DOI: 10.5061/dryad.zpc866thn
title: Data from: Smaller islands, bigger appetites: Evolutionary strategies of insular endemic skinks

description: Competitive dietary and morphological divergence among co-occurring
 species are fundamental aspects of ecological communities, particularly on
 islands. Cabo Verde (~570 km west of continental Africa) hosts several
 endemic reptiles descended from common ancestors, with sympatric species
 exhibiting wide morphological variation and competing for limited
 resources. To explore the mechanisms of resource partitioning between
 coexisting species, DNA metabarcoding was used to compare the diets of
 large and small skinks, Chioninia vaillantii and Chioninia delalandii in
 sympatric and allopatric contexts on Fogo Island and in a more competitive
 context on the small and resource-poor Cima Islet. The morphological
 variation of all populations was also examined to test the character
 displacement hypothesis and to compare the effect of different competitive
 scenarios. Results showed significant differences in diet and linear
 measurements between species and populations. The two sympatric
 populations of C. delalandii on Fogo and Cima showed similar changes in
 head morphology compared to the allopatric population, supporting
 character displacement. The effect of higher competitive pressure on Cima
 was evidenced by the increased morphological and dietary variation
 observed. This study demonstrates how sister species develop dietary
 adaptations/ morphologies to maintain stable coexistence, especially in
 highly competitive scenarios, providing useful insights for effective
 conservation strategies.  
description: Study area Fogo Island and the Rombo’s Islets
 are located in the Leeward group of Cabo Verde (Figure 1B). Fogo has 476
 km<sup>2</sup>, reaching the highest altitude of the country
 at 2829 m. The landscape is characterised by the active volcano, last
 erupted in 2014, and large agricultural and pasture areas within its wide
 caldera. Its climate varies from dry – in the lower areas – to sub-humid –
 in higher altitude areas. In 2020, it was declared a biosphere reserve by
 UNESCO due to its unique ecosystem and biodiversity.
 The uninhabited Rombo’s are composed of two main islets, Grande
 and Cima, surrounded by the smaller islets of Luiz Carneiro, Rei and
 Sapado (&lt;0.25 km<sup>2</sup>). These are classified as
 Integral Nature Reserve and constitute Important Bird Areas. Cima,
 approximately 15 km west of Fogo, is a dry flat islet with about 1.5
 km<sup>2</sup> and a small hill of approximately 77 m. This
 islet is a key biodiversity hotspot for seabird species. However, its
 biodiversity is severely affected by <em>Mus musculus
 </em>Linnaeus, 1758<em> </em>increasing
 populations. Study species
 Delalande’s skink <em>C. delalandii</em> (Cd hereon)
 is medium-sized (adults between 52–92 mm snout-vent length, SVL), and
 easily recognized by the presence of a black dot on the axilla and yellow
 eyelids. It is often found in stonewalls and beneath rocks within
 agricultural and livestock areas. On Fogo, it is also found in urban
 areas, including at higher altitudes (Figure 1C;). Despite its abundance
 and conspicuousness, little is known about its ecology, except for
 anecdotal reports of omnivory. It is classified as Least Concern according
 to the International Union for the Conservation of Nature (IUCN) Red List
 of Threatened Species, and as Low Risk on the National Red List at a
 national and main islands level, but Data Deficient on Rombo’s.
 The Vaillant's skink of Fogo <em>C. vaillantii
 xanthotis </em>(Cv hereon) is large-sized (adults between 87.5–105
 mm SVL), and distributed on Fogo and Rombo’s, more precisely on Cima [23].
 It can be identified by its three well-contrasted stripes along the body
 and the bright yellow scales around the ear opening. On Fogo, it is
 restricted to the north-western side (Figure 1C) and less abundant than
 Cd. It is typically found in stonewalls at high altitude in vegetated
 humid habitats. It is viviparous and its diet is thought to be composed
 mostly of insects and plants. It is classified as Endangered according to
 the IUCN and as Undetermined on a national level and on Fogo, and as Data
 Deficient on Rombo’s in the National Red List.
 Sampling Sampling took place on Fogo and Cima
 in May 2019, with additional samples of Cd collected on Fogo in September
 2021 in the allopatric area. On Fogo, 20 samples of Cd were collected in
 sympatric areas and 23 in allopatric areas, and 17 of Cv in sympatry. On
 Cima, a total of 10 and 17 faecal samples of<em> </em>Cd and
 Cv were collected, respectively. Specimens were captured using either hoop
 fishing or bucket traps with bait (banana and mango). Faecal samples were
 collected by abdominal massage and immediately preserved in 96% ethanol
 tubes. These were stored at -20ºC until further processing. Individuals
 were sexed, photographed and geolocated using a GPS device. Before
 release, morphological linear measures of both species were taken to the
 nearest 0.1 mm using digital callipers (Figure 2): snout-vent length
 (SVL), trunk length (TL), head length (HL), width (HW), and height (HH),
 eye-snout (ES) and eye-ear distance (EE), eye diameter (ED), and front
 (FLL) and hind limb length (HLL). Animals were placed in separate opaque
 tissue bags while others were processed to reduce stress.
 In addition, pitfall traps with water were placed close to the
 areas where skinks were captured to collect invertebrates to build a DNA
 reference database of available food items. Captured specimens were
 preserved in falcon tubes with 96% ethanol until examination under a
 magnifying lens with an assembled camera. Individuals were sorted into
 different high-level taxonomic groups based on morphological
 identification and photographed for higher-resolution identification by
 experts.Diet analysis Library preparation
 DNA from faecal samples was extracted using the Stool DNA
 Isolation Kit, following the manufacturer's instructions. Each batch
 of 23 samples included an extraction negative control. Extracted DNA was
 amplified using previously validated markers [47] for three diet groups:
 invertebrates with the IN16STK-1F-mod/IN16STK-1R-mod primers, targeting
 the mitochondrial 16S gene; plants with g/h primers targeting the short
 P6-loop of chloroplast <em>trnL</em> (UAA) gene and
 vertebrates with the 12sv5F/12Ssv5R primer pair, targeting V5-loop within
 the mitochondrial 12S gene. To prevent the amplification, with the 12S
 primers, of host DNA, a blocking primer was designed
 (5'-TCCTCTAGGTCGGTATGGGGCACCGCCA(C3 spacer)-3'). For this, 12S
 sequences of both <em>Chioninia</em> species were obtained
 from GenBank and aligned with sequences from birds, fish and reptiles
 known to occur in Cabo Verde. Its effectiveness for Cd and Cv was
 validated by comparing PCR amplification with and without the blocking
 primer, using previously extracted DNA from tail tissues. PCR products
 (see details in Supplementary material) were cleaned using Agencourt
 AMPure XP beads, quantified using Nanodrop, diluted to 15nM, and pooled
 for each marker. The three libraries were then quantified using qPCR and
 pooled to obtain a final library at 4nM, that was sequenced using the
 MiSeq Reagent Kit V2 for an expected average of 20,000 paired-end reads
 per PCR product. Invertebrate DNA for the reference
 library was extracted from legs or whole individuals (when very small),
 using saline extraction methods following and amplified using two primer
 sets. The IN16STK-mod primers, which follow the previously described
 methods and allow matching to dietary items, and the LCO1490/HC02198
 primers, which target the animal barcode region of cytochrome oxidase I
 (COI). PCR conditions used for the latter are described in previous
 studies. Barcode sequencing was undertaken to assist with taxonomic
 assignments. Bioinformatics filtering
 Sequences were bioinformatically processed using the OBITtools
 software, which in summary performs the alignment of the sequences
 obtained and the filtering of PCR/sequencing errors to obtain molecular
 operational taxonomic units (MOTUs; see details in Supplementary
 material). The LULU R package was used to combine MOTUs with &gt;84%
 similarity, removing remaining errors. Based on PCR blank counts, PCR
 products with less than 2000 reads were removed and within the remaining
 samples, MOTUs representing less than 1% of the total reads of that PCR
 product were also excluded. The remaining MOTUs were then compared against
 the NCBI Nucleotide Database and our reference collection, using the
 BLAST+ software, and classified to the lowest possible taxonomic rank. If
 a sequence matched more than one species with equal similarity values all
 belonging to the same genus, a genus-level assignment was considered. When
 a sequence matched more than one species or genus with similar
 probabilities, only species or genera known to occur in the archipelago
 were considered, otherwise a higher ranking would be attributed (e.g.,
 family). If different MOTUs corresponded to the same taxon, a number would
 be attributed to each one of them (e.g., Araneae_1; Araneae_2). MOTUs
 detected in extraction blanks, considered as contaminations or of sampling
 baits (banana and mango) were removed from the corresponding batch of
 samples. Data analysis The
 frequencies of occurrence (FO) of each of the three diet item groups
 (plant, invertebrate, vertebrate) were estimated for the sympatric
 populations from Fogo (Cd.F.s and Cv.F.s from hereon) and Cima Islet
 (Cd.c.s and Cv.c.s from hereon), and the allopatric population of Cd from
 Fogo (Cd.F.a from hereon). To test for differences in proportions of each
 diet item group between populations, FOs were compared using
 two-proportion z-tests with the function <em>prop.test</em> in
 R software version 4.3.2. Family-level identification
 was used for the analysis due to the inability to achieve species-level
 taxonomic resolution for several MOTUs. To visualize the dietary networks
 at the family level for the five populations, bipartite networks were
 generated using the function <em>plotWeb</em> from bipartite
 package v2.19. Adjacency plots were calculated at the order level using
 the function <em>metaComputeModules</em> and visualized using
 <em>plotModuleWeb</em>. Rarefaction curves were used to
 compare resource richness at the family level among the five populations.
 Richness values were estimated for the double of species with the lower
 sample size and differences were considered significant if the 95%
 confidence intervals did not overlap. The
 functions <em>iNEXT</em> and <em>ggiNEXT </em>from
 the INEXT package were used to perform and visualize this analysis,
 respectively. Dietary niche overlap within the two
 sympatric populations (Cd.F.s vs. Cv.F.s and Cd.c.s vs. Cv.c.s) was
 calculated using Pianka’s index at the family level. This index ranges
 from 0 to 1, where 0 indicates no common resources and 1 indicates
 complete resource overlap. To test whether the dietary niche overlap
 differed significantly from what would be expected by chance, null models
 were generated based on the RA3 randomization algorithm, generating 10,000
 null matrices that were compared to observed data, using the EcoSimR
 package. Non-metric multidimensional scaling (NMDS) was used to evaluate
 prey-use differences among populations. Samples were ordinated in a
 two-dimensional space according to diet dissimilarity, using the
 function<em> metaMDS</em> from the vegan package and based on
 a Jaccard distance matrix. Finally, generalised linear
 models (GLMs) were performed to test for significant differences in diet
 composition at the MOTU, family and order levels in the following
 contexts: within sympatric species (Cd.F.s vs. Cv.F.s and Cd.c.s vs.
 Cv.c.s); between species from different islands (Cd.F.s vs. Cd.c.s and
 Cv.F.s vs. Cv.c.s), and between Cd allopatric populations (Cd.F.s vs.
 Cd.F.a). GLMs for multivariate presence/absence data were fitted by
 implementing the <em>manyglm </em>function of the
 <em>mvabund</em> package. The complementary log-log
 distribution provided the best fit and was used for subsequent tests. The
 significance of the GLMs was tested using
 the <em>anova.manyglm</em> function that was then implemented
 with the argument <em>p.uni= 'adjusted'</em> to
 perform univariate tests for identifying prey items responsible for
 differences in diet composition between species and among
 populations.
description: # Data from: Smaller islands, bigger appetites: evolutionary strategies of
 insular endemic skinks
 [https://doi.org/10.5061/dryad.zpc866thn](https://doi.org/10.5061/dryad.zpc866thn) ## Description of the data and file structure **Data description** R scripts and corresponding data used to compare dietary composition (obtained using DNA metabarcoding methods) and body morphology of sympatric and allopatric populations of the endemic Cabo Verdean large and small skinks, *Chioninia vaillantii* and *Chioninia delalandii*. Results showed significant differences in diet and linear measurements between species and populations. ### Files and variables **File list (files found within Data_CdCv.zip)** CdCv_morpho_data.csv Morpho_CdCv_1.0.Rmd Diet_database_Cd-Cv.xlsx Diet_CdCv_1.0.qmd **File descriptions** *Morpho_CdCv_1.0.Rmd* – This file is an R markdown containing the code to reproduce the morphological analyses performed in the paper cited above, including annotations describing the steps. CdCv_morpho_data.csv – This file contains the morphological measures of all populations used for the statistical comparison. ·        Code – individual code assigned to each sample during fieldwork. ·        Group – codes of the five *Chioninia* populations (Cd, *Chioninia delalandii*; Cv, *Chioninia vaillantii*; F, Fogo Island; c, Cima Islet; s, sympatric; a, allopatric). ·        Species – indicates the two species: Cd, *Chioninia delalandii*; Cv, *Chioninia vaillantii*. ·        Island – code of the sampled islands: F, Fogo Island; c, Cima Islet. ·        Code_diet – individual code assigned during laboratory processing. ·        Loc – location of the groups within the islands: s, sympatric; a, allopatric. ·        Sex – sex of the individuals: F, female; M, male; unk, undetermined. ·        Weight – presented in grams ·        SVL – snout-vent length in millimetres ·        TL – trunk length in millimetres ·        HL – head length in millimetres ·        HW – head width in millimetres ·        HH – head height in millimetres ·        ES – eye-snout length in millimetres ·        EE – eye-ear distance in millimetres ·        ED – eye diameter in millimetres ·        FL – front limb length in millimetres ·        HLL – hind limb length in millimetres . n/a – not available data Diet_CdCv_1.0.qmd – This file is an R markdown containing the code to reproduce the dietary composition analyses performed in the paper cited above, including annotations describing the steps. *Diet_database_Cd-Cv.xlsx –* This file contains the dietary composition data of all populations generated using DNA metabarcoding, used for the statistical comparison. ·        Id – identification code of each sequence generated in the sequencing process. ·        haplotype – individual code assigned to each sequence. ·        seq_length – length of each sequence in base pairs. ·        Sequence – nucleotide sequence. ·        hap_total_count – total number of reads for each haplotype ·        status_1 – filter indicating to delete or maintain (“ok”) sequences. Sequences were excluded if the total read count (seq_reads) did not represent 1% of the total reads of the sample (sample_reads). ·        sample_status – filter by the distribution of reads per sample. Samples were excluded based on the blank reads. ·        Primer – genetic marker used for each sequence: 16S, mitochondrial 16S gene; trnl, short P6-loop of chloroplast trnL (UAA) gene; 12S,V5-loop within the mitochondrial 12S gene. ·        seq_reads – total read count for each sequence. ·        sample_reads – total read count for each sample. ·        sample_name – code of each sample, attributed during laboratory processing. ·        sample_name2 – code of each sample, used during bioinformatics and statistical analysis. ·        Island – code of the sampled islands: F, Fogo Island; c, Cima Islet. ·        location – location of the groups within the islands: s, sympatric; a, allopatric. ·        group1 – codes of the *Chioninia* populations excluding island indicative (Cd, *Chioninia delalandii*; Cv, *Chioninia vaillantii*; s, sympatric; a, allopatric) ·        group2 – codes of the five Chioninia populations including island indicative(Cd, *Chioninia delalandii*; Cv, *Chioninia vaillantii*; F, Fogo Island; c, Cima Islet; s, sympatric; a, allopatric). ·        Sex – sex of the individuals: F, female; M, male; 0, undetermined; J, juvenile. ·        Sp – indicates the two species: Cd, *Chioninia delalandii*; Cv, *Chioninia vaillantii*. ·        Date – collection date of the samples. ·        Final_ID – final taxonomic identification of each dietary haplotype. ·        Taxonomic classification of each dietary haplotype: o   Phylum o   Class o   Order o   Family o   Genus o   Species ·        Id_level – higher taxonomic classification reached for each haplotype. ·        Match – the percentage of the match during blast of the sequences. ·        Diet – if the dietary item is considered part of the diet (“yes”) or contamination (“no”). Items classified as “no” were excluded from the statistical analysis. ## Code/software Analyses were performed in R software version 4.3.2. Scripts including the description of loaded packages used to run files are provided in the uploaded files.