western Europe (Spain ples M28242 and M28250 clustered with indi- and Portugal), Africa (Nigeria, Ethiopia and viduals from eastern France. Kenia), South America (Brazil and Argentina), In Ljubljana, the molecular results confirmed and China (www.groupe-psa.com; fig. 2c). If our initial hypothesis based on phenotypic as- this hypothesis is correct, this also suggests sessment – the presence of non-native lin- that lizards could also easily be introduced into eages. Among the lizards sampled in Ljubl- Africa, Asia or America, following the steps of jana (fig. 1a, b), our results indicate that, ap- the car factory trade, if they will find a suitable parently, more than one introduction event of environment. The multiple origins of the popu- P. muralis occurred, since sampled individuals lation further suggest that the port may be acting showed a close genetic relationship with popu- as an invasion hub (Letnic et al., 2015). A neigh- lations from two geographically distant sites: boring touristic harbor where boats usually de- Serbia (Clade 4) and Italy (Clade 11). The prox- part to the nearby protected area of Atlantic Is- imity of two train stations near the collection lands from Galicia National Park (AIGNP), rep- sites, and the fact that the railway lines are resents a threat for subsequent invasion of these linked to both Serbia and Italy, suggest that small islands populated by endemic populations New introduced populations of Podarcis muralis 125 of Podarcis bocagei and Podarcis guadarramae we cannot exclude that the genetic variation that (Galán, 2003). result within hybrids between intra-specific lin- Man-made structures, mainly roads and rail- eages could have conservation value, particu- ways, as well as other trade and transportation larly in the case where adaptive potential in routes can be seen as corridors for both distribu- admixed populations would be observed (e.g. tion range expansions and colonization of new Becker et al., 2013); in the case of Vigo the in- areas, especially in cases of habitat degradation troduced population of P. muralis may outcom- (Hedeen and Hedeen, 1999). It has allowed the pete with the native species resulting in a loss success of invasive lizard species (Ficetola and of biodiversity. Therefore, especially in this lat- Padoa-Schioppa, 2009), not only of P. muralis ter case, conservation strategies should involve (Gherghel et al., 2009, this study), but also of controlling the pathway of these invasions, such other species (Kraus, 2009). as commercial cargo and trade. Recent intro- Results obtained in our study demonstrate duction events, as the case of Vigo, need an ur- that greater efforts are needed to recognize gent intervention to define, together with local the source of invasions, and to identify the authorities, strategies to eradicate the popula- pathways and timing of introductions. In cases tions. Otherwise, there is a risk of further invad- where introduction of non-native lineages is ing sensitive areas (as the AIGNP, see above). suspected, the molecular approach is the more Since they seem to be recent introductions they effective method to identify the origin of popu- are concentrated in small and restricted areas, lations and provide results for assessment of which facilitates the eradication process. Al- potential pathways of introduction (e.g. Silva- though this is the most efficient strategy and, Rocha et al., 2012, 2014; Salvi et al., 2014). when applied in the first stages of invasions is With this study, using a molecular approach, also better in both terms of costs and success of we effectively identified non-native populations probability, it should be combined with periodi- of P. muralis in Ljubljana and Vigo. The two cal monitoring to ensure a total eradication and cases of introductions shown are from multi- to prevent or allow early detection of new intro- ple population sources belonging to multiple ductions (Simberloff et al., 2013). lineages. Consequently, hybridization between Additionally, resources should be invested for closely related individuals is possible. This may collecting more data concerning the current dis- cause a disruption of specific gene combinations tribution and demographic status of the intro- that results either in a loss of genes adapted to duced populations, to determine the factors lim- the native habitat of both lineages or in a com- iting and/or promoting the success of their es- petitive advantage against native species (Al- tablishment and for identifying the impacts on lendorf et al., 2001; Schulte et al., 2012). On native populations and on other species (IUCN, the other hand, such hybrid populations can 2000). In sum, our study confirmed the human produce genetic pollution if they return to the induced dispersal potential of P. muralis,show- source population. Many introduced popula- ing the importance of investigating sources and tions develop different behavioral, physiologi- pathways of introductions to avoid further pos- cal or parasite resistance adaptations that can sible invasions. allow them to outcompete the native popula- tion and endemic species under specific con- Acknowledgements. The authors would like to thank Fran- ditions, resulting in a major threat to biodiver- cisco Garcia for sharing information regarding the possible sity (Ficetola and Padoa-Schioppa, 2009; Sim- presence of P. muralis in Vigo. All lizards were collected berloff et al., 2013). At this regard, we should under the special licenses 35601-14/2013-5 issued by the Slovenian Environment Agency and by licence 07/2015 is- distinguish the conservation implications of the sued by Consellería de Medio Ambiente e Ordenación do two cases of introduction. While in Ljubljana Territorio of Xunta de Galicia (Spain). AŽ, CR and DJH 126 J.L. Santos et al. were supported by Fundação para a Ciência e Tecnolo- Lee, C.E. (2002): Evolutionary genetics of invasive species. gia (FCT) under the Programa Operacional Potencial Hu- Trends Ecol. Evol. 17: 386-391. mano – Quadro de Referência Estratégico Nacional, Euro- Letnic, M.I., Webb, J.K., Jessop, T.S., Dempster, T. (2015): pean Social Fund and Portuguese Ministério da Educação e Restricting access to invasion hubs enables sustained Ciência (AŽ: PhD grant SFRH/BD/81324/2011; CR: post- control of an invasive vertebrate. J. Appl. Ecol. 52: 341- doctoral grant SFRH/BPD/92343/2013; DJH: IF-contract 347. IF/01627/2014). 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