Metabarcoding assessment of the diet of an introduced continental lizard to an oceanic island reveals dietary niche conservatism

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James Harris, Markus A. Roesch, Diana S. Vasconcelos, Chloé Bernet, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6830034/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Invasive species can have devastating effects when introduced into remote island ecosystems, and a fundamental aspect of this concerns the diet of these exotic taxa. Here we employed a DNA metabarcoding approach to determine the diet of the lizard Agama picticauda on Reunion Island, where it was introduced in 1995. Two separate markers were used to identify animal and plant components. The arthropod aspect was notably conservative, with the agama continuing to predominantly consume ants, as they do in their native range. A variety of other invertebrates were also preyed upon, the vast majority being introduced species. For plants again a wide variety were detected, and while most could not be identified fully, it seems that agamas are deliberately consuming many species, rather than accidentally intaking them along with targeted invertebrates. Agamas may play a role in seed dispersal of invasive plant species. We also detected some nematode groups, although with limited comparative sequences these could not be identified to the species level. Several records of invertebrates appear to be new records for Reunion Island, highlighting how reptiles can be considered as excellent biodiversity samplers, with barcoding diet studies providing novel data for poorly known invertebrate groups. The minimal identifications of endemic prey items may reflect that the agamas are still predominantly occupying anthropogenically disturbed parts of the island. Our study therefore provides baseline data that can be used to determine the impact of this introduced lizard as it spreads through the ecosystem. Biological sciences/Ecology/Evolutionary ecology Biological sciences/Zoology/Herpetology Biological sciences/Ecology/Invasive species Reunion Island trophic niche conservatism Agama picticauda invasive species Figures Figure 1 Figure 2 Figure 3 Introduction The main drivers of biodiversity loss include habitat fragmentation, climate change and invasive species. Furthermore, it has been well-documented that the majority of vertebrate extinctions in recent times have been on islands, and that the bulk of these were associated with the establishment of exotic species. Island reptiles are particularly threatened by introduced competitors, especially on islands that were previously predator-free (Case and Bolger 1991 ). Because of this, there have been extensive studies on the impact of invasive species such as rodents or cats on island endemic reptiles (e.g. Thibault et al. 2017 , Galão et al. 2025 ). Invasive reptiles have also been associated with extinctions, for example the introduced gecko Hemidactylus frenatus was linked to the decline and extinction of endemic night gecko Nactus populations on the Mascarene islands (Cole et al. 2005 ). However, there have been fewer studies on the impact of invasive introduced reptiles on consumed taxa, because this trophic interaction involving many different potential prey items is harder to determine, and disentangling this mechanism through which introduced species impact the ecosystem remains a key challenge in ecological research (Feit et al. 2020 ). The development of metabarcoding approaches, in which dietary items are identified using Next-generation genetic sequencing tools, offers a useful new technique to address this issue, particularly since prey items can be identified to much higher taxonomic levels than typically determined using microscopy assessments of stomach contents or pellets (e.g. Galão et al. 2025 , Pinho et al. 2023 ). The West African rainbow lizard Agama picticauda is a common and widespread lizard in sub-Saharan West Africa (Fig. 1 ), where it is often found in urban environments (Luiselli et al. 2011 ; Ofori et al. 2018 ). It is a highly-successful invasive species; for example, in Florida, USA, it is one of the most rapidly spreading non-native herpetofauna (Clements et al. 2025 ). In the Caribbean Lesser Antilles, A. picticauda is considered to have a major impact on numerous native species, and is spreading to new islands at an alarming rate (van den Burg et al. 2024 ). Recent molecular analyses have confirmed that the populations on Grande Comore and Reunion Island were independently introduced to these islands (Roesch et al. 2025 ). On Reunion Island, the first individuals were reported near the main maritime port, approximately 30 years ago (Guillermet et al. 1998 ), and are now spread across the coastal areas and recently into the higher elevation central region (Roesch et al. 2025 ). Agama picticauda in their native range prey predominantly on insects, with Hymenoptera (Formicidae) and Coleoptera being the most frequently consumed prey orders (Ofori et al. 2023a ). Morphological identification of stomach contents identified prey items from 14 orders, indicating a relatively wide prey spectrum (Ofori et al. 2023a ). Agama picticauda may also consume plant matter, small vertebrates and even scavenge opportunistically on anthropogenic processed foods such as bread (Ofori et al. 2018 ). The Mascarenes, consisting of the volcanic islands of Reunion, Mauritius, Rodrigues as well as various small coralline islands, form part of the world’s top biodiversity hotspots (Thebaud et al. 2009). Reunion Island is the largest island (2512 km 2 ), with dated lavas of around 2.1 million years. Although severely fragmented, about 25% of the estimated original extent of habitats on Reunion Island remain in a relatively good state. Like other similar island groups such as the Hawaiian Islands, the Mascarene biota includes notable levels of endemisms, including around 75% of native flowering plants and 90% of nonmarine molluscs (Thebaud et al. 2009). Of the terrestrial arthropods on Reunion Island, 31% are endemic to the island, and 40% endemic to the Mascarenes, although these are rough estimations given that around 60% of the arthropod fauna is predicted to remain unnamed (Legros et al. 2020 ). Given the rapid spread of A. picticauda across Reunion Island, and the high level of endemic and endangered potential prey items, an assessment of the trophic niche of this introduced agamid is clearly needed as part of the recently established “invasive species strategy and action plan”, which aims to mitigate its spread and associated risks (Nature Océan Indien, 2023). Here we used a metabarcoding approach to assess the diet from 66 adult individuals, using stomach contents collected from populations around the island, including the higher elevation central region (Fig. 1 ). We aimed to collect baseline data regarding prey items, and to determine if known endemic or endangered species were being preyed upon. The data can also be compared to the known diet from its native range, to assess levels of tropic niche conservatism. At the same time, dietary DNA or dDNA ( sensu Sousa et al. 2019 ) can often give notable new information about prey items themselves, helping to establish reliable barcoding databases of species presences. Furthermore, sequences of endoparasites such as nematodes can sometimes be identified in stomachs using this genetic approach, furnishing additional information for these poorly known species, potentially introduced along with their lizard host. Results The COI and trnL libraries generated ca. 6.4 million raw sequence reads, which were reduced to 171,419 reads during the bioinformatic processing and to 558 total OTUs. Non-target COI amplification from different sources was observed both in samples, extractions, and PCR negative controls representing approximately 50% of the total reads. Almost all of these corresponded to the host, Agama (48.19% of the total reads), while Nematoda represented around 1.25% of the total reads, and Fungi 0.04%. After negative controls, singletons, replicates, and taxa filtering the lizards’ final diet using the COI primers consisted in 20,655 reads and 68 animal OTUs from 3 phyla, Annelida, Chordata and Arthropoda. Prey items were considered to belong to 38 families of arthopods, 2 families of annelids, and 1 family of chordates (geckos) (Supp. Material Table S2 ). The most frequent OTUs were generally ants (Formicidae), and in particular Paratrechina longicornis (49.15% FO), Brachymyrmex cordemoyi (11.86%), Solenopsis geminate (8.47%) and Pheidole megacephala (6.78%). The most frequent prey OTUs after ants were the honey bee (Apidae) Apis mellifera (18.64%) and the beetle (Coccinellidae) Exochomus laeviusculus (15.25%). No other prey items had frequencies over 6%. As well as the prey items, 4 OTUs corresponded to nematodes from the orders Rhabditida and Spirurida. Regarding the chloroplast primers a total of 52,149 reads were obtained and 97 OTUs were detected (7 up to the species level), corresponding to 44 families (Supp. Material Table S3). Growth trajectories were similar for males and females, but males grew both bigger and heavier (Suppl. Material Figure S1 ). There were significant differences in SVL between districts (F = 2.35, p = 0.01) and sexes (F = 40.21, p = 0.00), but weight differences were only significant between the sexes (F = 32.80, p = 0.00) and not districts (F = 1.72, p = 0.08). Female agamas had higher dietary richness than males at both the prey OTU and family level, but the differences were not significant. Analysis of rarefaction and extrapolation curves (Fig. 2 ) indicate no significant differences between males and females, with overlapping dietary niches. According to the GLM analysis, there was significant differences in OTU richness between districts(District: df = 14, Deviance = 16.42, Residual Deviance = 36.66, p-value = 0.29; Sex: df = 1, Deviance = 0.88, Residual Deviance = 35.78, p-value = 0.35; logSVL: df = 1, Deviance = 0.01, Residual Deviance = 35.78, p-value = 0.94), but none of the pairwise comparisons were significant (Fig. 3 ). At the family level there were no significant results in richness ((District: df = 14, Deviance = 29.13, Residual Deviance = 54.82, p-value = 0.01; Sex: df = 1, Deviance = 1.88, Residual Deviance = 52.95, p-value = 0.17; logSVL: df = 1, Deviance = 0.21, Residual Deviance = 52.73, p-value = 0.64). Regarding the analysis on diet composition, there are significant differences only between districts at the OTU level, but not at a higher taxonomic level (Table 1 ). These significant results are not related to the dispersal of the data (F = 1.54, p = 0.15). Table 1 Results from PerMANOVA on the effect of District, Sex and logSVL on OTU and family diet composition. Df stands for degrees of freedom. Significant p -values are highlighted in bold. df Sum of squares R squared F model p -value OTU District 14 7.32 0.24 1.18 0.00 Sex 1 0.50 0.02 1.14 0.17 logSVL 1 0.42 0.01 0.95 0.57 Residual 51 22.51 0.73 NA NA Family District 14 5.93 0.22 1.07 0.19 Sex 1 0.36 0.01 0.91 0.57 logSVL 1 0.28 0.01 0.72 0.86 Residual 51 20.27 0.75 NA NA Discussion An assessment of the diet of A. picticauda along a transect of over 800 km within its native range along the west African coast showed that all populations were mainly insectivorous, and that food niche overlap was high between all populations (Akani et al. 2013 ). The most frequently identified prey item was ants (Formicoidea), with frequencies ranging from 20–53%. Coleoptera, Lepidoptera, Araneidae and Vespoidea were also found in all populations sampled. Dietary niche overlap decreased with increases in the difference of mean annual rainfall between sites, but there was no effect of geographic distance. Similarly, in a population from Nigeria, the most frequent prey items identified were black ants, Blattodea (termites), yellow ants, bees and wasps, with melon seeds also more frequent than some arthropod orders (Rabiu 2019 ). In this study, the diet of the introduced A. picticauda on Reunion Island show notable similarities with the populations from the native range, indicating considerable trophic niche conservatism. Again, the most frequently identified prey items were ants. Eleven OTUs corresponding to 8 genera within Formicidae were recorded (Suppl. Material Table S1 ), of which Paratrechina longicornis (49.15%), Brachymyrmex cordemoyi (11.86%), Solenopsis geminate (8.47%) and Pheidole megacephala (6.78%). were the most frequently recovered, representing 76.26% of all prey OTUs identified with these primers. Coleoptera, Lepidoptera, Araneidae and Vespoidea were also all found at high frequency. Other less widely identified prey items from the native range, such as Isopoda, Diptera, and Blattodea were also identified as prey items in Reunion Island. Differences included, for example, that in west Africa, scorpions were occasionally consumed (up to 1.8% frequency in 3 of 8 sampled populations), while these were not detected in Reunion Island, although this is unsurprising since only two introduced species are known from this island (Legros et al. 2020 ). An acknowledged advantage of the use of metabarcoding in lizard diet studies is the ability to determine prey items to a much higher level than typically obtained using microscopy, and this precision can sometimes uncover previously unknown aspects of the trophic niche (e.g. S’Khifa et al. 2023 ). Previous assessments of the invertebrate prey items of A. picticauda determined using microscopy had identified prey predominantly only to the order or family level (Akani et al. 2013 ; Rabiu 2019 ). A more recent extensive assessment of a population of A. picticauda from urban and rural populations in Ghana identified 14 Orders and 47 families (Ofori et al. 2023a ). In this study, 12 arthropod Orders and 41 families were identified, again demonstrating the wide breadth of the dietary niche of A. picticauda – while ants and a few other groups make up the bulk of the diet, an extremely wide diversity of arthropods and other small invertebrates are consumed. The precision of prey identification means that metabarcoding studies of lizard pellets can also provide new data regarding the prey items themselves, especially since distribution data for invertebrates is often imprecise. For example, in an assessment of diet of the wall lizard Podarcis lusitanicus in Northern Portugal, Simões et al. ( 2025 ) reported several invertebrate prey species that were apparently new records for this country. Fortunately, the terrestrial arthropod fauna of Reunion Island has been relatively well studied. A recent review identified 3,369 species, of which 31% were endemic to Reunion Island (Legros et al. 2020 ). Endemism rates, however, vary widely between groups, with for example 21% of spiders (Araneae) endemic, but only one ant (Formicidae) species out of 48 being endemic. Furthermore, it has been estimated that 62% of all the terrestrial arthropod fauna on Reunion Island remains to be named (Legros et al. 2020 ). Identified prey items may therefore be useful in highlighting undescribed diversity, as well as allowing identification of endangered or endemic species within the trophic niche, as opposed to introduced and widespread species. Identified prey items may also represent first reports of introduced species that can then be added to update the faunistic catalogues of the island. For example, the spider Oecobius putus is not in the most recent, exhaustive checklist for the island (Cazanove, 2022 ), or global databases such as GBIF ( http://www.gbif.org/ ) and may represent a first record for Reunion Island. Likewise, the earthworms Travoscolides chengannures and Pontoscolex corethrurus are neither in the GBIF database nor dedicated earthworm databases ( http://taxo.drilobase.org/ ) for Reunion Island. The same occurs with the milliped Leptogoniulus sorornus and the beetle Myrmechixenus vaporariorum . These potentially new records for Reunion Island are species that are poorly studied and likely introduced – regarding Oecobius on Reunion Island, Cazanove ( 2022 ) states that “some species may have gone unnoticed due to a lack of attention and specific sampling”. Similarly, P. corethrurus is widely introduced, and already reported as such to Mauritius, the Comoros and the Seychelles (Taheri et al. 2018 ). The characterization of ants in the diet of A. picticauda gives an example of the advantages of the barcoding technique over classic microscopy studies. All previous assessments had noted the high proportion of prey items of the family Formicidae. However, our study distinguished 11 OTUs, or presumed species, 10 of which were identified to the species level, within 8 genera. This means that of the 48 ant species known from Reunion Island, 22% were apparently part of the diet of A. picticauda . All of the identified ants were widespread, introduced species – the single known endemic ant species was not found. This is relatively good news regarding the impact of A. picticauda on native arthropods, as it appears to maintain a preference for ants, the vast majority of which are introduced on Reunion Island. Previous studies have suggested that A. picticauda may consume a high frequency of ants as they are among the most frequently encountered ground-dwelling insects (Ofori et al. 2023). However, our finding of such a wide diversity of ant species seems to rather indicate that ants are preferentially preyed upon, which may also help explain the conservatism of the trophic niche in this introduced population. Given that around one third of the terrestrial arthropods on Reunion Island are endemic, it might have been expected to find a similar proportion within the identified prey items. Instead, only one endemic species, the Hemiptera Deraeocoris howanus , was recognized, out of 20 identified to the species level. Although A. picticauda predominantly consumes introduced species, this is not to say that the lizard does not have a considerable impact on native terrestrial arthropods. Introduction of an exotic lizard has been shown to significantly alter ant community structure, reducing the abundance of some species and therefore having indirect effects on other species (Huang et al. 2008 ). Furthermore, the broad dietary niche of A. picticauda means that many species are consumed, and the unidentified prey items may correspond to additional endemic species. A more complete database of sequences from island endemics would be needed to further assess this. Another aspect of the threat caused by introduced species is that they can bring parasites with them, which may have severe impacts if they are transmitted to local hosts. An interesting bycatch of the COI primers employed in this study and widely used for assessing the diet of reptiles is that they also can amplify nematodes. In this study of A. picticauda introduced to Reunion Island, four distinct OTUs corresponded to nematodes. A previous morphological assessment of gastrointestinal helminths from A. picticauda within its native range in Ghana identified four helminth species, Ascaris spp., Enterobius spp., Pharyngodon spp. and Oxyurid spp. (Ofori et al. 2023b ). Unfortunately, none of the nematode sequences from this study had close matches on GenBank (83–93% matches), so we cannot ascertain with any degree of certainty which species they may correspond to. At the same time, morphological assessments of nematodes in other introduced populations of lizards have identified species typical of the local herpetofauna, indicating that the exotic species had acquired their helminth assemblage from the local helminth pool, rather than possessing species from the parasite fauna of the original population (Anjos et al. 2005 ; Criscione and Font, 2001 ). Although we cannot therefore currently identify these nematodes, as more sequence data becomes available for helminths from lizards, in the future these sequences may be useful for identifying these species. An additional potential threat from the introduced A. picticauda would be direct predation on endemic lizards, since agamas are known to occasionally prey on small vertebrates, and there are also records of cannibalism (Vasconcelos et al. 2014 ; Rabiu 2019 ). Cannibalism cannot be assessed with the metabarcoding approach used, as sequences corresponding to A. picticauda were automatically discarded as presumed belonging to the host individual. No sequences corresponding to Phelusma day geckos were identified, and although sampling locations do not overlap with the distribution of native Phelsuma spp. (Dubos et al. 2022a , b ), they do overlap heavily with introduced Phelsuma spp. (Dubos et al. 2023 ). The introduced gecko Hemidactylus frenatus was identified at low frequency (1.47%). Agama picticauda populations typically exhibit sexual size dimorphism (van den Burg et al. 2024 , Roesch et al. 2025 ), with both sexes showing a similar SVL/weight trajectory, but with males being both larger and heavier (Suppl. Mat. Fig. S 1). Given this, it might be presumed that the dietary niche of males would be larger than females, as their larger size would enable them to consume some prey items that would be too big for females to consume. Interestingly however, the females had higher diet richness when assessed both at the OTU and family level, although the difference was not significant. In other lizards with sexual dimorphism, males have been shown to select larger prey items, corresponding to their larger head size and bite force (Kaliontzopoulou et al. 2012 ). On the other hand, in dDNA assessments, the size of prey consumed remains unknown – females and males could be preying on the same species, but selecting specimens of different sizes for example. Rarefaction and extrapolation curves (Fig. 2 ) indicate that males and females have considerably overlapping dietary niches, with no significant differences observed. Previous studies of diet variation along transects of A. picticauda populations have indicated that, while the preference for certain prey items such as ants and beetles remains constant, variation between populations does occur (Akani et al. 2013 ). This is expected, as the terrestrial arthropod community will vary with geological factors such as rainfall, and also anthropogenic influences such as levels of urban development (Akani et al. 2013 , Ofori et al. 2023a ). When assessing differences in dietary consumption between populations, significant differences were found in OTU richness between districts, but in pairwise comparisons none are significant probably because of the low sample numbers in some regions (Fig. 3 ). Further, no significant differences were found between populations when assessing family-level richness. While A. picticauda is spreading around the island, they are still heavily associated with anthropogenically disturbed areas (Nature Océan Indien, 2023). This may also explain why so much of the diet consists of introduced species, similarly associated with urbanised areas. The Honey bee Apis mellifera (18.64%) is the second most frequent arthropod OTU in the diet of A. picticauda and, while introduced, represents a species of significant economic importance. Long-term monitoring will be needed to see if this dietary aspect changes with continuing spread of agamas into more natural areas. Our assessment of plant material in the stomach contents identified 97 OTUs from 44 families, with seven identified to the species level. Although the most common OTUs could not be fully identified (Unidentified Solanaceae 25%, Unidentified Fabaceae 22%), the third most frequent OTU was Desmondium scorpiurus (13%), a perennial tropical legume widely used to feed livestock. Rabiu ( 2019 ) identified seeds in 37% of pellets within the native range of A. picticauda , but the most common were melon seeds, available through anthropogenic discards. In this study various potential fruits were also identified including Ficus sp. (figs), Rubus sp. (blackberries) and Musa sp. (banana). Combined with the high diversity of plant species detected, and the high nutritional value of many of them, it seems likely that A. picticauda are specifically targeting plants for consumption, rather than accidentally ingesting them while aiming at invertebrates. None of the plant species identified were endemic to Reunion Island. However, some may be seed dispersed by this lizard. For example, one of the species identified was the highly invasive Brazilian pepper tree, Schinus terebinthifolius. It is known that recruitment of this species is dependent upon frugivores –in Australia seed germination is minimal without pulp removal, a task that is accomplished by a native bird species (Panetta and McKee 1997 ). Previous studies of other introduced reptiles, including the Green Iguana in Puerto Rico (Govender et al. 2012 ) and the panther chameleon in Reunion Island (Sanchez et al. 2021 ) have found they also consume seeds of S. terebinthifolius . Agama may therefore play a role in the maintenance and spread of this invasive tree species, highlighting the complex interplay between introduced species in these small island communities. Despite the clear advantages that metabarcoding approaches for dietary assessments have over microscopy appraisals, there are also some limitations that need to be considered. As well as the inability to identify the size of prey specimens, life cycle stage cannot be determined, so it remains unclear for example if agamas are consuming predominantly the flying adult stages of Lepidoptera, or the crawling larval stages. Furthermore, this study is an estimate of the diet from a single time point, which may overlook important temporal effects. For example, Rabiu ( 2019 ) suggested that A. picticauda ate more plant material in the rainy season in its native range. Such variation could only be assessed by repeating the sampling during a different time of the year. Another potential issue with dDNA studies is the risk of “secondary predation” (da Silva et al. 2019 ), where the DNA from prey items of prey items are detected. For instance, the DNA from the thrips Frankliniella schultzei was detected, but these can also be consumed by beetles, and therefore the DNA may arrive only indirectly in the pellets of the agama. Plant DNA is also widely detected in insectivorous species (e.g. da Silva et al. 2025 ), highlighting the need for caution in interpreting the presence of an OTU with deliberate consumption of that taxa. While this could cause some inflation in the dietary spectrum identified, the considerable number of orders and families detected demonstrates that the dietary niche of these agamas is very wide, even if they do preferentially consume ants. Finally, the potential new records of invertebrates for the island, for example for the spider O. putus , still need to be confirmed – these assessments are based only on a single marker, mitochondrial DNA, and the similarity with sequences from GenBank. Errors in the published database, or evolutionary aspects such as mtDNA introgression, while unusual, mean that these results need morphological corroboration. Conclusions To conclude, this study shows that the dietary niche of A. picticauda is extremely wide, with a high number of orders and families detected. On the other hand, the diet remains remarkably conservative, with a strong preference for ants continuing in this introduced population. While the ants consumed are generally introduced, at least one endemic insect was also consumed, and this raises conservation concerns. The high taxonomic levels of prey identification, often to the species level, give additional insights into the terrestrial arthropod fauna found on Reunion Island, with some potential examples of new records for the island. Plants formed a notable part of the diet, and were more frequent than previously considered. Gastrointestinal helminths were detected, but could not be adequately identified due to limitations in the comparative genetic databases currently available. Still, the high diversity of genetic sequences reported in this study will provide a strong baseline for future assessments both of this introduced lizard, and also for other barcoding studies of the invertebrate fauna of Reunion Island. Materials and Methods Sampling took place during June to August 2022, across 16 districts within Reunion Island (Fig. 1 and Suppl. Material Table S1 ). As part of an invasive species control, agamas were captured with artisanal glue traps, where the animals have been lured onto the trap with live bait ( Pycnoscelus surinamensis ). The live bait was securely contained within a transparent box, ensuring that it remained inaccessible to the lizards and was not consumed. Snout-vent length (to 1mm) was measured with a ruler and animals were weighed (to 0.5g) with a Pesola scale. Since the agamas are a non-native invasive species, all captured individuals were chemically euthanised using a Lidocaine/Prilocaine cream (EMLA) and the stomach contents were immediately collected and stored in 90% ethanol and at -20°C prior to DNA extraction. Sex was determined by examining the presence of femoral pores and hemipenial bulges and confirmed by dissection. DNA from approximately 200 mg of each collected 66 stomach samples was extracted using an E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, U.S.A.), following the manufacturer’s instructions with a minor modification in the digestion step, using 800 µL of Gordon buffer instead of 200 µL of TL buffer to improve DNA extraction of both hard and soft tissues, following Simões et al. ( 2025 ). Samples were vortexed and digested during 3 hours. Extracted DNA was stored at -20ºC. Extraction blank samples were included to control for contaminants present in extraction kits and/or in the laboratory environment. A short fragment (~ 205 bp) of the mitochondrial Cytochrome C Oxidase subunit I (COI) was amplified using Polymerase Chain Reaction (PCR) with the Fwh2 primers from Vamos et al. ( 2017 ), which are among the most effective primers for terrestrial arthropods (Elbrecht et al. 2019 ), and have worked well in other insectivorous lizards (e.g. Simões et al. 2025 ). To assess plant consumption, the g/h primers (Taberlet et al. 2007 ) were used targeting the short P6-loop of chloroplast trnL (UAA) intron (up to 143 bp). The primers were modified to include Illumina adaptors and a 0–5 bp addition of N bases between the adaptor and the primer to increase sequencing diversity and quality. The different primer variations were then combined before PCR reactions, resulting in mixed forward and reverse primer single solutions. The PCR reactions for both plant and animal amplifications consisted of 5 µL of QUIAGEN Multiplex PCR Master Mix (Quiagen, Crawley, UK), 0.3 µL combination of six Forward primers, 0.3 µL combination of Reverse Primers, 2.4 µL of ultra-pure water, and 2 µL of DNA extract. Cycling conditions used consisted of an initial denaturation at 95°C for 15 min, followed by 45 cycles of 95°C for 30s, annealing at 52°C for 45s, extension at 72°C for 20s, and a final extension at 60°C for 5min. Negative controls were included to detect possible contaminations. Initial PCR clean-up to remove unused primers and primer dimers was performed employing the Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), with a proportion of 0.85 µL of magnetic beads to 1 µL of PCR product. To attach unique barcodes and Illumina sequencing indexes to each sample, a second PCR was performed using a distinct combination of index sequences per sample. This PCR was performed using 2.8 µL of ultra-pure water, 7µL of 2x Kapa HiFi, 1.4 µL of each index, and 2.8 µL of cleaned DNA. Indexing the PCR required an initial denaturation of 95°C for 3min, followed by 10 cycles of 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, and a final extension of 72°C for 5min. PCR products were examined in a 2% agarose gel knowing that amplicons should be ~ 100 bp longer than in the previous PCR. A second PCR clean-up was then performed, under the same conditions as before, except that the beads’ ratio was 0.8. After the second PCR clean-up, all indexed PCRs were quantified using Epoch™ Microplate Spectrophotometer (BioTek Instruments, Inc.; Winooski, VT, USA), followed by normalisation to obtain the same concentration in all the samples, after which the samples were pooled. This pool was quality tested in a TapeStation 4200 High Sensitivity D1000 Assay (Agilent, Santa Clara, CA), and a cleaning was performed using a 0.78 ratio of beads. A second quality control test in the TapeStation confirmed the success of the cleaning. The final pool was sent to GENEWIZ Next Generation Sequencing laboratory with a final concentration of 27nM to be sequenced in an Illumina MiSeq sequencer with 2x250bp paired-end (PE) configuration and PhiX (≤ 30%) was used to increase sequencing diversity. Data analysis Bioinformatic processing of sequencing reads was done separately for each molecular marker. The software PEAR (Zhang et al. 2014 ) was used to merge the forward and reverse reads into single sequences, discarding single and unassembled reads, as well as cases with less than 26bp of overlap. Primers were removed, using the command ngsfilter from OBITools (Boyer et al. 2016 ), reads were counted to check for successful rates of the previous step with the grep command, and dereplicated into unique sequences using the obiuniq command. Sequence cleaning was performed to remove sequences with less than 10 reads and chimeras, using the obiclean command, and those with a length between 202 to 208bp (COI) and 30 to 120bp (trnL) were kept and clustered at a 99% similarity threshold to form Operational Taxonomic Units (OTUs) using VSearch (Rognes et al. 2016 ). OTUs that were represented by a read count < 1% of the total number of reads obtained for each sample were removed, as were all reads identified in the extraction and PCR controls of the corresponding sample batch. The obtained OTU table and sequences were further cleaned to remove redundant and unreliable OTUs using the R package LULU (Frøslev et al. 2017 ), and the taxonomic assignment of OTUs was performed using BOLDigger (Buchner and Leese 2020 ), followed by manual inspection and curation. We used the BLAST function from NCBI implemented in Geneious Prime 2021.1.1 (Drummond et al. 2010), when species-level assignments were not possible with BOLDigger. If similarity was > 95% assignment was made to the species level, or genus level in cases where more than one species from the same genus had a > 95% match. Assignment was made to the family level if between 90–95%, and to the class level if below 90% similarity. Prey OTUs that were represented by a read count < 1% of the total number of diet reads were removed. Additionally, all reads identified in the extraction and PCR controls were subtracted from the corresponding sample batch. For the statistical analysis, R 4.1.2 (R Core Team 2022) was used to assess differences in dietary descriptors (i.e., diet richness and composition) between localities and sexes. The log transformed body size (SVL) and mass (weight) variation between sexes across localities, was summarized using boxplots. Body condition was assessed by investigating the relationship between body mass and body length in both males and females, through a linear regression. Dietary analysis was based on two different taxonomic levels: OTU (all taxonomic units identified to the highest resolved taxonomic level) and Family. For the OTUs not identified to the species level, we built a neighbor-joining tree in Geneious Prime v. 2024.0.2 (Biomatters), visually inspected the corresponding alignment, and checked for patterns of genetic similarity (~ 98%) in order to cluster them into distinct taxa (e.g., Coleoptera 1, Coleoptera 2, and so on). The frequency of occurrence (FO) of each food item was calculated as the number of occurrences of the diet item (maximum 1 occurrence per sample) divided by the total number of sequenced reptile stomach samples. To assess the effects of locality, sex and size on the average number of prey taxa detected per sample (i.e., richness), a General Linear Model (GLM) was implemented. For this, the function glm was used and its significance tested with the anova function, both from the car package (Fox et al. 2012 ), fitting a Poisson error distribution. Graphical representation of the relationships depicted from the GLM results was performed using the effect function from the effects R package (Fox 2003 ), where richness means and standard errors were derived from the model’s parameters estimates. Pairwise comparisons were performed with the function emmeans (with a Tukey adjust) from the package emmeans (Lenth et al. 2018 ). To calculate the dietary niche-width between the sexes and among the different populations, prey rarefaction and extrapolation curves were built using the R package iNEXT v. 2.0.20 (Hsieh et al. 2016 ). Analyses were conducted with incidence frequencies for prey taxa. We compared the estimated richness considering completeness (i.e., sample coverage) instead of sample size (i.e., number of samples), to avoid biases of communities with different levels of richness requiring different sampling efforts in order to be sufficiently characterized (Chao and Jost 2012 ). Considering that the 95% confidence interval is a very conservative approach, we considered that differences were significant if the 84% confidence interval (a proxy for α = 0.05) of both estimates did not overlap (MacGregor-Fors and Payton 2013 ). Permutational multivariate analysis of variance (PerMANOVA) was used to compare the OTU and family diet composition between the sexes and the different localities with the vegan R package (function adonis2 ; Oksanen et al. 2013 ). Presence or absence of each prey item in each sample was used to build a Jaccard dissimilarity matrix using the vegdist function from the R package vegan. A homogeneity of dispersion test (function betadisper ) was also carried out to assess if the observed differences in PerMANOVA could be due to unequally dispersed values across the different groups (Anderson 2006 ). Declarations Acknowledgements We thank all team members of the Association Nature Océan Indien for administrative and operational support. Funding This project received funding from the Office Français de la Biodiversité (reference N°OFB-21-1920), Département de La Réunion (reference n° CP-2021-DEC-172-2, XXX), Direction de l’Environnement de l’Aménagement et du Logement de La Réunion (reference n°2023-29) and Fonds Européen de Développement Régional (reference n°20221409-0034656) awarded to the Association Nature Océan Indien. Author contributions D. James Harris: Writing – original draft, Writing – review & editing, Resources, Funding acquisition, Investigation, Conceptualization. Markus A. Roesch: Writing – review & editing, Project administration, Methodology, Conceptualization, Investigation. Diana S. Vasconcelos: Writing – review & editing, Data curation. Chloé Bernet: Writing – review & editing, Project administration, Methodology. Catarina Rato: Writing – review & editing, Validation, Investigation, Formal analysis, Data curation, Conceptualization. Ethics declaration The invasive species control programme follows order no. 2023-605 SG/SCOPP/BCPE in accordance with article L411.8 of the Environment Code on the territory of Reunion Island. Access and benefit-sharing was approved in accordance with Article17, paragraph 2, of the Nagoya Protocol and accessible under the reference number TREL2206915S/624. All methods were carried out in accordance with relevant guidelines and regulations. The entire experimental protocol was approved by the Ethics Committee of the University of Porto (https://www.up.pt/portal/pt/conhecer/organizacao/comissao-de-etica/). All work was conducted in accordance with ARRIVE guidelines. Data availability statement The raw data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB90254. 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We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6830034","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473232386,"identity":"5a5c4cb2-637c-4fd7-9259-e527407ae6ab","order_by":0,"name":"D. 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Associação Portuguesa de Herpetologia","correspondingAuthor":true,"prefix":"","firstName":"Catarina","middleName":"","lastName":"Rato","suffix":""}],"badges":[],"createdAt":"2025-06-05 14:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6830034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6830034/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85072988,"identity":"64d9507a-06b3-43ed-bf1c-b8b81632fe3e","added_by":"auto","created_at":"2025-06-20 15:57:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11505903,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the sampling localities of 66 \u003cem\u003eAgama picticauda\u003c/em\u003e from across Reunion Island.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/cfddcc36cf37f2ef480b667f.png"},{"id":85072984,"identity":"4377ce40-8698-40e0-b021-8875b2a734d2","added_by":"auto","created_at":"2025-06-20 15:57:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3042256,"visible":true,"origin":"","legend":"\u003cp\u003eRarefaction curves for males and females at the OTU (a) and family (b) levels, showing the observed (full line) and estimated (dashed line) richness, until double the reference sample size, and respective 84% confidence interval by sample coverage.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/24e1374e03d54b242437a69c.png"},{"id":85072985,"identity":"78be8354-ae62-41c9-a50d-4fcb7b755922","added_by":"auto","created_at":"2025-06-20 15:57:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6427291,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the General Linear Models results denoting the relationships between populations with OTU richness (a), and family richness (b).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/0a080edf8013ce6c37d7ceb1.png"},{"id":85074618,"identity":"57bf519b-daf2-4ea9-bf7d-9d7281e6bd54","added_by":"auto","created_at":"2025-06-20 16:21:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21585870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/2766b01f-609f-4a23-9db4-d9882a6c8368.pdf"},{"id":85072981,"identity":"3d547481-ae23-48bd-8c80-8e947a418398","added_by":"auto","created_at":"2025-06-20 15:57:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22720,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/b7bb7f720502d205e92eb5d1.xlsx"},{"id":85072982,"identity":"142db784-2cec-4070-97c5-53a0dd2bffe2","added_by":"auto","created_at":"2025-06-20 15:57:49","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":102345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1.\u003c/strong\u003e Linear regression of SVL logarithm (logSVL) against body mass logarithm (logWeight) in both males and females of \u003cem\u003eAgama picticauda\u003c/em\u003e from Reunion Island.\u003c/p\u003e","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-6830034/v1/a71f59a742617b56bb472b10.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabarcoding assessment of the diet of an introduced continental lizard to an oceanic island reveals dietary niche conservatism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe main drivers of biodiversity loss include habitat fragmentation, climate change and invasive species. Furthermore, it has been well-documented that the majority of vertebrate extinctions in recent times have been on islands, and that the bulk of these were associated with the establishment of exotic species. Island reptiles are particularly threatened by introduced competitors, especially on islands that were previously predator-free (Case and Bolger \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Because of this, there have been extensive studies on the impact of invasive species such as rodents or cats on island endemic reptiles (e.g. Thibault et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Gal\u0026atilde;o et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Invasive reptiles have also been associated with extinctions, for example the introduced gecko \u003cem\u003eHemidactylus frenatus\u003c/em\u003e was linked to the decline and extinction of endemic night gecko \u003cem\u003eNactus\u003c/em\u003e populations on the Mascarene islands (Cole et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, there have been fewer studies on the impact of invasive introduced reptiles on consumed taxa, because this trophic interaction involving many different potential prey items is harder to determine, and disentangling this mechanism through which introduced species impact the ecosystem remains a key challenge in ecological research (Feit et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The development of metabarcoding approaches, in which dietary items are identified using Next-generation genetic sequencing tools, offers a useful new technique to address this issue, particularly since prey items can be identified to much higher taxonomic levels than typically determined using microscopy assessments of stomach contents or pellets (e.g. Gal\u0026atilde;o et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Pinho et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe West African rainbow lizard \u003cem\u003eAgama picticauda\u003c/em\u003e is a common and widespread lizard in sub-Saharan West Africa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where it is often found in urban environments (Luiselli et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ofori et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is a highly-successful invasive species; for example, in Florida, USA, it is one of the most rapidly spreading non-native herpetofauna (Clements et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the Caribbean Lesser Antilles, \u003cem\u003eA. picticauda\u003c/em\u003e is considered to have a major impact on numerous native species, and is spreading to new islands at an alarming rate (van den Burg et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Recent molecular analyses have confirmed that the populations on Grande Comore and Reunion Island were independently introduced to these islands (Roesch et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). On Reunion Island, the first individuals were reported near the main maritime port, approximately 30 years ago (Guillermet et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and are now spread across the coastal areas and recently into the higher elevation central region (Roesch et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eAgama picticauda\u003c/em\u003e in their native range prey predominantly on insects, with Hymenoptera (Formicidae) and Coleoptera being the most frequently consumed prey orders (Ofori et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Morphological identification of stomach contents identified prey items from 14 orders, indicating a relatively wide prey spectrum (Ofori et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). \u003cem\u003eAgama picticauda\u003c/em\u003e may also consume plant matter, small vertebrates and even scavenge opportunistically on anthropogenic processed foods such as bread (Ofori et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Mascarenes, consisting of the volcanic islands of Reunion, Mauritius, Rodrigues as well as various small coralline islands, form part of the world\u0026rsquo;s top biodiversity hotspots (Thebaud et al. 2009). Reunion Island is the largest island (2512 km\u003csup\u003e2\u003c/sup\u003e), with dated lavas of around 2.1\u0026nbsp;million years. Although severely fragmented, about 25% of the estimated original extent of habitats on Reunion Island remain in a relatively good state. Like other similar island groups such as the Hawaiian Islands, the Mascarene biota includes notable levels of endemisms, including around 75% of native flowering plants and 90% of nonmarine molluscs (Thebaud et al. 2009). Of the terrestrial arthropods on Reunion Island, 31% are endemic to the island, and 40% endemic to the Mascarenes, although these are rough estimations given that around 60% of the arthropod fauna is predicted to remain unnamed (Legros et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the rapid spread of \u003cem\u003eA. picticauda\u003c/em\u003e across Reunion Island, and the high level of endemic and endangered potential prey items, an assessment of the trophic niche of this introduced agamid is clearly needed as part of the recently established \u0026ldquo;invasive species strategy and action plan\u0026rdquo;, which aims to mitigate its spread and associated risks (Nature Oc\u0026eacute;an Indien, 2023). Here we used a metabarcoding approach to assess the diet from 66 adult individuals, using stomach contents collected from populations around the island, including the higher elevation central region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We aimed to collect baseline data regarding prey items, and to determine if known endemic or endangered species were being preyed upon. The data can also be compared to the known diet from its native range, to assess levels of tropic niche conservatism. At the same time, dietary DNA or dDNA (\u003cem\u003esensu\u003c/em\u003e Sousa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) can often give notable new information about prey items themselves, helping to establish reliable barcoding databases of species presences. Furthermore, sequences of endoparasites such as nematodes can sometimes be identified in stomachs using this genetic approach, furnishing additional information for these poorly known species, potentially introduced along with their lizard host.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe COI and trnL libraries generated ca. 6.4\u0026nbsp;million raw sequence reads, which were reduced to 171,419 reads during the bioinformatic processing and to 558 total OTUs. Non-target COI amplification from different sources was observed both in samples, extractions, and PCR negative controls representing approximately 50% of the total reads. Almost all of these corresponded to the host, \u003cem\u003eAgama\u003c/em\u003e (48.19% of the total reads), while Nematoda represented around 1.25% of the total reads, and Fungi 0.04%. After negative controls, singletons, replicates, and taxa filtering the lizards\u0026rsquo; final diet using the COI primers consisted in 20,655 reads and 68 animal OTUs from 3 phyla, Annelida, Chordata and Arthropoda. Prey items were considered to belong to 38 families of arthopods, 2 families of annelids, and 1 family of chordates (geckos) (Supp. Material Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The most frequent OTUs were generally ants (Formicidae), and in particular \u003cem\u003eParatrechina longicornis\u003c/em\u003e (49.15% FO), \u003cem\u003eBrachymyrmex cordemoyi\u003c/em\u003e (11.86%), \u003cem\u003eSolenopsis geminate\u003c/em\u003e (8.47%) and \u003cem\u003ePheidole megacephala\u003c/em\u003e (6.78%). The most frequent prey OTUs after ants were the honey bee (Apidae) \u003cem\u003eApis mellifera\u003c/em\u003e (18.64%) and the beetle (Coccinellidae) \u003cem\u003eExochomus laeviusculus\u003c/em\u003e (15.25%). No other prey items had frequencies over 6%. As well as the prey items, 4 OTUs corresponded to nematodes from the orders Rhabditida and Spirurida. Regarding the chloroplast primers a total of 52,149 reads were obtained and 97 OTUs were detected (7 up to the species level), corresponding to 44 families (Supp. Material Table S3).\u003c/p\u003e \u003cp\u003eGrowth trajectories were similar for males and females, but males grew both bigger and heavier (Suppl. Material Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). There were significant differences in SVL between districts (F\u0026thinsp;=\u0026thinsp;2.35, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and sexes (F\u0026thinsp;=\u0026thinsp;40.21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00), but weight differences were only significant between the sexes (F\u0026thinsp;=\u0026thinsp;32.80, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00) and not districts (F\u0026thinsp;=\u0026thinsp;1.72, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08). Female agamas had higher dietary richness than males at both the prey OTU and family level, but the differences were not significant. Analysis of rarefaction and extrapolation curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicate no significant differences between males and females, with overlapping dietary niches. According to the GLM analysis, there was significant differences in OTU richness between districts(District: df\u0026thinsp;=\u0026thinsp;14, Deviance\u0026thinsp;=\u0026thinsp;16.42, Residual Deviance\u0026thinsp;=\u0026thinsp;36.66, p-value\u0026thinsp;=\u0026thinsp;0.29; Sex: df\u0026thinsp;=\u0026thinsp;1, Deviance\u0026thinsp;=\u0026thinsp;0.88, Residual Deviance\u0026thinsp;=\u0026thinsp;35.78, p-value\u0026thinsp;=\u0026thinsp;0.35; logSVL: df\u0026thinsp;=\u0026thinsp;1, Deviance\u0026thinsp;=\u0026thinsp;0.01, Residual Deviance\u0026thinsp;=\u0026thinsp;35.78, p-value\u0026thinsp;=\u0026thinsp;0.94), but none of the pairwise comparisons were significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the family level there were no significant results in richness ((District: df\u0026thinsp;=\u0026thinsp;14, Deviance\u0026thinsp;=\u0026thinsp;29.13, Residual Deviance\u0026thinsp;=\u0026thinsp;54.82, p-value\u0026thinsp;=\u0026thinsp;0.01; Sex: df\u0026thinsp;=\u0026thinsp;1, Deviance\u0026thinsp;=\u0026thinsp;1.88, Residual Deviance\u0026thinsp;=\u0026thinsp;52.95, p-value\u0026thinsp;=\u0026thinsp;0.17; logSVL: df\u0026thinsp;=\u0026thinsp;1, Deviance\u0026thinsp;=\u0026thinsp;0.21, Residual Deviance\u0026thinsp;=\u0026thinsp;52.73, p-value\u0026thinsp;=\u0026thinsp;0.64). Regarding the analysis on diet composition, there are significant differences only between districts at the OTU level, but not at a higher taxonomic level (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These significant results are not related to the dispersal of the data (F\u0026thinsp;=\u0026thinsp;1.54, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.15).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults from PerMANOVA on the effect of District, Sex and logSVL on OTU and family diet composition. Df stands for degrees of freedom. Significant \u003cem\u003ep\u003c/em\u003e-values are highlighted in bold.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e 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\u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elogSVL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFamily\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elogSVL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAn assessment of the diet of \u003cem\u003eA. picticauda\u003c/em\u003e along a transect of over 800 km within its native range along the west African coast showed that all populations were mainly insectivorous, and that food niche overlap was high between all populations (Akani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The most frequently identified prey item was ants (Formicoidea), with frequencies ranging from 20\u0026ndash;53%. Coleoptera, Lepidoptera, Araneidae and Vespoidea were also found in all populations sampled. Dietary niche overlap decreased with increases in the difference of mean annual rainfall between sites, but there was no effect of geographic distance. Similarly, in a population from Nigeria, the most frequent prey items identified were black ants, Blattodea (termites), yellow ants, bees and wasps, with melon seeds also more frequent than some arthropod orders (Rabiu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the diet of the introduced \u003cem\u003eA. picticauda\u003c/em\u003e on Reunion Island show notable similarities with the populations from the native range, indicating considerable trophic niche conservatism. Again, the most frequently identified prey items were ants. Eleven OTUs corresponding to 8 genera within Formicidae were recorded (Suppl. Material Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), of which \u003cem\u003eParatrechina longicornis\u003c/em\u003e (49.15%), \u003cem\u003eBrachymyrmex cordemoyi\u003c/em\u003e (11.86%), \u003cem\u003eSolenopsis geminate\u003c/em\u003e (8.47%) and \u003cem\u003ePheidole megacephala\u003c/em\u003e (6.78%). were the most frequently recovered, representing 76.26% of all prey OTUs identified with these primers. Coleoptera, Lepidoptera, Araneidae and Vespoidea were also all found at high frequency. Other less widely identified prey items from the native range, such as Isopoda, Diptera, and Blattodea were also identified as prey items in Reunion Island. Differences included, for example, that in west Africa, scorpions were occasionally consumed (up to 1.8% frequency in 3 of 8 sampled populations), while these were not detected in Reunion Island, although this is unsurprising since only two introduced species are known from this island (Legros et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn acknowledged advantage of the use of metabarcoding in lizard diet studies is the ability to determine prey items to a much higher level than typically obtained using microscopy, and this precision can sometimes uncover previously unknown aspects of the trophic niche (e.g. S\u0026rsquo;Khifa et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous assessments of the invertebrate prey items of \u003cem\u003eA. picticauda\u003c/em\u003e determined using microscopy had identified prey predominantly only to the order or family level (Akani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rabiu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A more recent extensive assessment of a population of \u003cem\u003eA. picticauda\u003c/em\u003e from urban and rural populations in Ghana identified 14 Orders and 47 families (Ofori et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). In this study, 12 arthropod Orders and 41 families were identified, again demonstrating the wide breadth of the dietary niche of \u003cem\u003eA. picticauda\u003c/em\u003e \u0026ndash; while ants and a few other groups make up the bulk of the diet, an extremely wide diversity of arthropods and other small invertebrates are consumed.\u003c/p\u003e \u003cp\u003eThe precision of prey identification means that metabarcoding studies of lizard pellets can also provide new data regarding the prey items themselves, especially since distribution data for invertebrates is often imprecise. For example, in an assessment of diet of the wall lizard \u003cem\u003ePodarcis lusitanicus\u003c/em\u003e in Northern Portugal, Sim\u0026otilde;es et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported several invertebrate prey species that were apparently new records for this country. Fortunately, the terrestrial arthropod fauna of Reunion Island has been relatively well studied. A recent review identified 3,369 species, of which 31% were endemic to Reunion Island (Legros et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Endemism rates, however, vary widely between groups, with for example 21% of spiders (Araneae) endemic, but only one ant (Formicidae) species out of 48 being endemic. Furthermore, it has been estimated that 62% of all the terrestrial arthropod fauna on Reunion Island remains to be named (Legros et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Identified prey items may therefore be useful in highlighting undescribed diversity, as well as allowing identification of endangered or endemic species within the trophic niche, as opposed to introduced and widespread species. Identified prey items may also represent first reports of introduced species that can then be added to update the faunistic catalogues of the island. For example, the spider \u003cem\u003eOecobius putus\u003c/em\u003e is not in the most recent, exhaustive checklist for the island (Cazanove, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), or global databases such as GBIF (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gbif.org/\u003c/span\u003e\u003cspan address=\"http://www.gbif.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and may represent a first record for Reunion Island. Likewise, the earthworms \u003cem\u003eTravoscolides chengannures\u003c/em\u003e and \u003cem\u003ePontoscolex corethrurus\u003c/em\u003e are neither in the GBIF database nor dedicated earthworm databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://taxo.drilobase.org/\u003c/span\u003e\u003cspan address=\"http://taxo.drilobase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for Reunion Island. The same occurs with the milliped \u003cem\u003eLeptogoniulus sorornus\u003c/em\u003e and the beetle \u003cem\u003eMyrmechixenus vaporariorum\u003c/em\u003e. These potentially new records for Reunion Island are species that are poorly studied and likely introduced \u0026ndash; regarding \u003cem\u003eOecobius\u003c/em\u003e on Reunion Island, Cazanove (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) states that \u0026ldquo;some species may have gone unnoticed due to a lack of attention and specific sampling\u0026rdquo;. Similarly, \u003cem\u003eP. corethrurus\u003c/em\u003e is widely introduced, and already reported as such to Mauritius, the Comoros and the Seychelles (Taheri et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe characterization of ants in the diet of \u003cem\u003eA. picticauda\u003c/em\u003e gives an example of the advantages of the barcoding technique over classic microscopy studies. All previous assessments had noted the high proportion of prey items of the family Formicidae. However, our study distinguished 11 OTUs, or presumed species, 10 of which were identified to the species level, within 8 genera. This means that of the 48 ant species known from Reunion Island, 22% were apparently part of the diet of \u003cem\u003eA. picticauda\u003c/em\u003e. All of the identified ants were widespread, introduced species \u0026ndash; the single known endemic ant species was not found. This is relatively good news regarding the impact of \u003cem\u003eA. picticauda\u003c/em\u003e on native arthropods, as it appears to maintain a preference for ants, the vast majority of which are introduced on Reunion Island. Previous studies have suggested that \u003cem\u003eA. picticauda\u003c/em\u003e may consume a high frequency of ants as they are among the most frequently encountered ground-dwelling insects (Ofori et al. 2023). However, our finding of such a wide diversity of ant species seems to rather indicate that ants are preferentially preyed upon, which may also help explain the conservatism of the trophic niche in this introduced population.\u003c/p\u003e \u003cp\u003eGiven that around one third of the terrestrial arthropods on Reunion Island are endemic, it might have been expected to find a similar proportion within the identified prey items. Instead, only one endemic species, the Hemiptera \u003cem\u003eDeraeocoris howanus\u003c/em\u003e, was recognized, out of 20 identified to the species level. Although \u003cem\u003eA. picticauda\u003c/em\u003e predominantly consumes introduced species, this is not to say that the lizard does not have a considerable impact on native terrestrial arthropods. Introduction of an exotic lizard has been shown to significantly alter ant community structure, reducing the abundance of some species and therefore having indirect effects on other species (Huang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, the broad dietary niche of \u003cem\u003eA. picticauda\u003c/em\u003e means that many species are consumed, and the unidentified prey items may correspond to additional endemic species. A more complete database of sequences from island endemics would be needed to further assess this.\u003c/p\u003e \u003cp\u003eAnother aspect of the threat caused by introduced species is that they can bring parasites with them, which may have severe impacts if they are transmitted to local hosts. An interesting bycatch of the COI primers employed in this study and widely used for assessing the diet of reptiles is that they also can amplify nematodes. In this study of \u003cem\u003eA. picticauda\u003c/em\u003e introduced to Reunion Island, four distinct OTUs corresponded to nematodes. A previous morphological assessment of gastrointestinal helminths from \u003cem\u003eA. picticauda\u003c/em\u003e within its native range in Ghana identified four helminth species, \u003cem\u003eAscaris\u003c/em\u003e spp., \u003cem\u003eEnterobius\u003c/em\u003e spp., \u003cem\u003ePharyngodon\u003c/em\u003e spp. and \u003cem\u003eOxyurid\u003c/em\u003e spp. (Ofori et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Unfortunately, none of the nematode sequences from this study had close matches on GenBank (83\u0026ndash;93% matches), so we cannot ascertain with any degree of certainty which species they may correspond to. At the same time, morphological assessments of nematodes in other introduced populations of lizards have identified species typical of the local herpetofauna, indicating that the exotic species had acquired their helminth assemblage from the local helminth pool, rather than possessing species from the parasite fauna of the original population (Anjos et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Criscione and Font, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Although we cannot therefore currently identify these nematodes, as more sequence data becomes available for helminths from lizards, in the future these sequences may be useful for identifying these species.\u003c/p\u003e \u003cp\u003eAn additional potential threat from the introduced \u003cem\u003eA. picticauda\u003c/em\u003e would be direct predation on endemic lizards, since agamas are known to occasionally prey on small vertebrates, and there are also records of cannibalism (Vasconcelos et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rabiu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cannibalism cannot be assessed with the metabarcoding approach used, as sequences corresponding to \u003cem\u003eA. picticauda\u003c/em\u003e were automatically discarded as presumed belonging to the host individual. No sequences corresponding to \u003cem\u003ePhelusma\u003c/em\u003e day geckos were identified, and although sampling locations do not overlap with the distribution of native \u003cem\u003ePhelsuma\u003c/em\u003e spp. (Dubos et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003eb\u003c/span\u003e), they do overlap heavily with introduced \u003cem\u003ePhelsuma\u003c/em\u003e spp. (Dubos et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The introduced gecko \u003cem\u003eHemidactylus frenatus\u003c/em\u003e was identified at low frequency (1.47%).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgama picticauda\u003c/em\u003e populations typically exhibit sexual size dimorphism (van den Burg et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Roesch et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), with both sexes showing a similar SVL/weight trajectory, but with males being both larger and heavier (Suppl. Mat. Fig. S\u003c/p\u003e \u003cp\u003e1). Given this, it might be presumed that the dietary niche of males would be larger than females, as their larger size would enable them to consume some prey items that would be too big for females to consume. Interestingly however, the females had higher diet richness when assessed both at the OTU and family level, although the difference was not significant. In other lizards with sexual dimorphism, males have been shown to select larger prey items, corresponding to their larger head size and bite force (Kaliontzopoulou et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the other hand, in dDNA assessments, the size of prey consumed remains unknown \u0026ndash; females and males could be preying on the same species, but selecting specimens of different sizes for example. Rarefaction and extrapolation curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicate that males and females have considerably overlapping dietary niches, with no significant differences observed.\u003c/p\u003e \u003cp\u003ePrevious studies of diet variation along transects of \u003cem\u003eA. picticauda\u003c/em\u003e populations have indicated that, while the preference for certain prey items such as ants and beetles remains constant, variation between populations does occur (Akani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This is expected, as the terrestrial arthropod community will vary with geological factors such as rainfall, and also anthropogenic influences such as levels of urban development (Akani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ofori et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). When assessing differences in dietary consumption between populations, significant differences were found in OTU richness between districts, but in pairwise comparisons none are significant probably because of the low sample numbers in some regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Further, no significant differences were found between populations when assessing family-level richness. While \u003cem\u003eA. picticauda\u003c/em\u003e is spreading around the island, they are still heavily associated with anthropogenically disturbed areas (Nature Oc\u0026eacute;an Indien, 2023). This may also explain why so much of the diet consists of introduced species, similarly associated with urbanised areas. The Honey bee \u003cem\u003eApis mellifera\u003c/em\u003e (18.64%) is the second most frequent arthropod OTU in the diet of \u003cem\u003eA. picticauda\u003c/em\u003e and, while introduced, represents a species of significant economic importance. Long-term monitoring will be needed to see if this dietary aspect changes with continuing spread of agamas into more natural areas.\u003c/p\u003e \u003cp\u003eOur assessment of plant material in the stomach contents identified 97 OTUs from 44 families, with seven identified to the species level. Although the most common OTUs could not be fully identified (Unidentified Solanaceae 25%, Unidentified Fabaceae 22%), the third most frequent OTU was \u003cem\u003eDesmondium scorpiurus\u003c/em\u003e (13%), a perennial tropical legume widely used to feed livestock. Rabiu (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) identified seeds in 37% of pellets within the native range of \u003cem\u003eA. picticauda\u003c/em\u003e, but the most common were melon seeds, available through anthropogenic discards. In this study various potential fruits were also identified including \u003cem\u003eFicus\u003c/em\u003e sp. (figs), \u003cem\u003eRubus\u003c/em\u003e sp. (blackberries) and \u003cem\u003eMusa\u003c/em\u003e sp. (banana). Combined with the high diversity of plant species detected, and the high nutritional value of many of them, it seems likely that \u003cem\u003eA. picticauda\u003c/em\u003e are specifically targeting plants for consumption, rather than accidentally ingesting them while aiming at invertebrates. None of the plant species identified were endemic to Reunion Island. However, some may be seed dispersed by this lizard. For example, one of the species identified was the highly invasive Brazilian pepper tree, \u003cem\u003eSchinus terebinthifolius.\u003c/em\u003e It is known that recruitment of this species is dependent upon frugivores \u0026ndash;in Australia seed germination is minimal without pulp removal, a task that is accomplished by a native bird species (Panetta and McKee \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Previous studies of other introduced reptiles, including the Green Iguana in Puerto Rico (Govender et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and the panther chameleon in Reunion Island (Sanchez et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have found they also consume seeds of \u003cem\u003eS. terebinthifolius\u003c/em\u003e. Agama may therefore play a role in the maintenance and spread of this invasive tree species, highlighting the complex interplay between introduced species in these small island communities.\u003c/p\u003e \u003cp\u003eDespite the clear advantages that metabarcoding approaches for dietary assessments have over microscopy appraisals, there are also some limitations that need to be considered. As well as the inability to identify the size of prey specimens, life cycle stage cannot be determined, so it remains unclear for example if agamas are consuming predominantly the flying adult stages of Lepidoptera, or the crawling larval stages. Furthermore, this study is an estimate of the diet from a single time point, which may overlook important temporal effects. For example, Rabiu (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) suggested that \u003cem\u003eA. picticauda\u003c/em\u003e ate more plant material in the rainy season in its native range. Such variation could only be assessed by repeating the sampling during a different time of the year. Another potential issue with dDNA studies is the risk of \u0026ldquo;secondary predation\u0026rdquo; (da Silva et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where the DNA from prey items of prey items are detected. For instance, the DNA from the thrips \u003cem\u003eFrankliniella schultzei\u003c/em\u003e was detected, but these can also be consumed by beetles, and therefore the DNA may arrive only indirectly in the pellets of the agama. Plant DNA is also widely detected in insectivorous species (e.g. da Silva et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), highlighting the need for caution in interpreting the presence of an OTU with deliberate consumption of that taxa. While this could cause some inflation in the dietary spectrum identified, the considerable number of orders and families detected demonstrates that the dietary niche of these agamas is very wide, even if they do preferentially consume ants. Finally, the potential new records of invertebrates for the island, for example for the spider \u003cem\u003eO. putus\u003c/em\u003e, still need to be confirmed \u0026ndash; these assessments are based only on a single marker, mitochondrial DNA, and the similarity with sequences from GenBank. Errors in the published database, or evolutionary aspects such as mtDNA introgression, while unusual, mean that these results need morphological corroboration.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo conclude, this study shows that the dietary niche of \u003cem\u003eA. picticauda\u003c/em\u003e is extremely wide, with a high number of orders and families detected. On the other hand, the diet remains remarkably conservative, with a strong preference for ants continuing in this introduced population. While the ants consumed are generally introduced, at least one endemic insect was also consumed, and this raises conservation concerns. The high taxonomic levels of prey identification, often to the species level, give additional insights into the terrestrial arthropod fauna found on Reunion Island, with some potential examples of new records for the island. Plants formed a notable part of the diet, and were more frequent than previously considered. Gastrointestinal helminths were detected, but could not be adequately identified due to limitations in the comparative genetic databases currently available. Still, the high diversity of genetic sequences reported in this study will provide a strong baseline for future assessments both of this introduced lizard, and also for other barcoding studies of the invertebrate fauna of Reunion Island.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSampling took place during June to August 2022, across 16 districts within Reunion Island (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Suppl. Material Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As part of an invasive species control, agamas were captured with artisanal glue traps, where the animals have been lured onto the trap with live bait (\u003cem\u003ePycnoscelus surinamensis\u003c/em\u003e). The live bait was securely contained within a transparent box, ensuring that it remained inaccessible to the lizards and was not consumed. Snout-vent length (to 1mm) was measured with a ruler and animals were weighed (to 0.5g) with a Pesola scale. Since the agamas are a non-native invasive species, all captured individuals were chemically euthanised using a Lidocaine/Prilocaine cream (EMLA) and the stomach contents were immediately collected and stored in 90% ethanol and at -20\u0026deg;C prior to DNA extraction. Sex was determined by examining the presence of femoral pores and hemipenial bulges and confirmed by dissection.\u003c/p\u003e \u003cp\u003eDNA from approximately 200 mg of each collected 66 stomach samples was extracted using an E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, U.S.A.), following the manufacturer\u0026rsquo;s instructions with a minor modification in the digestion step, using 800 \u0026micro;L of Gordon buffer instead of 200 \u0026micro;L of TL buffer to improve DNA extraction of both hard and soft tissues, following Sim\u0026otilde;es et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Samples were vortexed and digested during 3 hours. Extracted DNA was stored at -20\u0026ordm;C. Extraction blank samples were included to control for contaminants present in extraction kits and/or in the laboratory environment. A short fragment (~\u0026thinsp;205 bp) of the mitochondrial Cytochrome C Oxidase subunit I (COI) was amplified using Polymerase Chain Reaction (PCR) with the Fwh2 primers from Vamos et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which are among the most effective primers for terrestrial arthropods (Elbrecht et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and have worked well in other insectivorous lizards (e.g. Sim\u0026otilde;es et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To assess plant consumption, the g/h primers (Taberlet et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) were used targeting the short P6-loop of chloroplast trnL (UAA) intron (up to 143 bp). The primers were modified to include Illumina adaptors and a 0\u0026ndash;5 bp addition of N bases between the adaptor and the primer to increase sequencing diversity and quality. The different primer variations were then combined before PCR reactions, resulting in mixed forward and reverse primer single solutions.\u003c/p\u003e \u003cp\u003eThe PCR reactions for both plant and animal amplifications consisted of 5 \u0026micro;L of QUIAGEN Multiplex PCR Master Mix (Quiagen, Crawley, UK), 0.3 \u0026micro;L combination of six Forward primers, 0.3 \u0026micro;L combination of Reverse Primers, 2.4 \u0026micro;L of ultra-pure water, and 2 \u0026micro;L of DNA extract. Cycling conditions used consisted of an initial denaturation at 95\u0026deg;C for 15 min, followed by 45 cycles of 95\u0026deg;C for 30s, annealing at 52\u0026deg;C for 45s, extension at 72\u0026deg;C for 20s, and a final extension at 60\u0026deg;C for 5min. Negative controls were included to detect possible contaminations.\u003c/p\u003e \u003cp\u003eInitial PCR clean-up to remove unused primers and primer dimers was performed employing the Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), with a proportion of 0.85 \u0026micro;L of magnetic beads to 1 \u0026micro;L of PCR product. To attach unique barcodes and Illumina sequencing indexes to each sample, a second PCR was performed using a distinct combination of index sequences per sample. This PCR was performed using 2.8 \u0026micro;L of ultra-pure water, 7\u0026micro;L of 2x Kapa HiFi, 1.4 \u0026micro;L of each index, and 2.8 \u0026micro;L of cleaned DNA. Indexing the PCR required an initial denaturation of 95\u0026deg;C for 3min, followed by 10 cycles of 95\u0026deg;C for 30s, annealing at 55\u0026deg;C for 30s, extension at 72\u0026deg;C for 30s, and a final extension of 72\u0026deg;C for 5min. PCR products were examined in a 2% agarose gel knowing that amplicons should be ~\u0026thinsp;100 bp longer than in the previous PCR. A second PCR clean-up was then performed, under the same conditions as before, except that the beads\u0026rsquo; ratio was 0.8.\u003c/p\u003e \u003cp\u003e After the second PCR clean-up, all indexed PCRs were quantified using Epoch\u0026trade; Microplate Spectrophotometer (BioTek Instruments, Inc.; Winooski, VT, USA), followed by normalisation to obtain the same concentration in all the samples, after which the samples were pooled. This pool was quality tested in a TapeStation 4200 High Sensitivity D1000 Assay (Agilent, Santa Clara, CA), and a cleaning was performed using a 0.78 ratio of beads. A second quality control test in the TapeStation confirmed the success of the cleaning. The final pool was sent to GENEWIZ Next Generation Sequencing laboratory with a final concentration of 27nM to be sequenced in an Illumina MiSeq sequencer with 2x250bp paired-end (PE) configuration and PhiX (\u0026le;\u0026thinsp;30%) was used to increase sequencing diversity.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eBioinformatic processing of sequencing reads was done separately for each molecular marker. The software PEAR (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) was used to merge the forward and reverse reads into single sequences, discarding single and unassembled reads, as well as cases with less than 26bp of overlap. Primers were removed, using the command \u003cem\u003engsfilter\u003c/em\u003e from OBITools (Boyer et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), reads were counted to check for successful rates of the previous step with the \u003cem\u003egrep\u003c/em\u003e command, and dereplicated into unique sequences using the \u003cem\u003eobiuniq\u003c/em\u003e command. Sequence cleaning was performed to remove sequences with less than 10 reads and chimeras, using the \u003cem\u003eobiclean\u003c/em\u003e command, and those with a length between 202 to 208bp (COI) and 30 to 120bp (trnL) were kept and clustered at a 99% similarity threshold to form Operational Taxonomic Units (OTUs) using VSearch (Rognes et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). OTUs that were represented by a read count\u0026thinsp;\u0026lt;\u0026thinsp;1% of the total number of reads obtained for each sample were removed, as were all reads identified in the extraction and PCR controls of the corresponding sample batch.\u003c/p\u003e \u003cp\u003eThe obtained OTU table and sequences were further cleaned to remove redundant and unreliable OTUs using the R package LULU (Fr\u0026oslash;slev et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and the taxonomic assignment of OTUs was performed using BOLDigger (Buchner and Leese \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), followed by manual inspection and curation. We used the BLAST function from NCBI implemented in Geneious Prime 2021.1.1 (Drummond et al. 2010), when species-level assignments were not possible with BOLDigger. If similarity was \u0026gt;\u0026thinsp;95% assignment was made to the species level, or genus level in cases where more than one species from the same genus had a\u0026thinsp;\u0026gt;\u0026thinsp;95% match. Assignment was made to the family level if between 90\u0026ndash;95%, and to the class level if below 90% similarity. Prey OTUs that were represented by a read count\u0026thinsp;\u0026lt;\u0026thinsp;1% of the total number of diet reads were removed. Additionally, all reads identified in the extraction and PCR controls were subtracted from the corresponding sample batch.\u003c/p\u003e \u003cp\u003eFor the statistical analysis, R 4.1.2 (R Core Team 2022) was used to assess differences in dietary descriptors (i.e., diet richness and composition) between localities and sexes. The log transformed body size (SVL) and mass (weight) variation between sexes across localities, was summarized using boxplots. Body condition was assessed by investigating the relationship between body mass and body length in both males and females, through a linear regression.\u003c/p\u003e \u003cp\u003eDietary analysis was based on two different taxonomic levels: OTU (all taxonomic units identified to the highest resolved taxonomic level) and Family. For the OTUs not identified to the species level, we built a neighbor-joining tree in Geneious Prime v. 2024.0.2 (Biomatters), visually inspected the corresponding alignment, and checked for patterns of genetic similarity (~\u0026thinsp;98%) in order to cluster them into distinct taxa (e.g., Coleoptera 1, Coleoptera 2, and so on).\u003c/p\u003e \u003cp\u003eThe frequency of occurrence (FO) of each food item was calculated as the number of occurrences of the diet item (maximum 1 occurrence per sample) divided by the total number of sequenced reptile stomach samples.\u003c/p\u003e \u003cp\u003eTo assess the effects of locality, sex and size on the average number of prey taxa detected per sample (i.e., richness), a General Linear Model (GLM) was implemented. For this, the function \u003cem\u003eglm\u003c/em\u003e was used and its significance tested with the \u003cem\u003eanova\u003c/em\u003e function, both from the car package (Fox et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), fitting a Poisson error distribution. Graphical representation of the relationships depicted from the GLM results was performed using the \u003cem\u003eeffect\u003c/em\u003e function from the effects R package (Fox \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), where richness means and standard errors were derived from the model\u0026rsquo;s parameters estimates. Pairwise comparisons were performed with the function emmeans (with a Tukey adjust) from the package emmeans (Lenth et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo calculate the dietary niche-width between the sexes and among the different populations, prey rarefaction and extrapolation curves were built using the R package iNEXT v. 2.0.20 (Hsieh et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Analyses were conducted with incidence frequencies for prey taxa. We compared the estimated richness considering completeness (i.e., sample coverage) instead of sample size (i.e., number of samples), to avoid biases of communities with different levels of richness requiring different sampling efforts in order to be sufficiently characterized (Chao and Jost \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Considering that the 95% confidence interval is a very conservative approach, we considered that differences were significant if the 84% confidence interval (a proxy for α\u0026thinsp;=\u0026thinsp;0.05) of both estimates did not overlap (MacGregor-Fors and Payton \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePermutational multivariate analysis of variance (PerMANOVA) was used to compare the OTU and family diet composition between the sexes and the different localities with the vegan R package (function \u003cem\u003eadonis2\u003c/em\u003e; Oksanen et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Presence or absence of each prey item in each sample was used to build a Jaccard dissimilarity matrix using the \u003cem\u003evegdist\u003c/em\u003e function from the R package vegan. A homogeneity of dispersion test (function \u003cem\u003ebetadisper\u003c/em\u003e) was also carried out to assess if the observed differences in PerMANOVA could be due to unequally dispersed values across the different groups (Anderson \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all team members of the Association Nature Oc\u0026eacute;an Indien for administrative and operational support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project received funding from the Office Fran\u0026ccedil;ais de la Biodiversit\u0026eacute; (reference N\u0026deg;OFB-21-1920), D\u0026eacute;partement de La R\u0026eacute;union (reference n\u0026deg; CP-2021-DEC-172-2, XXX), Direction de l\u0026rsquo;Environnement de l\u0026rsquo;Am\u0026eacute;nagement et du Logement de La R\u0026eacute;union (reference n\u0026deg;2023-29) and Fonds Europ\u0026eacute;en de D\u0026eacute;veloppement R\u0026eacute;gional (reference n\u0026deg;20221409-0034656) awarded to the Association Nature Oc\u0026eacute;an Indien.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD. James Harris: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Resources, Funding acquisition, Investigation, Conceptualization. Markus A. Roesch: Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology, Conceptualization, Investigation. Diana S. Vasconcelos: Writing \u0026ndash; review \u0026amp; editing, Data curation. Chlo\u0026eacute; Bernet: Writing \u0026ndash; review \u0026amp; editing, Project administration, Methodology. Catarina Rato: Writing \u0026ndash; review \u0026amp; editing, Validation, Investigation, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe invasive species control programme follows order no. 2023-605 SG/SCOPP/BCPE in accordance with article L411.8 of the Environment Code on the territory of Reunion Island. Access and benefit-sharing was approved in accordance with Article17, paragraph 2, of the Nagoya Protocol and accessible under the reference number TREL2206915S/624. All methods were carried out in accordance with relevant guidelines and regulations. The entire experimental protocol was approved by the Ethics Committee of the University of Porto (https://www.up.pt/portal/pt/conhecer/organizacao/comissao-de-etica/). All work was conducted in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB90254.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that there have been no involvements that might raise the question of bias in the work reported or in the conclusions, implications, or opinions stated.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkani, G. C., Petrozzi, F., Rugiero, L., Segniagbeto, G. H., \u0026amp; Luiselli, L. (2013). Effects of rainfall and geography on the comparative diets of eight rainbow lizard populations across Togo, Benin and Nigeria (West Africa). Amphibia-Reptilia, 34(2), 185-192.\u003c/li\u003e\n\u003cli\u003eAnderson, M. J. (2006). Distance-based tests for homogeneity of multivariate dispersions. Biometrics, 62(1), 245-253.\u003c/li\u003e\n\u003cli\u003eAnjos, L. A., Rocha, C. F. D., Vrcibradic, D., \u0026amp; Vicente, J. J. (2005). Helminths of the exotic lizard \u003cem\u003eHemidactylus mabouia\u003c/em\u003e from a rock outcrop area in southeastern Brazil. \u003cem\u003eJournal of Helminthology\u003c/em\u003e, \u003cem\u003e79\u003c/em\u003e(4), 307-313.\u003c/li\u003e\n\u003cli\u003eBoyer, F., Mercier, C., Bonin, A., Le Bras, Y., Taberlet, P., \u0026amp; Coissac, E. (2016). obitools: A unix‐inspired software package for DNA metabarcoding. \u003cem\u003eMolecular ecology resources\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(1), 176-182.\u003c/li\u003e\n\u003cli\u003eBuchner, D., \u0026amp; Leese, F. (2020). BOLDigger\u0026ndash;a Python package to identify and organise sequences with the Barcode of Life Data systems. 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Non-native herpetofauna continue to proliferate in the world\u0026rsquo;s most invaded herpetofauna community: Evidence against community saturation.\u003c/li\u003e\n\u003cli\u003eCole, N. C., Jones, C. G., \u0026amp; Harris, S. (2005). The need for enemy-free space: the impact of an invasive gecko on island endemics. Biological Conservation, 125(4), 467-474.\u003c/li\u003e\n\u003cli\u003eCriscione, C. D., \u0026amp; Font, W. F. (2001). The guest playing host: colonization of the introduced Mediterranean gecko, \u003cem\u003eHemidactylus turcicus\u003c/em\u003e, by helminth parasites in southeastern Louisiana. Journal of Parasitology, 87(6), 1273-1278.\u003c/li\u003e\n\u003cli\u003eda Silva, L. P., Mata, V. A., Lopes, P. B., Pereira, P., Jarman, S. N., Lopes, R. J., \u0026amp; Beja, P. (2019). Advancing the integration of multi‐marker metabarcoding data in dietary analysis of trophic generalists. 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(2013): Contrasting diversity values: statistical inferences based on overlapping confidence intervals. PLoS One \u003cstrong\u003e8\u003c/strong\u003e: e56794.\u003c/li\u003e\n\u003cli\u003eNature Océan Indien (2023) Plan Opérationnel de Lutte contre l\u0026rsquo;Agame des roches à La Réunion. 2024 - 2028. Éd. Chloé Bernet, Steven Calesse, Sébastien Dervin, Marion Neymeyer, Markus Roesch \u0026amp; Jérémie Souchet. Association Nature Océan Indien pour l\u0026rsquo;Office Français de la Biodiversité et le Département de La Réunion. 58 pp + annexes.\u003c/li\u003e\n\u003cli\u003eOfori, B. Y., Martey, P., \u0026amp; Attuquayefio, D. K. (2018). Observations of the African rainbow lizard (\u003cem\u003eAgama picticauda\u003c/em\u003e Peters 1877) from Ghana feeding on bread. Herpetology Notes, 11, 955-957.\u003c/li\u003e\n\u003cli\u003eOfori, B. Y., Martey, P., Anderson, R. S., Mensah, J. B., Quartey, J. K., \u0026amp; Attuquayefio, D. K. (2023a). 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(2014). \u003cem\u003eAgama agama\u003c/em\u003e: a charter tourist in the Cape Verde Islands? African Journal of Herpetology, 63(1), 34-46.\u003c/li\u003e\n\u003cli\u003eZhang, J., Kobert, K., Flouri, T., \u0026amp; Stamatakis, A. (2014). PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics, 30(5), 614-620.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Reunion Island, trophic niche conservatism, Agama picticauda, invasive species","lastPublishedDoi":"10.21203/rs.3.rs-6830034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6830034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInvasive species can have devastating effects when introduced into remote island ecosystems, and a fundamental aspect of this concerns the diet of these exotic taxa. Here we employed a DNA metabarcoding approach to determine the diet of the lizard \u003cem\u003eAgama picticauda\u003c/em\u003e on Reunion Island, where it was introduced in 1995. Two separate markers were used to identify animal and plant components. The arthropod aspect was notably conservative, with the agama continuing to predominantly consume ants, as they do in their native range. A variety of other invertebrates were also preyed upon, the vast majority being introduced species. For plants again a wide variety were detected, and while most could not be identified fully, it seems that agamas are deliberately consuming many species, rather than accidentally intaking them along with targeted invertebrates. Agamas may play a role in seed dispersal of invasive plant species. We also detected some nematode groups, although with limited comparative sequences these could not be identified to the species level. Several records of invertebrates appear to be new records for Reunion Island, highlighting how reptiles can be considered as excellent biodiversity samplers, with barcoding diet studies providing novel data for poorly known invertebrate groups. The minimal identifications of endemic prey items may reflect that the agamas are still predominantly occupying anthropogenically disturbed parts of the island. Our study therefore provides baseline data that can be used to determine the impact of this introduced lizard as it spreads through the ecosystem.\u003c/p\u003e","manuscriptTitle":"Metabarcoding assessment of the diet of an introduced continental lizard to an oceanic island reveals dietary niche conservatism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 15:57:44","doi":"10.21203/rs.3.rs-6830034/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-07T06:02:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-12T04:49:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291326695604888085173498144108068118347","date":"2025-09-02T06:28:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258280174314259139369905112286287899804","date":"2025-07-30T08:00:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-21T17:32:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T18:12:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46915422472875468264797957257913564146","date":"2025-06-24T12:44:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188711953433081936719439411166016548175","date":"2025-06-18T17:39:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118699454113212881274094849047695643118","date":"2025-06-18T17:35:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T17:31:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T14:18:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-12T05:04:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T14:28:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-10T14:24:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4fc2d2b6-c0b6-4cce-bf9a-833452b92f5b","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50258773,"name":"Biological sciences/Ecology/Evolutionary ecology"},{"id":50258774,"name":"Biological sciences/Zoology/Herpetology"},{"id":50258775,"name":"Biological sciences/Ecology/Invasive species"}],"tags":[],"updatedAt":"2026-05-20T11:24:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 15:57:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6830034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6830034","identity":"rs-6830034","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}