A monogenean parasite reveals the widespread translocation of the African clawed frog in its native range

The Anthropocene is characterised by the unparalleled human-mediated passage of organisms from their natural ranges to ecosystems across world (Capinha et al., 2015; Ricciardi, 2007). If these non-native manage disperse initial introduction point and seed new populations in range, they are regarded as invasive (Blackburn 2011; Richardson 2011). This reshuffling world's biota a decisive agent global change, evidenced fact that species implicated cause third recent animal extinctions 2019). Clearly, development methods trace both origin expansion biological invaders an urgent task. Genomic tools can be informative for reconstruction invasion histories, prediction potential range success control strategies (e.g. de Busschere 2016; Dlugosch & Parker, 2008; Dufresnes 2017; Hiller Lessios, Hudson 2022; Lee Gelembiuk, Prentis 2009; Stouthamer Zahiri Nonetheless, many cases available molecular data or chosen markers insufficient resolution themselves movement Ahyong Criscione 2006). Thus, despite accessibility genomic widespread use phylogeographic analyses science, certainly have limitations (Fitzpatrick 2012; Rius Turon, 2020). A more holistic approach solving shifts involves cointroduced parasites (Gagne 2021). Such integrative has been proposed investigate invasions difficult (Clavero 2016). For example, long-debated cryptogenic history European marine snail Littorina littorea (Linnaeus, 1758) was solved with help associated digenean (Platyhelminthes: Digenea) parasite (Blakeslee 2008). Likewise, intraspecific morphometric variation monogenean flatworm Monogenea) identified introduced life stage clupeid fish Africa (Kmentová 2019), shed light on population odontobutid Europe (Ondračková 2012) provided information route gobiid (Huyse 2015). Moreover, genetic among gill used illuminate cichlid (Geraerts Jorissen 2022). Matters complicated even further when we wish conspecific between different populations. phenomenon, also known cryptic invasion, describes process where one lineage spreads into parts species' native another already present, often human (Morais Reichard, 2018; Saltonstall, 2002). Once again, cotranslocated hold great promise provide lineages hosts. Monogenean particular exhibit sufficient levels structuring assess spatial patterns hosts (Baldwin Hahn Huyse Kmentová 2018). At very least, support evidence revealed host dataset historic records, such well-documented anthropogenic salmonid stocks reflected phylogeography gyrodactylid (Hahn ideal tags genealogy due short generations, direct cycles generally high specificity (Nieberding Olivieri, 2007; Pariselle Yet, comparative analysis never applied within species. African Clawed Frog Xenopus laevis (Daudin, 1802) (Anura: Pipidae) arguably most widely distributed well-studied amphibian (Measey van Sittert Measey, Previous studies morphology genetics X. several differentiated South segregated large mountain climatic regions, notable divergence northern southern (de du Preez Furman Grohovaz 1996; Measey Channing, 2003). In opposition clear stands occurrence translocation region. consequence naturally occurring overland migration, domestic export frog teaching learning adopted practice live bait recreational angling Villiers Davies, Weldon One would expect translocations leave (van over long distances major geographic barriers not confirmed previous We posit bladder polystomatid Protopolystoma xenopodis (Price, 1943) Bychowsky, 1957 (Monogenea: Polystomatidae) tool hitherto unconfirmed contact its Africa. sub-Saharan Africa, P. xenopodis, Protopolystoma, restricted single group, leavis sympatry other (Tinsley Jackson, 1998a, 1998b). Furthermore, morphological exists infecting those northerly pattern echoed smaller regional scale phylogenetic parasitise (Schoeman combination suggests could useful holobiont bio-invasion study, since any inferred mediated host. Therefore, present intimately system proof concept introductions lineages. hypothesise will magnify laevis, which allow us employ this context translocation. postulate reveal distinct events might lacking insights pathways beyond. 46 adult were captured March 2017 April 2019 liver baited funnel traps 30 localities (Figure 1; see Appendix S1 locality details). representative frog, based upon assignment al. (2016) (see S2 classification). 13 collected 12 western France Portugal during summers 2017, 2018 France, frogs members EU LIFE CROAA team part eradication programme amphibians. Frogs obtained locally laboratory stock University Lisbon. All underwent double euthanasia anaesthesia 6% ethyl-3-aminobenzoate methanesulfonate (MS222) (Sigma-Aldrich Co., USA) followed pithing required institutional ethics committee. During subsequent dissection, 59 (one per frog) retrieved bladders analyses. Polystomatids tissue corresponding preserved 70% ethanol. analyses, sequences mitochondrial barcoding genes cytochrome c oxidase subunit I (COX1) small 12S ribosomal RNA (rrnS) polystomatids. DNA extracted PCRBIO Rapid Extract PCR kit (PCR Biosystems Ltd., UK) Quick-DNATM™ Miniprep Plus (Zymo Research, USA). Subsequently, amplification reactions COX1 rrnS polystomatids prepared 2–5 μl DNA, 1.25 forward primer [0.2 μm], reverse 12.5 master mix PCR-grade water final volume 25 μl. 2× HS Taq Mix Red OneTaq® Master Standard Buffer (New England Biolabs Inc., temperature elongation steps supplier instructions (68 72°C). COX1, ‘LCO1490’/‘HCO2198’ (Folmer 1994) ‘VF1’/‘VR1’ (Ivanova 2006; Ward 2005) pairs implemented following thermocycling profile: denaturation at 95°C 2 min, 35 s, annealing s 50°C first pair 54°C second terminated step 7 min. case ‘12SA-F’/‘12SB-R’ (Kocher 1989) obtain rrnS, profile Amaro (2013) followed. Héritier (2015) ‘L-CO1p’/‘H-Cox1R’, ‘L-CO1p’/‘H-Cox1p2’, ‘12SpolF1’/‘12SpolR1’ ‘12SpolF1’/‘12SpolR9’ (Héritier Littlewood 1997). purification sequencing, products sent commercial company (Inqaba Biotec, RSA) ExoSAP protocol Ltd.) BigDye® Terminator version 3.1 Cycle Sequencing, utilising reaction, ABI3500XL analyzer. Sequences assembled manually edited Geneious Prime 2021.0.3 (www.geneious.com). 236 deposited GenBank (accession numbers OP038566–OP38624, OP046050–OP046169). An investigation sequence diversity exploratory relative differentiation clusters. It give indication markers' applicability alignments each four gene sets: polystomatid. Clustal Omega algorithm under default settings (Sievers Higgins, 2017) alignment all datasets. protein-coding gene, translation vertebrate code (table 2) echinoderm 9) Genetic indices calculated specimens combined clusters samples. Number haplotypes polymorphic sites, haplotype diversity, nucleotide mean percentage pairwise differences Arlequin 3.5 (Excoffier Lischer, 2010). Three groups defined collection full details), namely southwest Great Escarpment, geographical barrier dispersal region (SA1, SA2, SA3, SA4 SA7; n = 30), northeast Escarpment (SA5 SA6; 16), (n 12). specimen included separate group because sample size. relationships represented trees derived genes, formed basis analysis. through removal duplicates, addition outgroups rooting, re-alignment concatenation. Duplicate CD-HIT (Fu Li Godzik, 2006) online (Huang Only unique retained analysis, leading combinations (23 10 rrnS) 44 (34 xenopodis. order root trees, congeneric Müller's clawed muelleri (Peters, 1844) (GenBank accession EU599031, AY581657; Evans 2004, 2008) occidentalis Tinsley 1998 KR856179, KR856121; 2015) added outgroups. only infects reportedly reproduces sterile offspring hybridisation occurs sympatric congener (Fischer 2000). selected applicable host–parasite system. resulting datasets, after duplicates outgroups, re-aligned detailed above. two aligned concatenated individual Prime. Ultimately, 31 total 1,153 bp (717 436 rrnS), containing 189 sites gaps 183 variable 88 parsimony informative, dataset. 45 consisting 934 (423 511 799 complete 225 subset 174 infer phylogeny parasite. Prior inference, optimal models evolution partition determined. Initially, comprised partitions each, three codon positions partial gene. ModelFinder selection routine (Kalyaanamoorthy 2017), conjunction FreeRate heterogeneity model (Soubrier 2012), IQ-TREE 2.1.2 (Minh 2020), employed select partition. To partitioning scheme suitable evolutionary alignment, sequentially merged (Chernomor 2016) until fit, evaluated Bayesian criterion (BIC), ceased improve. Finally, phylogenies using maximum likelihood (ML) approach. ML TIM2 (Posada, 2003) edge-unlinked proportions invariant (Gu 1995) discrete gamma (Yang, partitions. Similarly, tree TPM3 (Kimura, 1981) performed assessment branch ultrafast bootstrapping (UFboot2; Hoang 2018) Shimodaira–Hasegawa approximate ratio test (SH-aLRT; Guindon 2010), 10,000 replicates. rooted respect TreeGraph2 2.15.0-877 beta (Stöver Müller, Clades UFboot2 lower than 95% SH-aLRT 80% collapsed polytomies same programme. fit level framework interpret mismatches incongruencies certain links indications distance switching congruence assessed implementation Procrustean Approach Co-phylogeny (PACo) (Balbuena 2013) fourth-corner statistical ParaFit (Legendre Both consider contribution interactions 2013; Legendre 2002; Poisot Stouffer, accommodate fully resolved instances uneven terminal nodes parasites. PACo, dependency asymmetrical superimposition matrices, while tested dependence removed phylogenies. converted matrices patristic package ape (Paradis Schliep, 2019) R 4.0.2 (R Core Team, These served input along association matrix, assigned original individuals, identical individual. principal coordinates PACo r paco (Hutchinson 0.4.2 Procrustes Cailliez correction (Cailliez, 1983) avoid production negative eigenvalues non-Euclidean distances. performed, asymmetric statistic tracking host, permutations, conserved row sums number interactions. dynamics interacting clades explored identifying contribute possible concordance jackknife procedure paco. Those smallest residual interpreted stronger supporters overall congruence, larger likely phylogeny. 2002) carried out permutations 1983). ParaFitLink tests significance 2002), pointed signify incongruencies. Visualisation achieved tanglegram weighted interaction network, residuals yielded procedure. constructed phytools (Revell, function cophylo(), internal rotates optimally match tips. Due duplicate sequences, involved multiple Haplotype genealogies bifurcarting Fitch (1970) alternative way visualising giving reason, visualised inclusion genealogies. Two phylogenies, parasites, no described Fitchi (Matschiner, 2015), produced graphs. As (Weldon 2007) anglers anecdotal fishing practices stakeholders conducting our fieldwork asked about surrounding release activities. Nine subsistence Eastern Cape, Limpopo, Northwest, KwaZulu-Natal Gauteng Provinces communities vicinity some sampling localities. higher orthologous greater samples (Table 1). northeast, marked necessary patterns. Tree showed northeastern SA5 SA6 form well-supported clade, (clade NE Figure 95%/SH-aLRT 95.8%). addition, unresolved sampled southwestern (clades SWcoast 81%/SH-aLRT 86.1% SWinland 98%/SH-aLRT 99.7% 2). On whole, clade originate closer coastline SA1, SA7 localities) localities). Portugal, grouped together phylogeny, thus placing Jonkershoek 1), source majority exports. harboured clustered clade. manner inference single, localities, ne 97%/SH-aLRT 87.6%). Several basal emerged sw1, sw2, sw3 sw4 earliest diverging grouping SA3 SA7, sw1 94%/SH-alrt 88.1%). sw2 53%/SH-alrt 90.7%) SA2 61%/SH-aLRT 86.6%). Another sister ne, contained 100%/SH-aLRT 100%). However, low values tree, especially sw3, lineages, hampered interpretation clustering Consequently, clades. exhibited significant (PACo m xy 0.1034, p 0.0018, 10,000; ParaFitGlobal 0.0202, < 0.0001, 10,000), suggesting tracked laevis. According fall contributed congruence. establishment allowed hone signified past divergent Based visual inspection 2), few explained SW spill-over frog-polystomatid (Dullstroom, Hopetown, Johannesburg, Verkykerskop, Adelaide, Ugie Wakefield), counterparts. frog–polystomatid conveying zone near Sisters (SA4) Wakefield (SA6) demonstrate reported 48 0.05. Out 18 did significantly 11 switches occurred SA4, (Johannesburg, Sisters, Wakefield). 3) unsurprisingly demonstrated clearly together, except (SA5), clades, (SA4), situation somewhat polystomatids, illustrated cluster (ne) nine 16 seven remaining (Adelaide, Dullstroom, Ugie, Verkykerskop Wakefield) spread remainder therefore (sw1, sw3). (sw2) Our informal discussions five provinces, confirming existence areas. usually capture black bass Micropterus Lacepède, 1802 sharp-toothed catfish Clarias gariepinus (Burchell, 1822). Black inhabitants bodies (Hargrove Khosa areas south (Weyl Seven indicated caught locally, specimens. admitted buying breeders pet shops urban (Potchefstroom Johannesburg) transporting them rural bait. fishermen active samp