Journal of Parasitology

MOLECULAR SURVEY OF IN PODARCIS WALL DETECTS , AND --Manuscript Draft--

Manuscript Number: GE-2908R2 Full Title: MOLECULAR SURVEY OF APICOMPLEXA IN PODARCIS WALL LIZARDS DETECTS HEPATOZOON, SARCOCYSTIS AND EIMERIA SPECIES Short Title: HARRIS ET AL. - SURVEY OF APICOMPLEXA IN PODARCIS LIZARDS Article Type: Regular Article Corresponding Author: David James Harris CIBIO Vairao, Vila do Conde PORTUGAL Corresponding Author Secondary Information: Corresponding Author's Institution: CIBIO Corresponding Author's Secondary Institution: First Author: David James Harris First Author Secondary Information: All Authors: David James Harris João P. M. C. Maia Ana Perera All Authors Secondary Information: Abstract: The occurrence of Apicomplexan parasites in Podarcis wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from three genera, Hepatozoon, Sarcocystis and Eimeria were detected. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera island, in the Balearic Islands, appears related to Sarcocystis gallotiae, known only from endemic Gallotia lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite form presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae. Eimeria is reported for the first time from Podarcis lizards. This study shows the power of detecting multiple different Apicomplexa parasites through screening of tail tissue samples and blood drops that are often collected in for other purposes.

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1 RH: HARRIS ET AL. – SURVEY OF APICOMPLEXA IN PODARCIS LIZARDS

2 MOLECULAR SURVEY OF APICOMPLEXA IN PODARCIS WALL LIZARDS

3 DETECTS HEPATOZOON, SARCOCYSTIS AND EIMERIA SPECIES

4 D. James Harris, João P. M. C. Maia*, and Ana Perera

5 CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de

6 Vairão, 4485-661 Vairão, Portugal. e-mail: [email protected]

7 ABSTRACT: The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the

8 Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the

9 18S rRNA gene. Species from 3 genera, Hepatozoon, Sarcocystis, and Eimeria, were found.

10 The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the

11 evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part

12 of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in

13 Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis

14 gallotiae, known only from endemic Gallotia sp. lizards from the Canary Islands. Based on

15 the lack of snake predators on this island, this parasite presumably presents an atypical

16 transmission cycle that uses the same host species as both intermediate and final host through

17 cannibalism, like S. gallotiae. Eimeria sp. is reported for the first time from Podarcis spp.

18 lizards. This study shows the power of detecting multiple different apicomplexan parasites

19 through screening of tail tissue samples and blood drops that are often collected in reptiles for

20 other purposes.

21 The Apicomplexa includes a diverse group of unicellular parasites that is possibly the

22 poorest studied group of all in terms of biodiversity (Morrison, 2009). Yet, according

23 to the European Cooperation in Science and Technology (COST), apicomplexan protozoa

24 caused more human deaths than any other group of infectious agents and are also the most

25 significant parasites of livestock. Although molecular techniques have now become

26 established as standard tools for monitoring parasite populations (Beck et al., 2009), this is

27 heavily biased to certain groups within Apicomplexa, such as spp., and is also

28 directed primarily towards humans or commercially important groups. However,

29 parasites also have dual interests for conservation biologists. On the one hand, parasite-driven

30 declines in wildlife are becoming common (Pedersen et al., 2007), while on the other hand,

31 parasites themselves are also a major component of biodiversity (Poulin and Morand, 2004).

32 Due to their obligate relationship with a host, parasites are especially at risk through co-

33 extinction, where the loss of 1 species leads to the loss of another, so that an extinction

34 cascade can occur. Models suggest that co-extinction may be very common (Dunn, 2009), but

35 lack of knowledge hinders assessments.

36 Reptiles are hosts to a wide variety of apicomplexan parasites, including families with

37 human medical importance such as (which includes Toxoplasma), ,

38 e.g., parvum, and Haemosporidae, e.g., Plasmodium spp. The most common

39 and widely distributed apicomplexans found in reptiles are species of Hepatozoon (Telford,

40 2009). Traditionally, assessment of gametocyte morphology in the vertebrate host and

41 sporogonic stages in the invertebrate host was used for diagnosis and species description. This

42 situation is complicated, however, when only information from the vertebrate host is

43 available, which is often the case. Fortunately, for Hepatozoon species, universal primers are

44 available that can amplify part of the 18S rRNA gene. These have been used to assess

45 prevalence, estimate phylogenies, and confirm distinctiveness in a variety of vertebrate hosts,

46 including mammals (Merino et al., 2009) and reptiles (Harris et al., 2011; Maia et al., 2011).

47 However, although for mammals there is a wide knowledge regarding other apicomplexan

48 groups, much less information is available for reptiles. Eimeria species, for example, cause

49 coccidiosis and are the most important protozoan pathogens of poultry (Beck et al., 2009).

50 Not surprisingly, there is considerable molecular data available for both domestic (Barta et al.,

51 1997; Miska et al., 2010) and wild (Honma et al., 2007) birds, but only a single 18S rRNA

52 sequence is available from a species that infects reptiles, i.e., Eimeria arnyi (Upton and

53 Oppert, 1991). Similarly, Sarcosporidia are prevalent around the world, affecting amniotic

54 vertebrates, including humans. Species of Sarcocystis have a diheteroxenous cycle and, for

55 most described species, mammals and reptiles are both intermediate and final hosts. However,

56 some insular species such as Sarcocystis gallotiae or S. dugesii that infect lizards endemic to

57 the Canary and Islands, respectively, are atypical since the same species is both

58 intermediate and final host, which occurs via cannibalism (Matuschka and Bannert, 1987;

59 Matuschka, 1988). Various other species have a snake-lizard life cycle (reviewed in

60 Matuschka, 1987). Again, however, despite a wide variety of species from mammals and birds

61 having been sequenced for the 18S rRNA gene, often to confirm the distinctiveness of new

62 species (Xiang et al., 2010; Kutkiene et al., 2010, 2011), only a couple of studies have

63 included such data from species (Slapeta et al., 2001, 2003).

64 The aim of the present study was to screen populations of Podarcis spp. wall lizards

65 () from the Iberian Peninsula and the Balearic Islands for apicomplexan parasites

66 using 18S rRNA primers. Several Podarcis spp. from the Iberian Peninsula have been shown

67 to have high levels of infection with hemogregarines using traditional blood smears (Álvarez-

68 Calvo, 1975; Amo et al., 2005; Roca and Galdón, 2010), and 18S rRNA primers are known to

69 amplify Hepatozoon spp. from lacertids in North Africa, including Podarcis vaucheri (Maia et

70 al., 2011). At the same time, these lizards were assessed using autotomized tail samples that

71 are typically used in phylogeographic studies of the host (Pinho et al., 2011). Sarcocystis spp.

72 have been reported to infect several species of Podarcis, including Podarcis lilfordi endemic

73 to the eastern Balearic Islands (Mayo et al., 1988) and classified as endangered on the IUCN

74 red list (Pérez-Mellado and Martínez-Solano, 2009) among others (see a complete review in

75 Odening, 1998). Since sarcocysts are typically observed in tail muscle in lizards (Abdel-

76 Ghaffar et al., 2009), there is also a reasonable possibility that these will be detected by

77 molecular methods. Additionally, a possible amplification of an apparent member of the

78 Eimeriidae was reported using these primers within reptiles from the Seychelles (Harris et al.,

79 2011), and thus the possibility exists of detecting these, or even other, apicomplexan parasites.

80 In the present study, we use molecular methods to assess the existence of different

81 apicomplexan species using 18S rRNA primers. Positive samples were sequenced and

82 included in a phylogenetic assessment using previously published sequences to help

83 determine the evolutionary history and patterns of genetic diversity within these parasite

84 species.

85 MATERIALS AND METHODS

86 Sample collection

87 Samples from lizard specimens were collected from 2 localities in the Iberian Peninsula

88 (April 2008 in Beja, Portugal, and June 2009 in Alba de Tormes, Spain) and from Cabrera

89 island, Balearic Islands (September 2010). Tail tips from the lizards were preserved in 96%

90 ethanol for molecular analysis and blood drops were placed in Whatman paper. When lizards

91 autotomize their tails, very little blood is typically lost and, in most cases, there was not

92 enough to make blood slides. After samples were collected, the lizards were released at the

93 site of capture. A total of 44 tissue samples was collected, i.e., 10 from Cabrera (Podarcis

94 lilfordi), and 34 from the 2 localities on the Iberian Peninsula (Podarcis hispanica) (see Table

95 I).

96 DNA extraction, amplification, and sequencing

97 DNA was extracted from tissue and blood drops using standard High Salt methods

98 (Sambrook et al., 1989). Detection of parasites was made using PCR reactions with the

99 primers HEMO1 and HEMO2 (Perkins and Keller, 2001) targeting part of the 18S rRNA

100 region, and using the primers HepF300 and HepR900 (Ujvari et al., 2004), targeting another

101 partially overlapping part of the 18S rRNA region. Conditions of the PCR are detailed in

102 Harris et al. (2011). Briefly, PCR cycling for the HEMO primers consisted of 94 C-30 sec, 48

103 C-30 sec, 72 C-1 min (35 cycles), while for the Hep primers annealing temperature was 61 C.

104 Negative and positive controls were run with each reaction. The positive PCR products were

105 purified and sequenced by a commercial sequencing service (Macrogen Inc., Seoul, Korea).

106 All sequences were performed in both directions. Sequences were deposited in GenBank

107 under the accession numbers *** to *** (to be added after final acceptance).

108 Phylogenetic analysis

109 Due to the considerable genetic differences between the parasites detected, and since

110 different parasites were detected with the alternative primer pairs (see results), separate

111 phylogenetic analyses were performed for each of the parasite groups recovered. For each

112 dataset new sequences generated in this study were aligned with related sequences retrieved

113 from GenBank. All sequences for which the full length was available were included in the

114 analyses, except for cases in which many closely related sequences are available for some

115 taxa, e.g., Eimeria reichenowi. In these cases, single exemplars were arbitrarily used.

116 Sequences were aligned using ClustalW software implemented in the program BioEdit (Hall,

117 1999). The final 3 datasets contained 47, 21, and 12 taxa that were 798, 609, and 636 bp in

118 length, respectively.

119 Maximum Likelihood (ML) analysis with random sequence addition (100 replicate

120 heuristic searches) was used to estimate their evolutionary relationships using the software

121 PAUP (Swofford, 2002). Support for nodes was estimated using the bootstrap technique

122 (Felsenstein, 1985) with 1,000 replicates. The model of evolution employed, in all cases GTR

123 with an estimate of invariant sites and a gamma distribution of site variation, was chosen

124 using the AIC criteria carried out in Modeltest 3.06 (Posada and Crandall, 1998). Bayesian

125 analysis was implemented using Mr. Bayes v.3.2 (Huelsenbeck and Ronquist, 2001) with

126 parameters estimated as part of the analysis. The analysis was run for 1 x 107 generations,

127 saving 1 tree every 1,000 generations. The log-likelihood values of the sample point were

128 plotted against the generation time and all the trees prior to reaching stationary were discarded

129 as burn-in samples. Remaining trees were combined in a 50% majority consensus tree.

130 Following Maia et al. (2011) and Morrison (2009), Adelina bambarooniae was used as an

131 outgroup for rooting the phylogenetic tree for the Hepatozoon spp. sequences. Following

132 Slapeta et al. (2003), species of and Hylokossia were used to root the estimate of

133 relationships of the Sarcocystis spp., while, following Jirku et al. (2009), Choleoeimera sp.

134 and Eimeria tropidura were used to root the estimate of relationships of the Eimeria spp.

135 dataset.

136 RESULTS

137 Of the 44 individuals analyzed, 24 were found to be infected with Hepatozoon sp.

138 parasites, using the HEMO primers. This included infections in Podarcis hispanica (0% in

139 Portugal and 89.5% in Spain) and Podarcis lilfordi (70% in Cabrera) (Table I). Two

140 individuals negative for the HEMO primers were found to be infected with Hepatozoon sp.

141 using the HEP primers. Both phylogenetic methodologies produced the same estimate of

142 relationships among the Hepatozoon sp. sequences of the 18S rRNA gene (Fig. 1). All of the

143 Hepatozoon sp. lineages identified in Podarcis spp. from the Iberian Peninsula and the

144 Balearics formed part of a group composed of 1 of the major clades in North Africa, detected

145 in various lacertids (species of Timon, Podarcis and Scelarcis) and skinks (species of Eumeces

146 and Chalcides), and a clade of mammalian Hepatozoon sp. isolates (Fig. 1).

147 Within P. lilfordi, several individuals (80%) were found to be infected with Sarcocystis sp.

148 using the HEP primers, although no infection was reported in the other 2 localities analyzed

149 (Table I). Some of these (5) were also infected with Hepatozoon sp. parasites, but there was

150 no obvious double amplification, i.e., with the HEMO primers only Hepatozoon sp. was

151 amplified, and with the HEP only Sarcocystis sp. were amplified. The sequences amplified

152 group with the ones available in GenBank for other lacertid species (Fig. 2). Interestingly, the

153 Sarcocystis sp. from P. lilfordi was most closely related to S. gallotiae from Gallotia sp.

154 lizards, rather than S. lacertae that infects P. muralis (Fig. 2).

155 Also using the HEP primers, a single individual of P. hispanica from Portugal was found to

156 be infected with Eimeria sp. (Table I). Although there is limited available comparative data

157 for Eimeria spp. from reptiles, the sequence was determined to be most closely related to E.

158 arnyi from a snake, and then to E. ranae from a frog (Fig. 3). To our knowledge, this is the

159 first report of Eimeria sp. infection in Podarcis sp. lizards.

160 Thus, in this study HEP primers amplified Hepatozoon, Sarcocystis and Eimeria species,

161 while HEMO primers only amplified Hepatozoon sp. In dual infected P. lilfordi, HEP primers

162 preferentially amplified Sarcocystis sp., while HEMO amplified Hepatozoon sp.

163 DISCUSSION

164 For the first time, Hepatozoon sp. from Podarcis spp. from the Iberian Peninsula and the

165 Balearic Islands can be placed in a phylogenetic framework. Despite the shorter sequences

166 used in this study (about 800 bp), the overall estimate of phylogeny is essentially the same as

167 that found by Maia et al. (2011), using about 1400 bp, although with lower levels of statistical

168 support. All the newly sequenced parasites form part of a group that includes a variety of

169 lacertids and skinks from North Africa (Maia et al., 2011). The 4 Hepatozoon spp. haplotypes

170 recovered in this study do not form a clade, but are instead related to various haplotypes from

171 North Africa and to mammalian isolates. Although this increases the known geographic

172 distribution of this group, and the number of known host species, the inclusion of new data

173 has little influence on the overall phylogeny of Hepatozoon spp. This lineage, previously

174 known only from reptiles, appears to be related to a lineage that includes H. canis, H.

175 americanum, and H. felis, which usually use carnivores, including cats and dogs, as their

176 intermediate hosts. The present study also confirms that closely related Hepatozoon spp.

177 haplotypes can be found in unrelated hosts, i.e., in this case, species of Chalcides, Eumeces,

178 Atlantolacerta, Scelarcis, and Timon, and at least 3 Podarcis species. Clearly, there is a

179 difference in specificity between the pairs of primers used, with Hep primers amplifying

180 samples that failed to amplify with the partially overlapping HEMO primers. The Hep primers

181 are less specific, also amplifying other apicomplexans. It is, therefore, inappropriate to

182 directly compare prevalence across studies using these different markers.

183 Mayo et al. (1988) first reported Sarcocystis dugesii from P. lilfordi on Cabrera Island.

184 Until then, S. dugesii had been only known from the Madeiran endemic lacertid Teira dugesii

185 (Matuschka and Mehlhorn, 1984), and later reviews report only a form "similar to S. dugesii "

186 in P. lilfordi (Odening, 1998). Given that Sarcocystis spp. show some signs of co-evolution

187 with their snake hosts (Slapeta et al., 2003), it could be expected that the form in P. lilfordi

188 would be more similar to Sarcocystis lacertae, known from Podarcis muralis. However, in

189 our estimate of relationships, Sarcocystis spp. from P. lilfordi are clearly more closely related

190 to S. gallotiae from Gallotia spp. lizards endemic to the Canary Islands. Sarcocystis gallotiae

191 and S. dugesii are notable because they use the same lizards as both intermediate and final

192 host, a life cycle described as dihomoxenous (Matuschka, 1988), whereas Sarcocystis lacertae

193 has a more typical snake (Coronella austriaca)-lizard (P. muralis) life cycle (Volf et al.,

194 1999). Given that there are no snake or mammalian predators on Cabrera Island, the

195 Sarcocystis sp. found in P. lilfordi is likely to similarly use the lizard as both intermediate and

196 final host. Indeed, cannibalism is in general more common in island lizards where resources

197 are limited (Pafilis et al., 2009). Although these are the only species known to have always the

198 same intermediate and final host, 2 other species, Sarcocystis rodentifelis and S. muris, can

199 occasionally exhibit transmission from an intermediate host to a paratenic host through

200 cannibalism (Slapeta et al., 2001). In a concatenated analysis of large and small subunit rRNA

201 genes, Slapeta et al. (2003) found these 2 species to be sister taxa, and related to a lineage of

202 S. lacertae and S. gallotiae, although in our analysis the phylogenetic position of S.

203 rodentifelis is not well supported. The finding of another dihomoxenous form related to S.

204 gallotiae raises important evolutionary questions. First, it implies that dihomoxeny may have

205 arisen just once as a facultative strategy that became fixed in island host species that had no

206 alternative predator to fill the niche of final host. This would mean that a reversal to a

207 diheteroxenous life cycle occurred in S. lacertae. Alternatively, dihomoxeny could have arisen

208 twice, once in the lineage leading to S. muris and S. rodentifelis, and again in the lineage

209 leading to S. gallotiae and the form found in P. lilfordi. Second, the relationship of a parasite

210 lineage found in 2 geographically separated island-endemic hosts implies that the lineage is

211 probably old. Gallotia spp.separated from other ancestral lacertids when it colonized the

212 Canary Islands perhaps 17 to 20 MYA (Cox et al., 2010). Similarly, P. lilfordi has been

213 separated since the time of the Messinian Salinity Crisis, approximately 5.3 MYA (Brown et

214 al., 2008). To fully address both these questions relating to the origins of dihomoxeny and its

215 age, other closely related (and possibly conspecific) Sarcocystis species need to be included in

216 the phylogenetic framework, in particular S. dugesii from T. dugesii in Madeira and

217 Sarcocystis podarcicolubris, another Sarcocystis species known from Podarcis spp. that has a

218 diheteroxenous life cycle (Matuschka, 1985).

219 A single specimen of P. hispanica from Portugal was shown to be infected with Eimeria sp.

220 using the HEP primers. This is again relevant for various reasons. First, it is, to our

221 knowledge, the first report of Eimeria sp. from Podarcis sp. There is an old record of Eimeria

222 sp. in another lizard that occurs in the Iberian Peninsula, Zootoca vivipara (formerly

223 vivipara) from 1935 (Yakimoff and Gousseff, 1935). Additionally, there are a few reports of

224 Eimeria sp. in lacertids from North Africa (Acanthodactylus sp.; Sakran et al., 1994; Al Yousif

225 et al., 1997), Asia (Eremias sp. and Takydromus sp. [Davronov, 1985; Telford, 1992,

226 respectively]), and from the Canary Islands (Gallotia sp. [Matuschka and Bannert, 1987]).

227 Second, there were previously almost no molecular data regarding Eimeria sp. from

228 amphibians and reptiles, with only a couple of sequences previously published (Slapeta et al.,

229 2001, 2003).

230 The estimate of relationships presented here (Fig. 3) indicates that the species of Eimeria

231 from Podarcis spp. is most closely related to E. arnyi, the only other analyzed specimen from

232 a reptile host. In our analysis, Eimeria spp. from poikilothermic hosts form a monophyletic

233 group, while those from endothermic hosts are paraphyletic since the clade that includes E.

234 gruis and E. reichenowi, both from cranes, is basal to all ingroup taxa, although these

235 relationships are only weakly supported. This differs from the estimate of relationship of

236 Jirku et al. (2009), who found that the Eimeria spp. from endothermic hosts formed a

237 monophyletic group. Jirku et al. (2009) unfortunately did not include any members of the E.

238 gruis/E. reichenowi clade. Although Honma et al. (2007) also found the clade of E. gruis and

239 E. reichenowi as sister taxa to the remaining eimeriid coccidian with Stieda bodies, in this

240 assessment no Eimeria sp. from poikilothermic hosts was included. Clearly, additional data

241 regarding all available taxa will be needed to resolve this aspect of eimeriid relationships.

242 Finally, this is also an atypical example of Eimeria sp. detection, i.e., fecal material is almost

243 always used to amplify and sequence the DNA of these parasites (Motriuk-Smith et al., 2009;

244 Power et al., 2009). Since they autotomize naturally and typically regenerate, tail samples are

245 widely available in museum collections and stored in ethanol for genetic analyses of the

246 lizards. The present study shows that not only Hepatozoon and Sarcocystis species can be

247 detected from these tissues, but also Eimeria spp. In the present study, both blood and tissue

248 samples could be used to detect parasites. However, it is not yet clear if 1 source of DNA will

249 allow more accurate detection of parasites at low levels of parasitemia. This is something that

250 warrants further investigation.

251 Although molecular techniques are now standard tools for monitoring parasite populations

252 in humans and domestic animals, their use in wild animal populations has until recently been

253 limited. However, determining parasite distribution is crucial for conservation biologists,

254 especially in species whose existence are under challenge. The survey performed on Podarcis

255 spp. indicates that various apicomplexan parasites can be detected using tail tissue samples

256 that are often collected for other purposes. At the same time, by sequencing the 18S rRNA

257 gene, new insights into the evolutionary history of the parasites can be obtained. Depending

258 on the primers used, different apicomplexans will be detected, i.e., the cases of dual infection

259 with species of Hepatozoon and Sarcocystis in P. lilfordi clearly highlight this point. There is a

260 need, therefore, for further assessments of possible parasites using additional molecular

261 markers so that a clearer picture of the spectrum of parasites infecting these species can be

262 obtained.

263 ACKNOWLEDGMENTS

264 This project was supported by a grant from FCT to DJH (PTDC/BIA-BDE/74349/2006).

265 AP is supported by a FCT postdoctoral fellowship (SFRH/BPD/26546/2006), and JPMCM by

266 a FCT PhD grant (SFRH/BD/74305/2010). Thanks to our colleagues from CIBIO who helped

267 with the fieldwork, and to the people and entities that made it possible to obtain samples and

268 permits in Spain and Portugal. Thanks also to the anonymous reviewers and the Associate

269 Editor, Dr. Susan Perkins, for their helpful comments on an earlier draft of this manuscript.

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413 FIGURE 1. Tree derived from a Maximum Likelihood (ML) analysis of the Hepatozoon sp.

414 sequences. New haplotypes from this study are in bold. For all other codes refer to Maia et al.

415 (2011).

416 FIGURE 2. Tree derived from a Maximum Likelihood (ML) analysis of the Sarcocystis sp.

417 sequences. Bootstrap values for ML are given above relevant nodes, and Bayesian Posterior

418 Probability below them. When both values were 100%, this is indicated with a +. New

419 haplotypes from this study are in bold.

420 FIGURE 3. Tree derived from a Maximum Likelihood (ML) analysis of the Eimeria sequences.

421 Bootstrap values for ML are given above relevant nodes, and Bayesian Posterior Probability

422 below them. When both values were 100%, this is indicated with a +. New haplotypes from

423 this study are in bold.

424

425 *Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo

426 Alegre FC4 4169-007 Porto, Portugal.

427

Figure1 A. bambarooniae 0.001 subs/site H. sp - ex. Bandicota indica Click here89 H. tosp - ex.download Abrotrix olivaceus Figure: Fig1-Hepatozoon.pdf H. sp - ex. Abrotrix sanborni 92 98 H. sp - ex. Clethrionomys glareolus H. ayorgbor - ex. Python regius PTY01PoAL - H. sp - ex. Ptyodactylus oudrii 3126TmMO - H. sp - ex. Tarentola mauritanica PTY34PoMO - H. sp - ex. Ptyodactylus oudrii Q39QmMO - H. sp - ex. Quedenfeldtia moerens Rodent, lizard H. boigae - ex. Boiga fusca and snake hosts 100 35SHLsSE 100 41FGLsSE H. sp - ex. Lycognathophis seychellensis 87 59PLLsSE 87 1SPMwSE - H. sp - ex. Mabuya wrightii DB486TmAL H. sp - ex. Tarentola mauritanica DB487TmAL DB1606QmMO - H. sp - ex. Quedenfeldtia moerens LPA1TtMO - H. sp - ex. Timon tangitanus 100 H. sp - ex. Sciurus vulgaris 80 H. sp - ex. Martes martes 100 H. americanum - ex. Dusycion thous Rodent and 63 H. canis - ex. Vulpes vulpes 83 H. canis - ex. Dusycion thous carnivore hosts 100 H. felis - ex. Felis catus 165PvMO H. sp - ex. Podarcis vaucheri 168PvMO 317AmMO - H. sp - ex. Atlantolacerta andreanskyi LPA25TtMO - H. sp - ex. Timon tangitanus Pb7413PvMO - H. sp - ex. Podarcis vaucheri DB10519PlCA - H. sp - ex. Podarcis lilfordi 5220PhSP - H. sp - ex. Podarcis hispanica 167PvMO - H. sp - ex. Podarcis vaucheri 127EaMO H. sp - ex. Eumeces algeriensis 83 129EaMO Lizard hosts LPA4TtMO 95 LPA28TtMO H. sp - ex. Timon tangitanus 100 LPA31TtMO E16119CpMO - H. sp - ex. Chalcides polylepis 133SpMO - H. sp - ex. Scelarcis perspicillata 100 164PvMO H. sp - ex. Podarcis vaucheri Pb7421PvMO 5202PhSP H. sp - ex. Podarcis hispanica 82 5221PhSP H. sp - ex. Dromiciops gliroides Marsupial hosts H. catesbianae - ex. Rana catesbeiana Figure2 Hyaloklossia lieberkuehni - AF298623 Click100 here to download Figure: Fig2-Sarcocystis.pdf 99 Besnoitia besnoiti - FJ797432 S. sp - DB10319PlCA 100 98 96 S. gallotiae - AY015112 83 S. lacertae - AY015113 S. cruzi - AF176935 98 S. capracanis - L76472 76 100 72 86 S. gracilis - FJ196261

100 S. tarandi - EF056017 94 98 S. rangiferi - GQ251026 57 S. sinensis - AF176930 100 63 83 S. hominis - AF176945 S. scandinavica - EU282026 S. atheridis - AF120114

S. mucosa - AF109679

S. dispersa - AF120115 S. neurona - U07812

S. rodentifelis - AY015111

S. columbae - GU253883 S. microti - AF009244

S. glareoli - AF009245 0.01 substitutions/site Figure3 E. tropidura - AF324217 Click here to download Figure: Fig3-Eimeria.pdf Choleoeimeria sp. - AY043207

E. sp. - 667PhPO 89 73 100 E. arnyi - AY613853 96 E. ranae - EU717219 Poikilothermic hosts

E. pilarensis - AF324215

60 E. praecox - GQ421692

E. alabamensis - AF291427 65 100 93 colobi - AF111186 Eimeriid coccidia with Stieda bodies E. telekii - AF246717 Endothermic hosts

E. gruis - AB243082 100 99 E. reichenowi - AB243084 0.01 substitutions/site Table 1 Click here to download Table: GE-2908R2 Table 1.doc

TABLE I. Summary of the 44 samples analyzed in this study, including positives for parasites

and their geographical locations. Coordinates are in the WSG84 format. * Two samples were

positive with only the HEP primers and therefore were not included in the analyses.

Host species Podarcis hispanica Podarcis hispanica Podarcis lilfordi Portugal Spain Cabrera (Long 40.83º Lat -5.52º) (Long 38.02º Lat -7.86º) (Long 39.15º Lat 2.93º) Total

Hepatozoon 0 17* (89.5%) 7 (70%) 24

Sarcocystis 0 0 8 (80%) 8

Eimeria 1 (6.7%) 0 0 1

Total 15 19 10