An integrative approach to assess non‐native iguana presence on Saba and Montserrat: Are we losing all native <i>Iguana</i> populations in the Lesser Antilles?

Invasive alien species are among the main drivers of ongoing sixth mass extinction wave, especially affecting island populations. Although Caribbean is well-known for its high richness and endemism, also reptiles, equally important regional contribution non-native to biodiversity. The Lesser Antilles encompass genetic diversity in Iguana, though most native populations either have gone extinct or declining following competitive hybridization with invasive green iguanas. Here, we assessed presence two poorly-studied melanistic Iguana iguana using available tools explored utilizing size-dependent body measurements discriminate between Genetic samples from Saba Montserrat were genotyped across 17 microsatellite loci STRUCTURE, multivariate analyses indicating only on Saba. This was corroborated by mtDNA nDNA sequences, highlighting a origin Central America ABC islands. We identified preliminary evidence suggestive hybridization. Morphological variation characteristics showed that iguanas significantly larger subtympanic plates than Non-native individuals differed scalation coloration patterns. Overall, our findings demonstrate need continuous monitoring within remaining Antilles, as those not directly threatened restricted 8.7% historic range. data allow identification hybrid presence, this field-to-lab workflow time-consuming. Rapid in-situ crucial conservation management. In addition patterns coloration, highlighted utility variables rapid diagnosis. urge partners build morphometric databases allowing quick detection future incursions implementation effective countermeasures during early phase invasion. Les espèces exotiques envahissantes sont parmi les causes principales de la sixième vague d'extinction massive actuelle, affectant particulièrement insulaires. Bien que Caraïbes soient riches en endémiques, dont régionale des allochtones à biodiversité insulaire est. tout aussi importante. présentent une grande diversité génétique dans Petites bien plupart indigènes aient disparu ou déclinent suite l'hybridation compétitive avec iguanes envahissants. Pour détecter présence d'iguanes deux mélaniques d'I. sous-étudiées, nous avons utilisé outils génétiques disponibles et évalué l'utilité mesures morphométriques. Des échantillons ont été génotypés sur microsatellites STRUCTURE multivariées, indiqué uniquement séquences d'ADNmt d'ADNn corroboré origine Amérique centrale îles ABC. données préliminaires suggèrent l'hybridation. variations morphométriques montré plaques sous-tympaniques significativement plus grandes indigènes. individus différaient également leur motifs colorations d'écailles. Globalement, nos résultats démontrent nécessité d'une surveillance continue au sein restantes celles qui ne pas directement menacées par étant limitées 8,7% l'aire répartition historique. permettent actuellement d'identifier d'hybrides, ce travail laboratoire prend du temps. L'identification rapide cruciale pour gestion conservation, outre d'écailles, démontré l'importance un diagnostic rapide. Nous exhortons partenaires régionaux créer bases morphologiques indigènes, aideront rapidement futures permettront l'implémentation contre-mesures efficaces pendant initiale l'invasion. (IAS) prominent threats global biodiversity (Bax al., 2003; Butchart 2010), contributing what has been named event (McGeoch 2010; Bellard, Cassey, & Blackburn, 2016). natural introductions (e.g., hurricanes) shaped current biogeography (Heinicke, Duellman, Hedges, 2007; Fonte, Mayer, Lötters, 2019; Kennedy 2020), anthropogenically-mediated increase frequency redefining biogeographic (Capinha 2015). IAS can impact through range mechanisms, example, direct predation, competition, replacement, hybridization, their various combinations (Reaser 2007). Isolated insular vulnerable (Tershy 2015), including Greater region (Gleditsch 2022). Within region, human impact, terms inter-island transport poor biosecurity, drives occurrence species, reptiles (Cox 2022; Jesse addition, hurricane events translocate reptile other islands (Censky, Hodge, Dudley, 1998), recovery aid campaigns triggered disasters (van den Burg 2021a). being impacted recently-arrived (Knapp 2021). While Little Cayman intergeneric Cyclura nubila caymanensis alarming (Moss 2017), hotspot (Fig. 1). There, delicatissima iguanas, which considered major factor behind range-wide decline recent assignment Critically Endangered Burg, Breuil, Knapp, 2018a). currently competitively hybridizing outcompeting I. throughout French West Indies (Vuillaume 2015; Angin, Anguilla (Pounder St. Eustatius 2018b), recently Commonwealth Dominica Brisbane, 2020). Besides delicatissima, several complex occur Antillean (Stephen 2013; Taxonomy Working Group 2016, these received less attention, research interest gene pools increased recently. Iguanas Lucia, Vincent, Grenadines described subspecies later insularis, (Breuil 2019, IUCN-SSC Specialist Group's (ITWG) does recognize proposed full status, retaining status insularis i. sanctaluciae originally Breuil al. (2019) until such time at further may be published (Iguana Among southern islands, established hybridized Grenadine 1; Morton, 2008; For Grenada, some suggests but no analyzed so far confirm potential 2019). melanoderma (2020) Montserrat, while ITWG still considers distinct subpopulations iguana, calls revision become acknowledge taxonomic position under discussion now choose follow taxonomy. found based morphological characters limited sequence data. However, more extensive yet collected 2021 suggested few might already since arrived Madden, Debrot, Reports an apparent sudden numbers (pers. comm. Ernestine Corbett; pers. Stephen Mendes) give reason concern about possible spread higher reproductive potential. mostly conjunction Vuillaume van 2018b, 2021b; Pounder notably hatchling juvenile given similarity comparisons scale used (Breuil, 2013), underlying reference dataset cover small part numerous diagnostically informative absence quantitative lack definitions qualitative categories. These useful identify x hybrids 2020; correct characterization guaranteed Hence, difficult, additional should strengthen field identification. aimed assess last home this, implemented representing extended (spanning tropical South America). use assessing size-corrected Fieldwork conducted August–September July–December respectively. captured means lasso pole, hand, cage traps. then photographic images entire well all sides head, sample, blood (ventrally caudal vein) tissue (1 cm clip largest dorsal spine), animals. On both (58) (80), caught 2a,b). Morphology pre-identify characteristics: presence/absence patch eye plate, enlarged nasal scales Saba, sampling done opportunistically population assessment 2022), where measurements: snout-vent length, tail upper frontleg lower hindleg length 4th toe, head width, snout mouth depth, tympanum height, plate mid-body spine (‘horn’) (for methodology see Supplementary material); lateral facial measures taken right side. All measured knowledge SVL dependence allometry complex; topics will addressed separate manuscript prep.). first author. melanism defined one subsequent study very animals completely Therefore, assessment. Consequently, assessment, sufficed recording adult key distinguishing color characteristic (Gerber, 1999; Microsatellite laboratory procedures Labofarm-GenIndexe (France) amplify (see 2021b), identical methodologies presented Valette (2013). Subsequently, PCR product allele scoring performed (2021b) sample IGD277 standardize sizes. A subset five per processed University Amsterdam DNA isolation NADH dehydrogenase subunit 4 (ND4) MutL homolog 3 (MLH3) PCR, (2018b) Malone, Reynoso, Buckley (2017), selected morphology results origin. Successful amplification confirmed gel electrophoresis, whereafter resulting fragments reverse forward-sequenced. Chromatograms assessed, curated, aligned Geneious Prime (2021.1.1). Sequences uploaded GenBank (OQ556117-OQ556120), added IguanaBase database 2021c). <20% missing combinedly 2021c) extent clustering previously geographic First, predict.dapc (DAPC) function adegenet package (Jombart, 2008) R environment (R Core Team, assigns provided dataset, case IguanaBase. multiple PCA axes inclusion order test robustness. Thereafter, analysis (2021c) running (Pritchard, Stephens, Donnelly, 2000) GENODIVE (Meirmans, 2020) settings K (ranging 1 8) prepared visualizations bar plots Structure Harvester (Evanno, Regnaut, Goudet, 2005; Earl von Holdt, 2012) distruct (Rosenberg, 2004). Given large number compared sizes IguanaBase, ran half samples, specifically (partially) assigned DAPC analysis. ranges visually 2021c), regarded fixed populations; L3, L8, L13, L16, L17, L24. Lastly, relative allelic calculated separately GenPopReport (Adamack Gruber, 2014). Basemaps created QGIS 3.8.0 Zanzibar (QGIS.org, 2022) finalized Adobe Illustrator 25.3.1. Mitochondrial nuclear unpublished data). markers, included prior 2010. Pairwise differences sequences highest nucleotide regressing individual against size-dependence, residuals adults (>20 SVL) sexes sex-linked t-tests. Analyses dependence, prep). Variables initially checked normality equality variance. repeated regressions sex status. tested two-way ANOVA Kruskal-Wallis depending homogeneity variance tests. Data handling RStudio Version 1.2.5033 (RStudio Morphometric figshare 2023). Samples 57 76 Montserrat. posterior scores clustered except four morphospace minimally i 3). Specimen SAB58 rhinolopha, three specimens axes. Affinities latter follows: SAB04: aff. Clade I; SAB57: iguana; SAB08: mixed partial assignments I, consistently group Montserrat; Mont33, Mont39, Mont40 iguana. Results = 6 (Figs. 4a,b), clusters, indicated 2021-captured had >0.90 cluster (marked asterisk Fig. 4b). specimen rhinolopha (0.92), origins (SAB08, 0.42; SAB57, 0.29), I (SAB04, 0.13, 0.43). (Mont33, 0.35; 0.22; Mont40, 0.10) before 2021. Novel haplotypes 15 S1). Compared 23 new overlapped. 13 fell outside known size mean 2.57 1.67 when excluding 1.82 combining 53 six 2021-sampled 2.33, 2.50 14 samples. Generated ND4 (789 bp) MLH3 (778 matched 100% ND4, SAB02 CAR2 haplotype (GenBank accession HM352505, 2013). From SAB08, Central-American CA4 HM352508, SAB04 (SA16; MLH3, unreadable chromatogram specimens, SAB02, homozygous pre-2021 (H18; SAB08 SAB57 heterozygous H18 and, respectively, El Salvador (H16; data) Honduras (H15; Finally, H15 H16. Considering life stages each variable size-dependent, Table 1. female male significant 11 16 (Table Tail could percentage broken tail. native/non-native ten height 1, Figs 5 6). Nasal assessments SAB58) 6b,c), absent (SAB57 + 58) faintly present (SAB04 08) 6c). patch; however, Mont36 Mont42, feature limited. plagued continuing threat Here report believed Our integrated flags incursion patterns, highlight how morphometrics populations, analytic techniques. Passenger cargo mainly channeled Maarten, although private vessels often origins. reinstalled ferry service passes Eustatius, population. Additionally, biweekly delivers Martin County (Florida, USA), (Meshaka, Butterfield, Hauge, 2004; iNaturalist, extirpated, currently, present. Based microsatellite, data, ultimate America, connectivity Maarten same backgrounds there likely immediate Maarten. made even considering originating documented Hylkema, 2021d). traced back origins, mainland (Brazil), Salvador), Lucia 2018b; work others show must dispersed (neighboring) 2018b) past, it human-mediated means. demonstrated Saba/Montserrat particularly shows utilize markers trying cannot excluded introgressed Saban re-invade original (De Jesús Villanueva 2021) (the references above), stand out clades represented. 26 novel alleles represent undiscovered pool. As relatively surprising alleles. examined, L20 stands locus believe significance strongly differs. Namely, 182 coincides closely IguanaBase; constitutes rare haplotype. By contrast, suggest gap (but Estoup, Jarne, Cornuet, 2002). pre- post-2021 combined (n 63) 90), total polymorphic (L3, L17) exception aberrant S1) provides Eventhough complete overview lacking, highly complex. population, previous (horn) additionally fourth, genetically non-native, lacked clear black (though faint partially scales). roughly 50% scales, 10–20% scales. words, pronounced less-pronounced (2020), who melanistic. help distinguish invaded composite phenotypic pattern 2013) molecular Miller Mitchell, Welch, Size-dependent genetically-assigned (Figs power usage depend admixture individuals. short, SVL-dependent variable(s) different admixtu