The use of animal translocation as a conservation strategy is growing, and translocations involve founder events. Because resultant populations pass through genetic bottlenecks, it is anticipated that they should maintain less genetic variation than their source populations. In addition to the reduced evolutionary potential associated with decreased genetic variation, founder events can lead to increased expression of the genetic load due to inbreeding and give rise to inbreeding depression. Here, we describe the effects of serial translocations on the genetic diversity of critically endangered Anegada Rock Iguanas (Cyclura pinguis) in the British Virgin Islands in the 1980s and 1990s. Very few founders were involved (N = 8, N = 4) but they were extremely fecund and census sizes in these translocated iguana populations increased quickly. This implies a genetic paradox because these translocations were successful from a demographic perspective despite the fact that they are inbred. To quantify genetic variation in these bottlenecked populations, blood samples were collected from the source population and two translocated populations. Genetic diversity at 21 microsatellite loci was significantly lower in the translocated populations than that in the source population. However, statistical methods designed to detect molecular signatures of genetic bottlenecks failed to do so. Estimates of internal relatedness by age class suggest that inbreeding depression may be elevated in the translocated populations, likely reflecting the small initial population sizes. Anecdotally, our work shows that translocations may result in subtle genetic erosion, even when census size indicates demographic success.

Galápagos iguanas are a monophyletic group endemic to the Galápagos archipelago, comprising the marine iguana Amblyrhynchus cristatus and three species of land iguanas: Conolophus subcristatus, C. pallidus and C. marthae. The biogeographic history of the land species in relation to their current distributions remains uncertain, in particular the origins of C. marthae, which is restricted to a small area of the northern part of Isabela Island. The classification of C. pallidus as a separate species has also been debated. We analyzed DNA sequences (RADseq) to reconstruct demographic histories of selected local populations of all Galápagos iguana species and estimate their divergence times within a multispecies coalescent framework. Our results indicate an early date for the colonization of Galápagos by iguanas, relative to island formation, at ca. 10 Mya, and support a recent split of C. marthae via allopatric speciation, after the emergence of Isabela Island, at ca. 0.57 Mya. We find contrasting demographic histories in C. marthae and the syntopic population of C. subcristatus, suggesting competitive interaction between these species. We also confirm that the divergence of C. pallidus from C. subcristatus is recent (0.09 Mya) and close in time to the split between populations of C. subcristatus from different islands. Our genetic data support recent census estimates indicating a relatively small current effective population size (Ne) in all the studied populations. Our findings shed light on the evolutionary history of Galápagos iguanas and emphasize the need for targeted conservation strategies.

Short tandem repeat (STR) variation is rarely explored as a contributor to adaptive evolution. An intriguing mechanism involving STRs suggests that STRs function as “tuning knobs” of adaptation whereby stepwise changes in STR allele length have stepwise effects on phenotypes. Previously, we tested the predictions of the “tuning knob” model at the gene expression level by conducting an RNA-Seq experiment on natural populations of common sunflower (Helianthus annuus L.) transecting a well-defined cline from Kansas to Oklahoma. We identified 479 STRs with significant allele length effects on gene expression (eSTRs). In this study, we expanded the range to populations further north and south of the focal populations and used a targeted approach to study the relationship between STR allele length and gene expression in five selected eSTRs. Seeds from 96 individuals from six natural populations of sunflower from Nebraska and Texas were grown in a common garden. The individuals were genotyped at the five eSTRs, and gene expression was quantified with qRT-PCR. Linear regression models identified that eSTR length in comp26672 was significantly correlated with gene expression. Further, the length of comp26672 eSTR was significantly correlated with latitude across the range from Nebraska to Texas. The eSTR locus comp26672 was located in the CHUP1 gene, a gene associated with chloroplast movement in response to light intensity, which suggests a potential adaptive role for the eSTR locus. Collectively, our results from this targeted study show a consistent relationship between allele length and gene expression in some eSTRs across a broad geographical range in sunflower and suggest that some eSTRs may contribute to adaptive traits in common sunflower.

Lizards within the Iguana iguana species complex are among the most common reptilian pets, with the widest natural geographic range among iguanids. Deep phylogenetic divergence distinguishes multiple mitochondrial clades, and several taxonomic changes have recently been proposed. These small populations, typically island endemics, are threatened by numerous factors, including the international pet trade. Recent investigations reveal the absence of required CITES permits for lawful export of animals, providing evidence of ongoing illegal trade. Additional monitoring of trade in iguanas can be achieved through the application of forensic molecular techniques. In this study, two captive melanistic iguanas were genotyped for molecular markers for which geographic distributions of alleles have been established. Mitochondrial sequencing indicates that both animals carry a haplotype known to originate from the islands of Saba and Montserrat, populations taxonomically proposed to be Iguana melanoderma. Genotypes at 15 microsatellite loci are equally consistent with this origin, given the results of a principal component analysis. This first forensic genetic assessment within the extensive I. iguana pet trade highlights the presence of illegal activity. The need for additional forensic assessments of pet-trade iguanas is evident, especially given that their value is driven by variety and rarity, which is further intensified by recent taxonomic changes.

The genus Cyclura includes nine extant species and six subspecies of West Indian Rock Iguanas and is one of the most imperiled genera of squamate reptiles globally. An understanding of species diversity, evolutionary relationships, diversification, and historical biogeography in this group is crucial for implementing sound long-term conservation strategies. We collected DNA samples from 1 to 10 individuals per taxon from all Cyclura taxa (n = 70 ingroup individuals), focusing where possible on incorporating individuals from different populations of each species. We also collected 1-2 individuals from each of seven outgroup species of iguanas (Iguana delicatissima; five Ctenosaura species) and Anolis sagrei (n = 12 total outgroup individuals). We used targeted genomic sequence capture to isolate and to sequence 1,872 loci comprising of 687,308 base pairs (bp) from each of the 82 individuals from across the nuclear genome. We extracted mitochondrial reads and assembled and annotated mitogenomes for all Cyclura taxa plus outgroup species. We present well-supported phylogenomic gene tree/species tree analyses for all extant species of Cyclura using ASTRAL-III, SVDQuartets, and StarBEAST2 methods, and discuss the taxonomic, biogeographic, and conservation implications of these data. We find a most recent common ancestor of the genus 9.91 million years ago. The earliest divergence within Cyclura separates C. pinguis from a clade comprising all other Cyclura. Within the latter group, a clade comprising C. carinata from the southern Lucayan Islands and C. ricordii from Hispaniola is the sister taxon to a clade comprising the other Cyclura. Among the other Cyclura, the species C. cornuta and C. stejnegeri (from Hispaniola and Isla Mona) form the sister taxon to a clade of species from Jamaica (C. collei), Cuba and Cayman Islands (C. nubila and C. lewisi), and the eastern (C. rileyi) and western (C. cychlura) Lucayan Islands. Cyclura cychlura and C. rileyi form a clade whose sister taxa are C. nubila and C. lewisi. Cyclura collei is the sister taxon to these four species combined.

Microsatellites, also known as short tandem repeats (STRs), have long been considered non-functional, neutrally evolving regions of the genome. Recent findings suggest that they can function as drivers of rapid adaptive evolution. Previous work on the common sunflower identified 479 transcribed microsatellites where allele length significantly correlates with gene expression (eSTRs) in a stepwise manner. Here, a population genetic approach is used to test whether eSTR allele length variation is under selection. Genotypic variation among and within populations at 13 eSTRs was compared with that at 19 anonymous microsatellites in 672 individuals from 17 natural populations of sunflower from across a cline running from Saskatchewan to Oklahoma (distance of approximately 1600 km). Expected heterozygosity, allelic richness, and allelic diversity were significantly lower at eSTRs, a pattern consistent with higher relative rates of purifying selection. Further, an analysis of variation in microsatellite allele lengths ( lnRV ), and heterozygosities ( lnRH ), indicate recent selective sweeps at the eSTRs. Mean microsatellite allele lengths at four eSTRs within populations are significantly correlated with latitude consistent with the predictions of the tuning-knob model which predicts stepwise relationships between microsatellite allele length and phenotypes. This finding suggests that shorter or longer alleles at eSTRs may be favored in climatic extremes. Collectively, our results imply that eSTRs are likely under selection and that they may be playing a role in facilitating local adaptation across a well-defined cline in the common sunflower.

Three subspecies of Northern Bahamian Rock Iguanas, Cyclura cychlura, are currently recognized: C. c. cychlura, restricted to Andros Island, and C. c. figginsi and C. c. inornata, native to the Exuma Island chain. Populations on Andros are genetically distinct from Exuma Island populations, yet genetic divergence among populations in the Exumas is inconsistent with the 2 currently recognized subspecies from those islands. The potential consequences of this discrepancy might include the recognition of a single subspecies throughout the Exumas rather than 2. That inference also ignores evidence that populations of C. cychlura are potentially adaptively divergent. We compared patterns of population relatedness in a three-tiered host-parasite system: C. cychlura iguanas, their ticks (genus Amblyomma, preferentially parasitizing these reptiles), and Rickettsia spp. endosymbionts (within tick ectoparasites). Our results indicate that while C. c. cychlura on Andros is consistently supported as a separate clade, patterns of relatedness among populations of C. c. figginsi and C. c. inornata within the Exuma Island chain are more complex. The distribution of the hosts, different tick species, and Rickettsia spp., supports the evolutionary independence of C. c. inornata. Further, these patterns are also consistent with two independent evolutionarily significant units within C. c. figginsi. Our findings suggest coevolutionary relationships between the reptile hosts, their ectoparasites, and rickettsial organisms, suggesting local adaptation. This work also speaks to the limitations of using neutral molecular markers from a single focal taxon as the sole currency for recognizing evolutionary novelty in populations of endangered species.

Abstract Eurasian watermilfoil (Myriophyllum spicatum) is an invasive submersed macrophyte that has infested many waters in North America since its introduction. Eurasian watermilfoil has the ability to alter the structure and function of the ecosystems that it invades. Eurasian watermilfoil was first surveyed in the Clark Fork River, MT in 2008, alongside the native northern watermilfoil (Myriophyllum sibiricum). Three reservoirs (Cabinet Gorge, Noxon Rapids and Thompson Falls) on the lower Clark Fork River had entire lake surveys conducted using the point intercept method. Morphological data from these surveys showed that only Eurasian watermilfoil and northern watermilfoil were present during the time of the surveys in 2008. The results of the morphological identification were supported by molecular identification at three different laboratories. In 2015, a genetic survey of watermilfoil species was conducted on the Noxon Rapids Reservoir and found that hybrid watermilfoil (Myriophyllum spicatum × sibiricum) was present. This hybridization event poses a number of issues for aquatic plant management. Hybrid watermilfoil is much more difficult to identify morphologically than its parent types. The hybrid is also more invasive and may be differentially susceptible to some herbicides than the parental type Eurasian watermilfoil.

Radio telemetry is a commonly used technique in conservation biology and ecology, particularly for studying the movement and range of individuals and populations. Traditionally, most radio telemetry work is done using handheld directional antennae and either direction‐finding and homing techniques or radio‐triangulation techniques. Over the past couple of decades, efforts have been made to utilize unmanned aerial vehicles to make radio‐telemetry tracking more efficient, or cover more area. However, many of these approaches are complex and have not been rigorously field‐tested. To provide scientists with reliable quality tracking data, tracking systems need to be rigorously tested and characterized. In this paper, we present a novel, drone‐based, radio‐telemetry tracking method for tracking the broad‐scale movement paths of animals over multiple days and its implementation and deployment under field conditions. During a 2‐week field period in the Cayman Islands, we demonstrated this system's ability to localize multiple targets simultaneously, in daily 10 min tracking sessions over a period of 2 weeks, generating more precise estimates than comparable efforts using manual triangulation techniques.

Mutations that provide environment-dependent selective advantages drive adaptive divergence among species. Many phenotypic differences among related species are more likely to result from gene expression divergence rather than from non-synonymous mutations. In this regard, cis-regulatory mutations play an important part in generating functionally significant variation. Some proposed mechanisms that explore the role of cis-regulatory mutations in gene expression divergence involve microsatellites. Microsatellites exhibit high mutation rates achieved through symmetric or asymmetric mutation processes and are abundant in both coding and non-coding regions in positions that could influence gene function and products. Here we tested the hypothesis that microsatellites contribute to gene expression divergence among species with 50 individuals from five closely related Helianthus species using an RNA-seq approach. Differential expression analyses of the transcriptomes revealed that genes containing microsatellites in non-coding regions (UTRs and introns) are more likely to be differentially expressed among species when compared to genes with microsatellites in the coding regions and transcripts lacking microsatellites. We detected a greater proportion of shared microsatellites in 5′UTRs and coding regions compared to 3′UTRs and non-coding transcripts among Helianthus spp. Furthermore, allele frequency differences measured by pairwise FST at single nucleotide polymorphisms (SNPs), indicate greater genetic divergence in transcripts containing microsatellites compared to those lacking microsatellites. A gene ontology (GO) analysis revealed that microsatellite-containing differentially expressed genes are significantly enriched for GO terms associated with regulation of transcription and transcription factor activity. Collectively, our study provides compelling evidence to support the role of microsatellites in gene expression divergence.

The decision of females to nest communally has important consequences for reproductive success. While often associated with reduced energetic expenditure, conspecific aggregations also expose females and offspring to conspecific aggression, exploitation, and infanticide. Intrasexual competition pressures are expected to favor the evolution of conditional strategies, which could be based on simple decision rules (i.e., availability of nesting sites and synchronicity with conspecifics) or on a focal individual's condition or status (i.e., body size). Oviparous reptiles that reproduce seasonally and provide limited to no postnatal care provide ideal systems for disentangling social factors that influence different female reproductive tactics from those present in offspring-rearing environments. In this study, we investigated whether nesting strategies in a West Indian rock iguana, Cyclura nubila caymanensis, vary conditionally with reproductive timing or body size, and evaluated consequences for nesting success. Nesting surveys were conducted on Little Cayman, Cayman Islands, British West Indies for four consecutive years. Use of high-density nesting sites was increasingly favored up to seasonal nesting activity peaks, after which nesting was generally restricted to low-density nesting areas. Although larger females were not more likely than smaller females to nest in high-density areas, larger females nested earlier and gained access to priority oviposition sites. Smaller females constructed nests later in the season, apparently foregoing investment in extended nest defense. Late-season nests were also constructed at shallower depths and exhibited shorter incubation periods. While nest depth and incubation length had significant effects on reproductive outcomes, so did local nest densities. Higher densities were associated with significant declines in hatching success, with up to 20% of egg-filled nests experiencing later intrusion by a conspecific. Despite these risks, nests in high-density areas were significantly more successful than elsewhere due to the benefits of greater chamber depths and longer incubation times. These results imply that communal nest sites convey honest signals of habitat quality, but that gaining and defending priority oviposition sites requires competitive ability.

West Indian rock iguanas (genus Cyclura) represent the most endangered group of lizards in the world, with most taxa restricted to few or single islands. In addition to distribution and availability of resources, factors influencing optimal use of insular space in these large vertebrates may include a combination of social, ontogenetic, and long-term demographic factors. Life stage-specific dispersal and habitat use patterns have previously been characterized in Cyclura, but assessments across the lifespan are lacking. This study evaluates shifting patterns of movement in a single taxon, Cyclura nubila caymanensis. We used mark-recapture and radio tracking simultaneously to assess age- and sex-dependent variation in spatial ecology. Evidence for prolonged site fidelity among adults was consistent with territoriality-modulating space use in this long-lived taxon. Over 5-10 yr, some females were never resighted or recaptured outside of an 80-m perimeter. A notable exception was that gravid females migrated 1.17 +/- 0.91 km to utilize coastal areas during the nesting season. In contrast, neonates dispersed farther (2.32 +/- 2.26 km) and their trajectories were often nonlinear. Radio-tracked brother-sister pairs exhibited no tendency to disperse together during their first 4 wk, settling into final known locations at 4 pairwise distances of 2.16 +/- 1.74 km. Annual net displacement remained high over the first 1-3 yr of life. Our data are consistent with studies of related taxa wherein individual spatial requirements vary with population density and island size. Notably, panmixis of small island iguana populations appears largely driven by pre-reproductive movements.

Microsatellites are common in genomes of most eukaryotic species. Due to their high mutability, an adaptive role for microsatellites has been considered. However, little is known concerning the contribution of microsatellites towards phenotypic variation. We used populations of the common sunflower (Helianthus annuus) at two latitudes to quantify the effect of microsatellite allele length on phenotype at the level of gene expression. We conducted a common garden experiment with seed collected from sunflower populations in Kansas and Oklahoma followed by an RNA-Seq experiment on 95 individuals. The effect of microsatellite allele length on gene expression was assessed across 3,325 microsatellites that could be consistently scored. Our study revealed 479 microsatellites at which allele length significantly correlates with gene expression (eSTRs). When irregular allele sizes not conforming to the motif length were removed, the number of eSTRs rose to 2,379. The percentage of variation in gene expression explained by eSTRs ranged from 1%-86% when controlling for population and allele-by-population interaction effects at the 479 eSTRs. Of these eSTRs, 70.4% are in untranslated regions (UTRs). A gene ontology (GO) analysis revealed that eSTRs are significantly enriched for GO terms associated with cis- and trans-regulatory processes. Our findings suggest that a substantial number of transcribed microsatellites can influence gene expression.

In natural populations susceptible to inbreeding depression, behaviors such as female promiscuity and disassortative mating may enhance the production of outbred progeny and help maintain genetic variation at the population-level. However, empirical tests of such hypotheses have largely focused on mating systems in which female choice is known to play a large role. In insular reptile populations, cryptic choice may be important for overcoming constraints on precopulatory choice and diversifying breeder representation. We carried out pedigree reconstructions of 50 clutches of critically endangered Cyclura nubila caymanensis (Sauria: Iguanidae) to investigate the prevalence and efficacy of strategies theorized to optimize genetic compatibility among mate pairs. We found that females mating disassortatively and multiply, but not with respect to male heterozygosity, tended to produce more heterozygous offspring on average. Pair relatedness also had a negative effect on hatching success, whereas additional sires positively influenced clutch size. Despite evidence for direct and indirect benefits, females did not mate with more outbred or genetically dissimilar males than expected by chance. Our data imply strong reproductive skew among males, with geographic proximity and body size largely predicting siring success. Multiple-paternity occurred in a minimum of 38% of clutches. Paradoxically, females were not more promiscuous when confronted with more males, as demonstrated at a site supporting high local densities. Our data imply that while female-mating behaviors have the potential to confer adaptive benefits in this small population, the trajectory of mating system evolution may be largely constrained by sexual conflict.

Inbreeding depression, though challenging to identify in nature, may play an important role in regulating the dynamics of small and isolated populations. Conversely, greater expression of genetic load can enhance opportunities for natural selection. Conditional expression concentrates these opportunities for selection and may lead to failure of detection. This study investigates the possibility for age-dependent expression of inbreeding depression in a critically endangered population of rock iguanas, Cyclura nubila caymanensis. We employ heterozygote-fitness correlations to examine the contributions of individual genetic factors to body size, a fitness-related trait. Nonsignificant reductions in homozygosity (up to 7%) were detected between neonates and individuals surviving past their first year, which may reflect natural absorption of inbreeding effects by this small, fecund population. The majority of variation in neonate body size was attributed to maternal or environmental effects (i.e., clutch identity and incubation length); however, heterozygosity across 22 microsatellite loci also contributed significantly and positively to model predictions. Conversely, effects of heterozygosity on fitness were not detectable when adults were examined, suggesting that inbreeding depression in body size may be age dependent in this taxon. Overall, these findings emphasize the importance of taking holistic, cross-generational approaches to genetic monitoring of endangered populations.

The Lesser Antillean iguana, Iguana delicatissima Laurenti 1768, is one of the most endangered vertebrate taxa in the West Indies. This species faces significant threats, including introgressive hybridization with the introduced congener Iguana iguana. We deploy a combination of off-target sequence capture obtained from Illumina® reads and targeted Sanger reads to assemble the mitochondrial genome of I. delicatissima. The mitogenome is 16,616 bp in length and is comprised of 13 protein-coding genes, two ribosomal subunits (rRNAs), 22 transfer RNAs, and a control region. Gene order is identical to that of congener I. iguana and other closely related taxa, absent of any tandem repeat regions. We show the phylogenetic utility of the mitogenome with a maximum-likelihood analysis, which yields a topology concordant with previous studies of iguanine taxa. We are hopeful that this genomic resource will be useful in further informing applied conservation and management for this critically endangered species.

Abstract Cyclura ricordii is an endemic iguana from Hispaniola Island and is threatened on the IUCN Red List. The main threats are predation by introduced mammals, habitat destruction, and hunting pressure. The present study focused on two nesting sites from Pedernales Province in the Dominican Republic. The hypothesis that natal philopatry influences dispersal and nest‐site selection was tested. Monitoring and sampling took place in 2012 and 2013. Polymorphic markers were used to evaluate whether natal philopatry limits dispersal at multiple spatial scales. Ripley's K revealed that nests were significantly clustered at multiple scales, when both nesting sites were considered and within each nesting site. This suggests a patchy, nonrandom distribution of nests within nest sites. Hierarchical AMOVA revealed that nest‐site aggregations did not explain a significant portion of genetic variation within nesting sites. However, a small but positive correlation between geographic and genetic distance was detected using a Mantel's test. Hence, the relationship between geographic distance and genetic distance among hatchlings within nest sites, while detectable, was not strong enough to have a marked effect on fine‐scale genetic structure. Spatial and genetic data combined determined that the nesting sites included nesting females from multiple locations, and the hypothesis of “natal philopatry” was not supported because females nesting in the same cluster were no more closely related to each other than to other females from the same nesting site. These findings imply that nesting aggregations are more likely associated with cryptic habitat variables contributing to optimal nesting conditions.

The mechanisms by which natural populations generate adaptive genetic variation are not well understood. Some studies propose that microsatellites can function as drivers of adaptive variation. Here, we tested a potentially adaptive role for transcribed microsatellites with natural populations of the common sunflower (Helianthus annuus L.) by assessing the enrichment of microsatellites in genes that show expression divergence across latitudes. Seeds collected from six populations at two distinct latitudes in Kansas and Oklahoma were planted and grown in a common garden. Morphological measurements from the common garden demonstrated that phenotypic variation among populations is largely explained by underlying genetic variation. An RNA-Seq experiment was conducted with 96 of the individuals grown in the common garden and differentially expressed (DE) transcripts between the two latitudes were identified. A total number of 825 DE transcripts were identified. DE transcripts and nondifferentially expressed (NDE) transcripts were then scanned for microsatellites. The abundance of different motif lengths and types in both groups were estimated. Our results indicate that DE transcripts are significantly enriched with mononucleotide repeats and significantly depauperate in trinucleotide repeats. Further, the standardized mononucleotide repeat motif A and dinucleotide repeat motif AG were significantly enriched within DE transcripts while motif types, C, AT, ACC and AAC in DE transcripts, are significantly differentiated in microsatellite tract length between the two latitudes. The tract length differentiation at specific microsatellite motif types across latitudes and their enrichment within DE transcripts indicate a potential functional role for transcribed microsatellites in gene expression divergence in sunflower.

Abstract Inbreeding depression can have alarming impacts on threatened species with small population sizes. Assessing inbreeding has therefore become an important focus of conservation research. In this study, heterozygosity–fitness correlations (HFCs) were measured by genotyping 7 loci in 83 adult and 184 hatchling Lesser Antillean Iguanas, Iguana delicatissima, at a communal nesting site in Dominica to assess the role of inbreeding depression on hatchling fitness and recruitment to the adult population in this endangered species. We found insignificant correlations between multilocus heterozygosity and multiple fitness proxies in hatchlings and adults. Further, multilocus heterozygosity did not differ significantly between hatchlings and adults, which suggests that the survivorship of homozygous hatchlings does not differ markedly from that of their heterozygous counterparts. However, genotypes at two individual loci were correlated with hatching date, a finding consistent with the linkage between specific marker loci and segregating deleterious recessive alleles. These results provide only modest evidence that inbreeding depression influences the population dynamics of I. delicatissima on Dominica.