Austrolebias (Cyprinodontiformes: Rivulidae) represents a specious group of taxa following annual life cycles in the neotropical ichthyofauna. They live in temporary ponds and each generation must be completed in a few months, depending on environmental stochasticity. Annual fish survive the dry season through diapausing eggs buried in the substrate of these ponds. A hypothesized bimodal hybrid zone between two taxa of the genus, A. charrua and A. reicherti from Dos Patos Merin lagoon system, was recently proposed based on genetics and morphological analyses. However, hundreds of additional nuclear molecular markers should be used to strongly support this hypothesized bimodal pattern. In the present paper, we conducted RNA-seq-based sequencing of the transcriptomes from pools of individuals of A. charrua, A. reicherti and their putative natural hybrids from the previously characterized hybrid zone. As a result, we identified a set of 111,725 SNP (single nucleotide polymorphism) markers, representing presumably fixed allelic differences among the two species. The present study provided the first panel of 106 SNP markers as a single diagnostic multiplex assay and validated their capacity to reconstruct the patterns of the hybrid zone between both taxa. These nuclear markers combined with Cytb gene and morphological analyses detected a population structure in which some groups among the hybrid swarms showed different level of introgression towards one or the other parental species according to their geographic distribution. High-quality transcriptomes and a large set of gene-linked SNPs should greatly facilitate functional and population genomics studies in the hybrid zone of these endangered species.The genus  Hybrid zones are remarkable systems for understanding the origin and persistence of species diversity. They constitute reservoirs of polymorphism and are key to the maintenance of biodiversity . IdentifXiphophorus birchmanni and X. malinche in the Sierra Madre Oriental of eastern Mexico, where natural hybrids were found and characterized [Austrolebias (Cyprinodontiformes: Rivulidae): A. charrua and A. reicherti was characterized for the first time [Austrolebias genus represents a specious group of taxa with an annual life cycle among neotropical ichthyofauna, in South America. They live in temporary ponds and each generation must be completed in a few months, depending on environmental stochasticity. Annual fish survive the dry season through diapausing eggs buried in the substrate of these temporary ponds. A spectacular species radiation has been reported for Austrolebias from the Pampas [Austrolebias was associated with an unusually large genome size increase within the Aplocheiloidei, with an average DNA content of about 5.95 ± 0.45 picograms per diploid cell  [Austrolebias genomes [A. charrua and A. reicherti taxa included microsatellite loci, one mitochondrial marker and morphological studies [2 within the Patos-Merin drainage system (DMS). Previous phylogeographic studies had proposed scenarios of past allopatric fragmentation and range expansion involving secondary contact between both taxa followed by genetic and morphological divergence [A. charrua and A. reicherti was estimated to have occurred since 100,000 years ago (late Pleistocene). In fact, hybrid zones are traditionally assumed to form via secondary contact after geographic divergence in allopatry [Given the fact that the neotropical ichthyofauna is one of the most abundant and diverse on Earth, with more than 5600 species , it is scterized . More rerst time . Austrole Pampas ,11, where Pampas ,13,14. T2.98 pg) ,16 and a genomes ,18 were  genomes ,20. The vergence . Moreovellopatry ,23,24.A. charrua and A. reicherti, a hypothetic bimodal pattern was proposed. Hybrid swarms were predominantly individuals genetically similar to one or another of the parental genotypes, with no F1 hybrid progeny. In fact, some of the admixed individuals were morphologically not distinguishable from pure individuals of one or the other parental species [In the hybrid zone between  species . As sugg species  many add species , studies species . AdditioA. charrua and A. reicherti. To accomplish this objective, the sampling area was extended, at the genomic level numerous nuclear molecular markers were obtained and cytochrome b (Cytb) gene and morphometric analyses were included. To avoid problems with the large genome size of these diploid species and its high level of redundancy, we conducted RNA-Seq based sequencing of the transcriptomes from pools of individuals of A. charrua, A. reicherti and their putative natural hybrids to obtain more than 100 single nucleotide polymorphisms (SNPs) from expressed sequences. This methodological strategy was proposed by De Wit et al. [In this work, we attempted to address the open question about the existence of a possible bimodal pattern and levels of introgression involved in the hybrid zone between t et al.  and it wt et al. . High-quA. charrua and A. reicherti [A. charrua (pond CH66) and A. reicherti (pond CH43) and 6 of their putative hybrids detected in geographically intermediate ponds  on the southern and northern margins of the Corrales del Parao stream in the DMS  , that guarantees more than 120 million reads per sample  . Total R females . For tesr sample .A. charrua [http://www.htslib.org/) [A. charrua [Before the analysis, raw reads were trimmed using Trimmomatic 0.38  by remov charrua  using th charrua , and seaib.org/) . Mapping charrua .A. charrua and A. reicherti . SNPs that showed the consensus base  in one species, and the alternative allele in the other one, with both alleles present in the hybrid population read file , were carried forward as putative fixed-allele diagnostic SNPs.Since we used pooled samples, we focused particularly on the identification of SNPs with fixed or nearly fixed allelic differences between A. charrua from pond CH66 (N = 10), of A. reicherti from CH43 (N = 14) and from putative hybrid ponds of CH60 (N = 21), CH54-61 (N = 12), CHN3 (N = 8), CHN4 (N = 7), CHN6 (N = 12) and CH64 (N = 10),  were employed to validate a subset of 130 among the identified SNPs. Only SNPs with at least a 100 bp flanking region on either side of the polymorphic site were selected for the assay design. The final multiplex was tested on a plate including a subset of 94 DNA samples belonged to parental populations of 10), see . GenomicO), and the expected heterozygosity (He) were calculated for each locus per population and taxon using the GENEPOP 4.2  [IS) [Population genetic analyses included 94 individuals described in the  France) , ARLEQUI France) , and FST France) . GENEPOP France)  was usedce) [IS) . Linkagece) [IS)  and usince) [IS)  for multce) [IS) .The number of individuals per population used in the structure and patterns of hybridization analyses were described in p = 0.05) were obtained performing 1.000 permutations. All these analyses were performed using ARLEQUIN v. 3.5 software package [Considering all 103 polymorphic loci, different hypotheses of grouping as sources of variation were assessed in the analysis of molecular variance (AMOVA). Fst values for pairwise comparisons among the 8 ponds populations and their significant level for genetic differentiation  pure A. reicherti, (iii) F1 hybrid, (iv) F2 hybrid, (v) backcross towards A. charrua (Bx charrua), (vi) backcross towards A. reicherti (Bx reicherti).The number of individuals per population included in the estimates of recent introgression analysis were described in cytochrome b (Cytb) gene included a total of 159 individuals belonging to parental populations of A. charrua from pond CH66 (N = 16), of A. reicherti from CH43 (N = 20) and from putative hybrid ponds of CH60 (N = 35), CH54-61 (N = 21), CHN3 (N = 12), CHN4 (N = 7), CHN6 (N = 37) and CH64 (N = 11), (see Cytb gene was amplified using the CB3-H (5′-GGCAAATAGGAARTATCATTC-3′) and Gludg-L (5′-TGACTTGAARAACCAYCGTTG-3′) primers [2, 0.2 mM each dNTP, 0.5 μM of each primer, 0.5 units of Taq DNA polymerase (Invitrogen) and approximately 100 ng of template DNA. Cycling conditions included an initial denaturation of 3 min at 94 °C, followed by four cycles of: 94 °C for 1 min, 42 °C for 1 min, 72 °C for 1 min, followed by 29 cycles of: 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min and a final extension of 7 min at 72 °C. PCR products were purified and subsequently sequenced in both strand directions, using the same amplification primers, in a Perkin-Elmer ABI Prism 377 Automated Sequencer .The analysis based on the 11), see . Genomic primers . PolymerMultiple sequence alignment was performed by using the ClustalW tool implemented in MEGA v. 7.0  . NucleotCytb sequences was constructed with NETWORK v. 5.0.0.0  [A. charrua, A. reicherti and their putative hybrid populations was assessed using the variance components among hierarchical partitions in the dataset as implemented in the AMOVA analysis [CT), among populations within groups (ΦSC), and among individuals within populations (ΦST), disregarding either their original populations or their groups. The eight populations were ascribed to different groups and different grouping hypotheses were tested.The haplotype network based on England) . Geneticanalysis  using ARanalysis . The Eucanalysis  was usedA. charrua, 22 A. reicherti, and 45 putative hybrids) and 138 females , whereas 23 males and 20 females were included in the general morphological analyses. Specimens were deposited in the Fish Collection (ZVCP) of the Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.Morphological analyses were conducted to confirm the identity of species and their putative hybrids detected by the molecular markers. Specimens analyzed using the morphometric approach comprised 123 males . RNA-Seq data were mapped onto reference transcriptome of A. charrua [A. charrua and A. reicherti. These markers belong to a total of 22,490 contig transcripts. charrua . As a reA. charrua, A. reicherti and hybrids according to transcriptomic analysis and posterior genotyping of sub-samples per population. Remarkably, most loci were fixed in one of the parental and were polymorphic in the other parent and in the hybrid population.Of a subset of the randomly selected 130 SNPs to perform the diagnostic multiplex assay, one failed because of its high dimer potential formation for both forward and reverse primers. Another 5 loci were discarded due to their lower call rates, and 18 SNP loci failed to genotype most of the samples. Finally, the genotype data were generated using a panel of 106 SNP loci, with 44 SNPs called at a 100% success rate, 53 SNP called at 90% and 9 SNP at 70%. These success rates were calculated using 90 of the 94 samples . A. charrua (CH66), A. reicherti (CH43), and from hybrid populations from ponds CH60, CH54-61, CHN3, CHN4, CHN6, CH64 (IS) for each locus per population and taxon. Among 103 polymorphic SNPs, expected heterozygosity (He) estimate ranged from 0.386 in the putative hybrid population of CHN3 to 0.243 in the parental pond CH43 of A. reicherti. Departures from HWE were detected in markers for some populations (CH43 and CH60) although most of these SNP markers were at equilibrium after Bonferroni correction (p = 0.001) (IS) was the highest for all loci in A. reicherti (0.141) whereas A. charrua from CH66 showed the lowest (−0.338), but none of these values were significant after Bonferroni correction (p = 0.001) was detected in three putative hybrid populations between some pair of loci as follows: in pond CH60, between SNP_054 vs. SNP_095; in pond CH54-61, between loci SNP_055 vs. SNP_060; and in pond CHN6, between loci SNP_009 vs. SNP_019, and SNP_019 vs. SNP_050.In order to estimate the genetic diversity parameters, all 106 SNPs were genotyped for polymorphism in 94 samples belonging to parental populations of N6, CH64 . Table S= 0.001) . The intrrection . LinkageA. reicherti (pond CH43), a second group comprised putative hybrid ponds CH64, CH54-61, CHN3, CHN4 a third group included hybrid ponds CHN6 and CH60, and fourth group was integrated by the population from CH66 of A. charrua.AMOVA results in A. reicherti (CH43) to those of A. charrua (CH66), as well as between the first population and populations from ponds CH64, CHN3, CHN4 and CH54-61. On the other hand, the putative hybrid ponds CH60 and CHN6 remained differentiated from A. charrua. The lowest Fst values were detected in ponds CH64 and CHN4 in relation to pond CH54-61, as well as between ponds CH60 and CHN6.Pairwise Fst values among the 8 populations are presented in A. charrua.DAPC in A. charrua and A. reicherti respectively  = −7088.30; K = 4, ΔK = 0.08; Mean Ln(PX|K) = −6991.90; K = 5, ΔK = 43.94; Mean Ln(PX|K) = −6886.96),  = −7315.82), corresponding to the ‘pure’ populations of ectively A. This a886.96), B–D. Unde886.96), B, all thA. charrua in the first pond and toward A. reicherti in the last one. Interestingly, neither of the prior assumptions in NewHybrids detected F1 individuals. Under this analysis, ponds CH54-61, CHN3, CHN4 and CH64 were assigned to the A. charrua-like genotype. Among the hybrid individuals, a uniform prior assumption ; F2 (40.00%); backcrossed toward A. charrua (1.43%) and toward A. reicherti (5.71%).NewHybrids software was used to detect recent hybridization events with a threshold value of 0.5, which showed an efficiency to identify hybrids similar to that of the STRUCTURE analysis. Both Jeffrey and Uniform prior distribution showed the same inference about hybrid categories A, B. Onlsumption A displaysumption B displayCytb sequences from 159 individuals belonging to 8 populations from the three taxa was analyzed in the present work. All sequences were grouped into 29 haplotypes (H1–H29). Haplotype assignation per sample is listed in A matrix of 795-bp of Cytb polymorphism in each of 8 populations in the hybrid zone between A. charrua and A. reicherti from DMS in South America. The highest intra-population sequence distance using K2P model was observed in the hybrid population of pond CHN6 (3.8%) and the lowest (0.1%), in the parental A. reicherti (from pond CH43). The lowest inter-population pairwise K2P sequence divergence was detected between CHN3 and CH64 whereas the highest values were observed inpond CH43 of A. reicherti in relation to A. charrua from pond CH66, and the remaining ponds CH64, CHN3, CHN4, CH54-61 . In paired comparisons, after the Bonferroni correction of p values, only A. charrua exhibited significant differences with A. reicherti , CH60 , and CHN6 . In females, differences among all groups also resulted statistically significant . As observed in males, the only paired comparison with significant differences corresponded to A. charrua in relation to A. reicherti , to CH60 , and to CHN6 .Multivariate analysis of covariance in males generally resulted in significant differences , by a deeper and smaller head, and from CH60 and CHN6 along Root 2 (28%) by a shallower body and an anterior displacement of the anal fin origin  by a deeper body; and, from CH60 and CHN6 along root 2 (26%) by a deeper body and larger head . This hybrid zone was only on the southern margin of the Corrales del Parao stream, a tributary of the Cebollatí River basin in the DMS [A. charrua [reicherti-like [The present work represents a robust contribution to the analysis of the hybrid zone between populations of  America . Our stu the DMS . After 1 charrua . By contrti-like .A. charrua and A. reicherti based on seven microsatellite loci and one mitochondrial marker [STRUCTURE results with K = 2, allowed us to support a probable hypothesis of a bimodal structure for this hybrid zone A. This rl marker . HoweverCytb polymorphism in relation to parental taxa. Additionally, departure from HWE in pond CH60 and the LD detected between some pairs of loci in both ponds could be consistent with their hybrid swarm status. On the other hand, Newhybrid analysis detected that most individuals belonged to F2 ancestry class and some of them corresponded to backcross with A. charrua in pond CH60 whereas almost half of specimens in pond CHN6 evidenced backcross with A. reicherti. All these new analyses corroborated the existence of bidirectional backcrossing between both taxa as was previously reported [One of these groups comprised hybrid ponds CH60 and CHN6, located on both margins of the Corrales del Parao stream. These ponds presented high levels of heterozygosity and reported .A. charrua genome, as supported by Cytb analysis, Newhybrid and STRUCTURE (K = 2) results. On the other hand, Cytb results, AMOVA, DAPC, STRUCTURE (from K = 3 to K = 5), and morphological analyses could suggest that the combination of hybrid genotypes and introgression have generated in this group a new genomic entity, which is different from A. charrua and from the remaining hybrid pondsIn fact, the pairwise Fst comparisons based on the Cytb gene showed a high level of differentiation between this group of populations and A. charrua. In contrast a lowest differentiation in relation to the hybrid pond of CH60 was observed. Additionally, it cannot be ruled out in this group the possible occurrence of past introgression events (or re-colonization by A. charrua-like populations), which could explain their closeness with this parental taxon in some of the implemented analyses.Other putative hybrid ponds  are located in the Southern of the Corrales del Parao stream and around pond CH60. Remarkably, these ponds showed the highest expected heterozygosity values. According to Twyford and Ennos  when intA. charrua genome would be observed.Therefore, in present paper, the ponds CH60 and CHN6 could represent the start of hybridization and introgression processes toward one or another parental species, whereas in the remaining ponds CH54-61, CHN3, CHN4 and CH64 a higher introgression towards the A. charrua in relation to A. reicherti and from individuals of hybrid ponds CH60 and CHN6. In fact, morphological analysis showed evidence of subtle changes in some body characters in males and females of the CH60 and CHN6 ponds in relation to the parental A. charrua. This fact could indicate divergent selection acting in such environments and/or the occurrence of possible character displacement among these taxa. Following Brown and Wilson [Moreover, DAPC and multivariate analysis of covariance in males and females were consistent in detecting statistically significant differences between d Wilson  in the ad Wilson ,60, and d Wilson ,62. TherAustrolebias hybrid zone. In this last case, hybrids of different generations seem to be segregating in more or less nearby ponds depending on rainfall levels and their potential connection.Movement and stability may be plausible scenarios for the hybrid zone because contact among populations of these Austrolebias taxa inhabiting in para/sympatry depend on seasonal flooding, when ponds may collapse in an extensive area. These unstable and heterogeneous environments represent major challenges where the genomic/phenotypic plasticity are required to overcome selective constraints in each temporal pond. Following Abbott , hybrid A. charrua and A. reicherti which has been subject to spatial/temporal movements from the North to South and in the reverse direction, reaching the lowland region near the Atlantic Ocean, in accordance with geological and climatic changes in the recent past [Moreover, previous analyses suggested the long term existence of the hybrid zone between ent past . As a reCytb analysis showed that some haplotypes H_2, H_9, H_10, H_12, H_16 located on the southern margin of this stream were shared by individuals from both ponds CH60 and CHN6 as well as by individuals of A. charrua (CH66) and the remaining ponds . In contrast, the H_17 haplotype situated on the northern margin of this stream was shared by individuals belonging to hybrids ponds CHN6 and CH60 and A. reicherti, whereas H_16 was common among specimens from CHN6 and CH60. This fact constitutes robust support for the recent past movement of the hybrid zone and the separation of the patches through the Corrales del Parao stream. This hybrid zone movement could be associated with major basin transformation. In this sense, some authors have reported the capture of alluvial fans located in the South as part of the Cebollatí river in the DMS drainage, during the Holocene, as a clear demonstration of its recent activity and the extensive reorganization of this basin [The extensive geological instability of this area could explain the separation of the present hybrid zone in two patches (ponds CH60 and CHN6) through the actual geographical barrier constituted by the Corrales del Parao stream. Interestingly, network is basin ,64.Austrolebias genomics, remains extremely challenging. The availability and high-quality transcriptomes and a large set of gene-linked SNPs, should greatly facilitate functional and population genomics studies in these species allowing the identification of traits and processes under selection during introgressive hybridization. At the same time, in present work the 106 validated SNP markers revealed the existence of a peculiar structure that does not support the previously hypothesized bimodal pattern for the hybrid zone between A. charrua and A. reicherti. In fact, all hybrid populations analyzed showed a different degree of introgression towards one or another parental species. Moreover, the nuclear dataset and the morphological analyses showed that hybrid populations are different entities from both parents. Since hybrids’ mosaic genomes aggregating genes from different parental lineages can create novel phenotypes [A. charrua and A. reicherti to preserve in situ biodiversity by increasing the observed phenotypic variation in each temporal pond where these two endangered killifish species inhabit.Mainly due to giant genome size ,17,18, Aenotypes , hybridienotypes . Moreoveenotypes  suggest enotypes . TherefoA. charrua and A. reicherti inhabiting the biosphere reserve and Ramsar sites in South America. The present hybrid zone remains localized and spatially reduced in two patches in both margins of the Corrales del Parao stream in the DMS basin. High-quality transcriptomes and a large set of gene-linked SNPs should greatly facilitate functional and population genomics studies in these endangered species. The analyses performed, such as population genomic based on 106 validated SNPs loci, the Cytb gene and morphology detected a complex population structure in the hybrid zone between A. charrua and A. reicherti that was not concordant with a typical bimodal pattern. All combined methods allowed unmasking the existence of different groups among the hybrid swarms involved in the contact area, with different levels of introgression towards one or other parental taxa, according to their geographic distribution. Moreover, these analyses corroborated the existence of bidirectional backcrossing between both species as was previously reported [A. charrua and A. reicherti to preserve in situ biodiversity of these endangered taxa.The present work represents a robust contribution to the analysis of the hybrid zone between reported . Finally


Phrynosoma cornutum) is native to the western United States (US) and was historically introduced to several locations in the southeastern US. We studied three introduced populations in South Carolina, US to determine if they exhibit dietary, morphological and genetic divergence from the native western US populations. We expected little divergence from western populations because P. cornutum is a specialist whose biology is largely shaped by its diet of Pogonomyrmex harvester ants. We show that the introduced populations have mixed ancestry between south Texas and more northern areas and experienced founder effects and genetic bottlenecks resulting in decreased genetic diversity. South Carolina lizards primarily consume ants (94%), but surprisingly, they did not eat harvester ants. Introduced lizards primarily eat Dorymyrmex ants, but each introduced population complements Dorymyrmex with significantly different amounts of other species of ants, insects and plant matter. Introduced populations have smaller body size and have different limb and head shapes compared to western populations. This study demonstrates successful persistence of an introduced vertebrate that may be attributed to phenotypic change, even in the face of reduced genetic diversity.Introduced species can diverge from their source population when they become established in a new ecosystem. The Texas Horned Lizard ( The main concern with non-native species is that they are often invasive, which occurs when they have detrimental impacts on native species and ecosystem processes2. However, not all introductions are detrimental, with some non-native species causing changes to ecosystem dynamics that benefit native species and others causing little or no effect5.The number of non-native species is estimated at >15,000 worldwide and the rate of invasion is increasing8. This can result in the non-native species effectively competing for a limiting resource or it could result in a shift to a novel resource10. For example, morphological changes often occur after invasion of a new area14. Collectively, these changes may result in increased fitness and rapid population growth15.Non-native species must effectively deal with suites of environmental and biological factors if they are to become established. Non-native species may find competition, novel predators, and few or different resources in the invaded habitat. These factors can exert selection and this may cause phenotypic and/or genetic changes in the non-native species to enable their persistence16. The invaded area may have a resource pool similar to the source population, there may be few competitors for those resources, predators may not target the non-native species and environmental factors may be similar18. This may result in very little phenotypic or genetic change in the non-native species when compared to the source population19. Quantifying potential changes in non-native species compared to its own native range is important because it could give insight into their impact on native ecosystems.Conversely, non-native species may already have phenotypes well-suited for the invaded area; they are pre-adapted20. However, while genetic variation is often decreased after invasion, quantitative trait variation is rarely altered20. Decoupling genetic and quantitative trait variation occurs for several reasons. First, quantitative traits are affected by many genes and are often robust to loss of rare alleles21. Second, loss of genetic variation is sometimes recovered because dominance and epistatic variance may be converted to additive genetic variance during the bottleneck; this can be especially high for founder events with few individuals24. In addition, mutations can rapidly accumulate and impact quantitative traits, especially in species with rapid, post-introduction population growth21. Third, the molecular markers used to quantify genetic variation are neutral and may have little or no direct impact on quantitative phenotypic traits27. Thus, loss of total genetic variation might be coupled with a stable or increased additive genetic variation and maintenance of quantitative trait variation after a bottleneck. Although reduction of genetic variation may not impact quantitative trait variation, it often results in inbreeding depression and reduced fitness. When coupled with small population size and new selective pressures, reduced genetic variation might limit that ability of many non-native species to persist28.Although selection and phenotypic change are clearly important in determining the success of many non-native species; genetic bottlenecks and founder effects reduce genetic variation which can slow the response to selection29. Barrier island ecosystems provide unique habitats that support a variety of species. However, they also have been greatly impacted by humans and are prone to flooding and wind-associated damage caused by tropical cyclones31. These factors collectively cause disturbance to barrier island ecosystems which may encourage the establishment of non-native species32. In the southeastern United States, the fire ant (Solenopsis invicta) and beach vitex (Vitex rotundifolia) are invasive on barrier islands34.Barrier islands are elongate landmasses that are parallel to the shoreline and separated from the mainland by bays or lagoons and from each other by inlets; they are a major geographical feature of the coastline of the southeastern United StatesPhrynosoma cornutum) was introduced in the Carolinas and Florida37. This species was first introduced to these areas during the 1920’s–1940’s and was thought to be released as a type of pest control in Florida and as released ‘pets’ by soldiers stationed at military bases in the Carolinas39. Since the introduction, P. cornutum has been reported in several locations along the east coast including Sullivan’s Island, Isle of Palms, Edisto Island, and Murrells Inlet in South Carolina40. The native range of P. cornutum extends from Kansas and Colorado at the north, through Oklahoma, Texas, New Mexico, and parts of Arizona into northern Mexico37. It is unknown if the populations in the southeastern US represent a single introduction and subsequent spread, multiple introductions or some combination of both. Discovering the source population(s) is particularly important for making comparisons between native and introduced ranges41.The Texas Horned Lizard  inhabits the sandy dune habitats and is the only harvester ant present51 and thus may represent a food source that has sustained introduced populations of P. cornutum.The persistence of P. cornutum populations at three different South Carolina (SC) locations and compare them to each other and to the native populations in the western United States. Specifically, we test the following hypotheses (1) SC populations represent a subset of genetic variation relative to Texas, (2) SC populations were established from few (versus multiple) introduction events, (3) SC populations exhibit no differences among each other or compared to western populations in the body size, limb shape or head shape, and (4) the diet of SC lizards is composed of at least 51% Pogonomyrmex harvester ants by number, similar to the diet of western populations. We expect little differentiation in morphology and ecology because P. cornutum is a highly specialized myrmecophage and a terrestrial habitat generalist.In this study we confirm the presence of P. cornutum from May 2014 – July 2015 at three study sites in southeastern, coastal South Carolina . We marked them via a unique toe-clip pattern, and later released them at the GPS coordinates of capture. We also captured additional lizards at Chaparral Wildlife Management Area (WMA) in southern Texas (N = 5) and Irion and Crockett counties in western Texas (N = 21). Lizards captured in Texas were only used for the morphological analysis (see below).We hand-captured 52. We also amplified a 353 bp section of the mitochondrial control region (d-loop) using the primers PcCR_F: 5′-CTTATGATGGCGGGTTGCT-3′ and PcCR_R: 5′-GGCTGTTAAATTTATCCTCTGGTG-3′. Polymerase chain reactions (PCR) (10 µL) contained 10–50 ng DNA, 0.5 µM of each primer, 1X Qiagen Multiplex PCR Master Mix with HotStarTaq, Multiplex PCR buffer with 3 mM MgCl2 pH8.7, and dNTPs. Reactions were cycled in an ABI 2720 thermal cycler. The cycling parameters were one cycle at 95 °C for 15 min, followed by 30 cycles of 30 s at 94 °C, 90 s at 55 °C, 90 s at 72 °C, and then a final extension at 72 °C for 5 minutes. We sequenced products using ABI Big Dye Terminator Cycle Sequencing v3.1 Chemistry (Life Technologies) using the PCR primers. Sequences were electrophoresed on an ABI 3130XL Genetic Analyzer (Life Technologies); edited and trimmed using Sequencher v5.0 (Gene Codes USA); and then aligned in MEGA 6.053 using Muscle54. We identified haplotypes using GenAlEx v6.555.Texas Horned Lizard tissue samples  were obtained in Texas, New Mexico, Colorado, and Oklahoma between 2009–2017 by a large number of volunteers and from other research projects. Additionally, we obtained tissue samples from a total of 91 horned lizards in SC in Isle of Palms (N = 37), Sullivan’s Island (N = 29), and Edisto Beach (N = 25). DNA was extracted from samples and genotyped at 10 microsatellite lociPhrynosoma and can be impacted by environmental variation58. Five individuals were caught as a juvenile and then recaptured as an adult in the next season; we counted these as separate data points. We excluded 10 individuals from morphological analysis because they had broken tails.We measured, massed, and sexed all lizards captured in SC and TX. We used digital calipers to take the following 13 measurements: body (snout-vent length and tail length), fore limb , hind limb  and head . We took all measurements on the lizard’s right side unless there was damage, in which case we measured the left side. We chose these specific measurements because they are the morphological traits commonly studied in P. cornutum fecal pellets in the field  from each of the three SC study sites and preserved them in 95% ethanol. We analyzed ten fecal pellets from each of the SC sites for a total of 30 samples. In addition, we obtained stomach contents via gastric lavage from nine Isle of Palms lizards59. Additionally, we obtained seven historical P. cornutum specimens from the University of Michigan Museum of Zoology (UMMZ) that originated in the Charleston, SC area from the years 1954–1982. We removed the gut contents from these historical specimens. We dissected all samples and visually identified prey items to genus for ants and order for other insects.We collected fresh et al. (2019): Matador WMA (wildlife management area) , Chaparral WMA (N = 63), Yoakum Dunes WMA (N = 36), Rolling Plains Quail Research Ranch (RPQRR) (N = 79), Seminole Canyon State Park (N = 17), and Matagorda Island WMA (N = 30) in Texas. Previous analysis revealed that the Matagorda Island WMA barrier island population has lower genetic diversity than the mainland areas suggesting they have been bottlenecked60. We used GENEPOP v4.5 to test for Hardy-Weinberg and genotypic linkage equilibrium61. We used sequential Bonferroni correction to determine significance for these tests. MICRO-CHECKER 2.2.362 was used to determine the presence of null alleles, large allele dropout, or issues with scoring due to stuttering. As there was some evidence of null alleles, we used the ENA correction method63 implemented in the software FreeNA to calculate FST. We calculated the number of alleles, observed heterozygosity (Ho), expected heterozygosity (He), the inbreeding coefficient (F),using GenAlEx v6.555. We calculated allelic richness using HP-Rare to control for sample size differences between the populations64. We estimated the effective population sizes using the linkage disequilibrium method in NeEstimator v2.0165. The lowest allele frequency used was 0.01 and the 95% confidence intervals were estimated using the jack-knife method.We compared the SC populations to five populations from large natural protected areas in Texas that were analyzed in Williams 66 to assign South Carolina samples to previously defined genetic regions in Williams et al. (2019). Previous analyses have found two major mitochondrial clades and three major genetic groupings at nuclear microsatellite loci, a western group corresponding with the western mitochondrial clade and a northern and southern group within the eastern mitochondrial clade60. We chose a subset of individuals from that study (N = 484) that had high ancestry (q > 0.90) in each of the three microsatellite genetic groups  and used them as training samples to assign the SC samples to a particular region. We assumed admixture, correlated allele frequencies, updated the allele frequencies using only the training samples, and allowed ALPHA to vary between clusters. We used a burn-in of 50,000 iterations and ran the MCMC  with log10 geometric mean ‘size’ as the response and study site as the main effect. Locations were compared using Tukey HSD tests. An identical analysis was run using log10 snout-vent length as the response.We tested for population differences in body size using two different methods. First, we computed the geometric mean of the 13 morphological measurements and this value was used as an estimate of size68. To do this, we subtracted the log10 geometric mean ‘size’ from each of the log10 transformed morphological measurements, resulting in 13 morphological ‘shape’ variables68. We then used principal components analysis to reduce the dimensionality of the morphological data set, because some populations/sex/age categories had relatively small samples sizes  Texas populations differed from South Carolina populations, (2) Edisto differed from Sullivan’s Island, (3) Sullivan’s’ Island differed from Isle of Palms and (4) Isle of Palms differed from Edisto. We plotted the first two canonical axes from the MANCOVA to help visualize how populations were positioned in multivariate morphological shape space and how the principal components axes explained that positioning.We tested for populations differences in morphological ‘shape’ by first converting all 13 morphological measurements using Mosimann’s methodWe created species accumulation curves with cumulative number of prey types as the response and sample number as the predictor for fecal pellets from the three SC study sites and stomach contents from Isle of Palms. We also created curves with cumulative proportion of prey individuals sampled (representing each new prey type) as the response and sample number as the predictor. These figures helped us to determine that we had sufficient sampling using 10 fecal pellets.69, we performed Kruskal-Wallis tests of each prey type by location for the fecal pellet data. We also performed comparisons with the historical data. Kruskal-Wallis tests were done on the following: (1) historical gut contents from Isle of Palms, present day stomach contents from Isle of Palms, and present-day fecal pellet data from Isle of Palms and (2) historical gut contents from Sullivan’s Island and present-day fecal pellet contents from Sullivan’s Island. We performed post-hoc Steel-Dwass comparisons with sequential Bonferroni corrections. Additionally, we performed a canonical correspondence analysis (CCA) on the fecal pellet data and then ran an ANOVA permutation test on the model using the ‘vegan’ package in R 3.1.371.Using JMP v12.1.0All methods were approved by the College of Charleston Institutional Animal Care and Use Committee (protocols: 2012–007 and 2015–007) and followed all applicable laws, rules, and regulations of the United States Government and the State of South Carolina.O) of 0.58 ± 0.05 (SE) at Isle of Palms, 0.53 ± 0.05 at Sullivan’s Island, and 0.41 ± 0.07 at Edisto Beach  at Isle of Palms. After sequential Bonferroni correction, none of the loci showed heterozygote deficits, but MICRO-CHECKER indicated null alleles were present at two loci PcD01 and PcD09 in Isle of Palms . There was low allelic richness in the SC populations compared to the populations in the west . The effective population sizes in the SC populations were 23.5 (95% CI: 8.6–665.1) at Edisto, 26 (95% CI: 16.1–47.3) at Isle of Palms, and 25.2 (95% CI: 11.4–110.5) at Sullivan’s Island which are lower than the point estimate for Matagorda WMA .The three SC populations had low genetic diversity at microsatellite loci with average observed heterozygosity . These mitochondrial data are consistent with the results of the microsatellite assignment test which indicated the lizards in SC have a mix of southern and northern ancestry.One haplotype (H08) occurred in 90 of 91 individuals from all three locations in SC. One individual from Sullivan’s Island had another haplotype (H36). Compared to most of the protected sites in Texas, haplotype diversity in Sullivan’s Island, Isle of Palms, and Edisto Beach was low . Matagorda Island WMA also had only one haplotype Fig. . The com10 geometric mean size significantly differed across populations for females , males  and juveniles . Tukey HSD tests showed several between population differences in geometric mean size , males  and juveniles . Tukey HSD tests showed several between population differences in snout-vent length . Females from South Carolina had significantly different morphological shape compared to females from Texas  and this was predominantly due to PC3 . Differences among females in South Carolina populations was primarily due to PC2 and to a lesser extent PC4 and PC5. PC2 loads positively with thigh, brachium and shank and negatively with jaw length . Males from South Carolina had significantly different morphological shape compared to males from Texas . PC2 was the most important at separating Texas and South Carolina males . The difference between Sullivan’s Island and Isle of Palms was driven by PC1 and PC4  or when compared to Isle of Palms males .Male morphological shape significantly differed across locations . Among the three South Carolina populations, only Edisto had significantly different morphological shape when compared to Isle of Palms . The difference between Edisto and Isle of Palms juveniles was driven primarily by PC1 . Isle of Palms juveniles did not differ from Sullivan’s Island juveniles .Juvenile morphological shape significantly differed across locations . CCA1 explained 10.25% and CCA2 explained 7.71%, for a total of 17.96% of the variation in prey explained by site , further suggesting that loss of genetic diversity has not impaired population persistence. The surprising part of these results is that this specialized lizard species, even after a severe loss of genetic diversity, has found success after introduction on barrier islands in SC.Loss of genetic diversity due a major bottleneck can eventually lead to inbreeding depression which can reduce the ability of a population to adapt and lead to population extinction81. But, how do non-native species survive after loss of genetic diversity? First, loss of diversity does not necessarily equate to loss of fitness. For example, loss of genetic diversity in argentine ants (Linepithema humile) is related to reduction of aggressive behavior such that colonies from introduced populations do not attack each other and can build super-colonies that overwhelm native ants73. We do not know if fitness  in SC differs from the native range because we lack such data. Second, there might be recovery of genetic variation after the initial bottleneck during a ‘lag phase’6. Mechanisms such as purging of deleterious alleles, accumulation of advantageous mutations, or genetic admixture due to repeated introductions can occur during the lag phase6. There is evidence for admixture between southern and northern regions in SC populations although this does not appear to have enhanced the genetic diversity of these populations compared to native populations. The role of purging is unclear and mutation seems unlikely given the short time since introduction and very low genetic diversity in SC. Third, empty niches may offer the opportunity for establishment of non-native species, even with low genetic diversity and its associated risks. Wall lizards  were established in Cincinnati, Ohio, USA from a founding population of ~ 10 individuals and show the genetic signature of a severe bottleneck79. This population subsequently expanded throughout the city resulting in additional bottlenecks and yet these populations have persisted. This result was, in part, explained by an open niche that was unoccupied by native lizards82.There are many other examples of successful establishment of non-native species after severe bottlenecks or extremely small founder eventsP. cornutum consume mostly ants (94%) which is not surprising as P. cornutum is an ant specialist in the native western range83. However, no harvester ants (Pogonomyrmex spp.) were found in any of the present day or historical samples from SC and this was unexpected because harvester ants are the main prey of P. cornutum in the lizard’s native range (Table Pogonomyrmex badius) does inhabit the SC study area, but they do not appear to be very abundant on the parts of the dunes where we routinely captured P. cornutum (EJM unpublished data). During the two summers of sampling, we only saw two harvester ant mounds on Sullivan’s Island and three on Edisto Beach, and lizards were not found in the immediate area of the mounds. It is unclear if the lack of harvester ants in the lizards’ diet is due to lack of available prey or if the introduced lizard has decimated local harvester ant populations. It is worth noting that harvester ants are abundant on the entire dune system at Kiawah Island, SC, a barrier island just north of Edisto and south of Sullivan’s and Isle of Palms . However, it is clear that introduced P. cornutum are persisting on SC barrier islands without eating harvester ants and this represents a shift in resource use.Dietary analysis revealed that SC ly ants 9% which iP. cornutum living in SC have shifted their diet to prey that are available. While we do not have data on the insect abundance on the dunes, the most common prey eaten (Dorymyrmex) were also the most commonly observed insect on the dunes (EJM unpublished data). Previous studies have found that P. cornutum will consume other ants and insects in the absence of harvester ants86. A study on the desert horned lizard (Phrynosoma platyrhinos) suggests that horned lizards are opportunistic feeders that consume ants based on availability and size instead of specifically preferring harvester ants87. Similarly, the coast horned lizard (Phrynosoma coronatum) has been known to shift its diet when harvester ants become limited. In southern California, the invasive Argentine ant (Linepithema humile) has displaced the native harvester ants, and the lizards shifted to eating insects and other smaller ants in these areas89. Interestingly, Dorymyrmex insanus was the ant most commonly eaten by P. coronatum when harvester ants were absent88. These results may suggest that Phrynosoma species have greater dietary flexibility than is commonly thought and can persist without harvester ants given that suitable substitute ant species are present.In the absence of harvester ants, Dorymyrmex ants may be such a species. Dorymyrmex colonies are found in open, dry environments with many species distributed across the U.S. and several species occupying sand dune habitats in the southeast51. Dorymyrmex ants are very active with workers foraging during the day51. The activity pattern of Dorymyrmex coincides with P. cornutum activity and these ants are abundant, making Dorymyrmex a suitable prey item for the lizards. These results suggest that P. cornutum may be opportunistic myrmecophages and that they do not require harvester ants as the main component or even as part of their diet.Gastrophryne carolinensis) and antlions . Narrow-mouth toads are nocturnal and primarily consume nocturnally active ants90. They are found in the swales between dune ridges and in maritime forests adjacent to the dunes (EJM pers. obs). Narrow-mouth toads and horned lizards likely have little dietary overlap because Phrynosoma cornutum is diurnal and is found on the dune ridges and drier soils. Antlions  are abundant on the sand dunes and can have significant effects on ant ecology and behavior91. Antlion species compete both intra- and interspecifically93. Thus, it is likely that antlions and horned lizards compete for the same ants. However, given the success of P. cornutum at establishing populations on at least three barrier islands, it seems unlikely that antlion distributions could limit horned lizard establishment success. Other dune dwelling lizard species  include relatively low percentages of ants in their diets96. The lack of ant predators coupled with an abundance of ants and the coarse similarity in structural habitat to Western US deserts and grasslands may prime barrier island sand dune communities for establishment of ant-eating desert lizards, such as horned lizards. Thus, the invasion success of a dietary specialist may depend on an appropriate resource pool with little competition from native species, i.e. an open niche.Two other myrmecophages are commonly found on or near sand dunes in SC: eastern narrow-mouth toads  and where domestic dogs  frequently run off leash, both of which are known predators of horned lizards99. Additionally, diet differed across the three SC populations suggesting that horned lizards are adjusting their dietary preferences to local prey, which may exert selection on limb and head morphology100. However, genetic drift may also be driving population level morphological divergence in SC, which is a possibility given small populations founded from a few individuals.The morphological differentiation of the three SC populations may be evidence of local adaptation, a plastic response to local selection or random drift due to founding effects. Other studies on phrynosomatid lizards have found that morphology is correlated with microhabitat useWe found significant differences in body size and shape between the SC and Texas populations. Although the data are limited, it is clear that females from South Texas are morphologically distinct from all SC populations and that both males and females from West Texas differ from SC populations. Studies of additional Texas populations are needed to better link population level genetic variation to morphological differentiation. Measurements of habitat use, predation pressure and climate would be useful as population-level differences in these variables might indicate different selective regimes. Additionally, a common garden study among populations (both SC and TX) could differentiate plasticity vs. adaptation as the cause of population level morphological differentiation.Supplementary Information


Trifolium pratense L.) from plots in a field experiment in southern Norway were genetically characterized using genotyping by sequencing, and compared with the original population and each other. Genetic differentiation between populations was characterized on the basis of allele frequencies of single nucleotide polymorphisms (SNPs), using principal component analysis. SNPs that had been under selection, i.e., SNPs with significantly different allele frequencies in survivor populations relative to the original population, or between survivor populations that had received different treatments, were identified by analysis of FST values, using BayeScan and a simple and stringent FST-based test utilizing replicate populations from the field experiment. In addition, we tested the possibility of pooling DNA samples prior to sequencing, and pooling leaf samples prior to DNA extraction and sequencing, followed by allele frequency estimation on the basis of number of variant reads. Overall, survivor populations were more different from each other than from the original population, indicating random changes in allele frequency, selection in response to local variation in conditions between plots in the field experiment, or sampling error. However, some differentiation was observed between plots sown as pure stands or species mixtures, plots sown at different densities, and plots subjected to different harvesting regimes. Allele frequencies could be accurately estimated from pooled DNA, and SNPs under selection could be identified when leaf samples were pooled prior to DNA extraction. However, substantial sampling error required replicate populations and/or a high number of sampled individuals. We identified a number of chromosomal loci that had been under selection in pure stand plots relative to the original sown population, and loci that had been under differential selection in pure stands of red clover vs. red clover grown in species mixtures. These are all candidate loci for establishment success or persistence in red clover.Survivor populations of red clover ( Trifolium pratense L.) is a perennial legume used extensively in production of silage and hay in temperate regions, where it is cultivated in mixture with grasses such as timothy, meadow fescue and perennial ryegrass. It has high yields, can largely replace nitrogen fertilization through its symbiosis with nitrogen-fixing rhizobia, and it contributes to protein-rich forage and high intake , this equates to a red clover seeding rate of around 1 × 106 – 1.5 × 106 seeds ha-1. Only a fraction of the sown plants will survive the first years due to competition and stress. For example, in pure red clover stands sown at a rate of 18 kg ha-1, 40, 27 and 18% of the number of plants expected to germinate according to a germination test, had survived by the end of the first (establishment year), second and third growing season, respectively  of red clover, are commonly sown at a rate of 20–30 kg haectively . HoweverNatural selection can guide breeding , particuIn this paper, we utilized survivor populations to investigate whether non-random selection could be detected within one generation of red clover growing in the field for 2.5 years, and to what extent any such selection had acted on the population structure or on individual loci. We first aimed to identify loci controlling persistence by detecting loci with significantly altered allele frequencies in survivor populations compared to the originally sown population (study 1). We based our analyses on single nucleotide polymorphism (SNP) data obtained from genotyping by sequencing (GBS) of individuals from the original population and survivor populations. We then tested whether reliable allele frequencies could be obtained by sequencing pools of individual DNA samples rather than sequencing the individual samples themselves (study 2). Finally, we used GBS-derived SNP data from pools of leaf samples to investigate whether different loci had been selected in red clover survivors grown in pure stands as compared to red clover survivors grown in species mixtures (study 3).2, seeds were sown at a total seed rate of 10 (low) or 20 (high) kg ha-1, either as red clover pure stand (Ps) or as mixed stands (Ms) sown with equal amounts (seed weight) of red clover, white clover, perennial ryegrass and tall fescue. With an approximate thousand seed weight of diploid red clover of 2 g, this equals approximately 3750 or 7500 seeds per Ps plot, and 938 or 1875 seeds per Ms plot. Plots had been harvested either 3 or 5 times a year (3H and 5H). Leaf blades were sampled from survivor populations  in October 2012, and stored at -80°C.The diploid red clover cultivar “Lea”  was included in a larger field experiment, sown in two replicates at Ås, Norway, in June 2010 . The ploIn 2013, DNA was extracted from leaves of 48 or 47 survivor plants randomly selected from Ps survivor populations sown at high seeding rate and harvested 3 or 5 times a year  were performed in The Unscrambler X v.10.3  in order to visualize differentiation between populations as well as population structure. For the visualization of population differentiation MAF of each SNP in each population were used as input data. For visualization of population structure the genotype of each SNP and all individuals were used as input data.FST-based method, pairwise FST-values  were calculated for each SNP and pair of populations as q is the allele frequency of a SNP variant and all the averages are calculated over the two populations compared. Secondly, a chi-square test was used to identify SNPs with significant FST’s at different significance levels, using the test statistic 2 = 2NFSTX, where 2N = the sum of genotyped gametes in the two populations  were calculated for each significance level as l = number of SNP loci tested (4966), P = the significance level of the individual chi-square tests, n = the number of survivor populations tested against the original population (4), and d = the number of SNP loci identified with a significant FST in all n population pairs. For each SNP with a significant FST in all four survivor populations relative to the original population, the significance of the difference between the allele frequency in the original population and the combined population of 190 genotyped survivors  was confirmed with Fishers exact test, using the tool at http://lh3lh3.users.sourceforge.net/fisher.shtml. The 64 bp tag sequences containing significant SNPs were blasted against the red clover draft genome  and surrounding candidate genes.In order to identify SNP loci that had been under selection two different methods were used. With the first method, a simple ulations . Thirdlyt genome  at httpsFST-values into population-specific and SNP-specific components. Population-specific FST-values are based on the comparison between each population and the pool of all populations in the analysis. Outlier SNPs are identified as those, where the SNP-specific component is necessary to explain the observed variation. We ran the analysis with five defined populations  and three defined populations .Single nucleotide polymorphism outliers were also detected with BayeScan v 2.1 , using dFor the seven libraries made from pools of individual DNA samples , were used to characterize the genetic changes that had occurred in the field over the two and a half years. A PCA of MAF for each SNP and population showed that along the two first axes, the survivor populations had diverged from the original population in different directions . The twoPopulation structure was analyzed with PCA of all individuals and the 4966 SNPs. The same weak structure observed in the original population, with one major and one minor subpopulation, was observed in each of four survivor populations . The twoFST values and a chi square test combined with FDR. When testing individual survivor populations separately, or when combining all survivors in one common population, significant FDR values could not be obtained; they were in the range of 0.4 – 0.8. Thus, the number of SNP loci identified was only about twice of what could be expected from chance alone. However, it is very unlikely that a locus has a shift in allele frequency due to chance alone in all four survivor populations. We therefore looked for SNP loci that had a significant shift in allele frequency in all four populations, and calculated a modified FDR accounting for this reduced number of expected false positives . Twenty-seven SNPs had a shift in allele frequency at P < 0.1 in all four survivor populations with an overall corresponding FDR of 0.018. Thirteen of these were also significant at P < 0.05 in all survivor populations with an overall FDR of 0.002  when all five populations were defined, but when we combined the two 3H populations into one population, and the two 5H populations into another, in addition to the original population, one significant SNP was identified. This SNP, Tp3_16031875, was also identified with the simple d method . We alsoA comparison was made between the allele frequencies obtained by genotyping individuals from the original population, with the allele frequency estimates obtained when sequencing a pool of equal amounts of DNA from each individual (sample set 2 and 3). The inaccuracy of the allele frequency estimates based on GBS of pooled DNA was higher for SNPs with less than 50 reads or more than 600 reads from the sequencing of the pool . The bes2) was 0.90 in both populations. Principal component analysis (PCA)   revealedis (PCA) , indicatThe first principal component, explaining 24% of the variation in MAF between populations, separated Ms, Ps H, and Ps L populations , suggestFST-based method identified 11 SNPs at P < 0.1 (FDR 0.04), six SNPs at P < 0.05 (FDR 0.005) and one SNP at P < 0.01 (FDR 0.00005) . Testing each of these SNPs with analysis of variance showed that stand type had a significant effect on the allele frequency of 42 of these SNPs, while seeding density had an effect on 19, and 12 were affected by both factors  . The difThe identified SNPs under selection were spread across all seven red clover chromosomes, and were in some cases closely or moderately linked . We tookwithin one generation only, with no reproduction or migration occurring. This allows for the use of a simple FST-based test of changes in allele frequencies resulting from selection. We show that in spite of a large proportion of random mortality/survival, the use of several replicate survivor populations, sampled from replicate plots in a field experiment, improves the power of the test substantially, and makes it possible to remove these random effects and identify loci that have been under selection in all replicates. In study 1, BayeScan identified one of the SNP outliers identified by the simple FST-based method, after combining the 3H and 5H replicates. In contrast, in study 3, where a higher number of individuals were pooled in each population sample and the differentiation between populations was larger than in study 1, BayeScan identified more potential outliers than the simple FST-based method. Testing these SNPs further with analysis of variance made it possible to identify differential selection due to stand type and/or seeding density. In study 3, all outliers identified by the simple FST-based method were included among those identified by BayeScan.Several statistical methods have been developed to scan large numbers of loci across many individuals and link patterns of genetic variation to environmental variation . These m2, at distances of 100 Kb, ranged between 0.19 and 0.25 for the different chromosomes. At 500 Kb LD had decayed completely to background levels (R2 0.02–0.05). The likelihood of detecting a locus with significantly different allele frequency in different populations depends on the magnitude of the allele frequency difference, the distance between the gene conferring the effect on survival and a linked SNP, and the LD in that specific region. Here, we obtained an average density of one SNP per 85 kb or 37 kb in study 1 and in study 3, respectively. The studied variety is a synthetic population with several possible haplotypes at any given chromosomal segment, thus all nearby SNPs might not necessarily be diagnostic, that is, distinguish between alleles with different effects on survival. Therefore, with the SNP densities obtained in our study, we are likely to pick up a substantial amount of loci affecting survival, but not all, particularly not in study 1.In order to be able to detect all loci with differences in allele frequency, it is necessary to have a sufficient coverage of the genome, i.e., a sufficiently high SNP density. A high SNP density can be achieved by using a restriction enzyme in the GBS protocol which is a frequent cutter , combining several restriction enzymes, and by sequencing to a sufficient read depth to be able to call SNPs and determine allele frequencies for the majority of restriction sites. The required SNP density depends on the linkage disequilibrium (LD) of the population. The lower the LD, the higher the SNP density needed in order for all genes to be in some degree of linkage with at least one nearby SNP. Red clover has a relatively small genome (approximately 420 Mb), facilitating good read depth relative to the sequencing effort, but varieties tend to have limited LD. The LD along the different chromosomes in the original population studied here has previously been characterized by 2 was 0.97, while it was somewhat lower at lower and higher read depth. At the same MAF and read depth range, pooling of leaves of 100 plants prior to DNA extraction led to an average correlation of 0.87 and 0.90 in two sets of three replicates. This is slightly lower than that reported by R = 0.91 (R2 = 0.93) at MAF > 0.05 and read depth above 20x in replicate samples of leaves from around 200 perennial ryegrass seedlings. Pooling of individual leaf samples prior to DNA extraction reduces costs, but the accuracy of the allele frequency estimates is also reduced. Estimates could possibly have been improved if we had used more uniform leaf material and taken more care in sampling equal amounts of tissue from each individual. However, the use of several replicate populations compensates to some extent for the reduced accuracy of allele frequency estimates. The replicate samples from two of the populations in study 3 showed that there was considerable sampling error in our method. This could be overcome by sampling more individuals and/or by including replicate samples or populations in the study.Pooling of individual DNA samples, or of individual leaf samples prior to DNA extraction, can increase the allele frequency information obtained per sequencing effort, and allow for comparison of a large number of populations . While sThe 88 plants in the original population sample represent the sown populations while the survivor populations represent subsets remaining in each plot after selection  during 2.5 years of exposure to the prevailing field conditions and management. Such selection within one generation represents the environmental flexibility that the genetic variation within populations of outcrossing species can provide . Some alFST, in Ps survivor populations (high seeding rate) relative to the original population. These SNPs represent loci with alleles conferring a higher likelihood for survival under the conditions that are common to all four plots. They may be related to, e.g., establishment, competition in Ps, winter survival and the general environmental and management conditions. The absolute average allele frequency changes detected ranged from 0.22 to 0.09. Tp3_5909984 was the SNP with the largest allele frequency shift from the original population to the survivor populations in study 1. It is located in the middle of the proximal half of Tp3. Interestingly, this is also the approximate location of the only QTL for persistence detected in a red clover mapping population of red clover by If the original population has high genetic diversity and low LD , it cannot be expected that selection acting on a relatively limited number of loci will affect average genetic distance measured across the genome. In order to identify such selection, each individual locus must be considered. Indeed, by looking for allelic shifts of individual SNPs in several replicate survivor populations we identified loci that had been systematically selected under the prevailing conditions in the investigated field experiment . These aIn study 3, survivor populations were not compared with the originally sown population. Instead, survivors from Ps populations were compared to survivors from Ms populations, and survivors from populations sown at high seeding density was compared to survivors from populations sown at low density. A number of loci with allele frequencies indicating differential selection in Ps and Ms were identified. The absolute allele frequency changes detected were up to 0.36, suggesting that stand type exerted a relatively strong differential selection pressure. Red clover in mixture with perennial ryegrass and tall fescue experience earlier competition for light and possibly other resources, as the grasses grow and elongate earlier in the summer. Indeed, we have previously shown that offspring of survivor populations from Ms have earlier stem elongation than offspring from survivor populations from Ps , suggest-1) than those in our experiment, our results suggest that unintended selection occurring in Ps during breeding and seed multiplication may not necessarily be in favor of good persistence in practical farming, were Ms are used.Breeding, variety testing and seed multiplication of red clover occurs in Ps. Although seeding rates used usually are much lower (2–4 kg haFST-based test, it was possible to identify loci that had been under selection within one generation in a red clover variety grown in a field experiment for two and a half years. Pooling of individual DNA samples or leaf samples before sequencing and estimation of allele frequencies reduce costs substantially, allowing analysis of multiple populations and treatments simultaneously. Sampling error must be controlled, e.g., by sampling a large number of individuals and/or sampling from several replicate populations. Characterization of genomic changes in survival experiments may be utilized in identification of genomic regions, genes and alleles conferring survival in red clover and other species under various environmental conditions, which again can be utilized in breeding. In addition to identifying loci associated with survival under the conditions prevailing in our field experiment, we have shown that there is differential selection occurring in pure stands of red clover as compared to red clover growing in species mixtures, suggesting that the use of pure stands in breeding might not identify the best genotypes for development of varieties to be used in species mixtures.Making use of replicate populations and a simple OR initiated the research. ÅE designed the experiments and wrote the manuscript. ÅE and VS conducted the experiments. ÅE, LS, and VS analyzed the data. All authors corrected and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Conus miliaris, a species that exhibits geographic variation in prey utilization and dietary breadth, in order to determine the extent to which dietary differences are correlated with differences in venom composition, and if expanded niche breadth is associated with increased variation in venom composition. While populations showed little to no overlap in resource utilization, taxonomic richness of prey was greatest at Easter Island. Changes in dietary breadth were associated with differences in expression patterns and increased genetic differentiation of toxin-related genes. The Easter Island population also exhibited greater diversity of toxin-related transcripts, but did not show increased variance in expression of these transcripts. These results imply that differences in dietary breadth contribute more to the structural and regulatory differentiation of venoms than differences in diet.Species interactions are fundamental ecological forces that can have significant impacts on the evolutionary trajectories of species. Nonetheless, the contribution of predator-prey interactions to genetic and phenotypic divergence remains largely unknown. Predatory marine snails of the family Conidae exhibit specializations for different prey items and intraspecific variation in prey utilization patterns at geographic scales. Because cone snails utilize venom to capture prey and venom peptides are direct gene products, it is feasible to examine the evolution of genes associated with changes in resource utilization. Here, we compared feeding ecologies and venom duct transcriptomes of individuals from three populations of  Brassica rapa plants diverge in floral traits in response to exposure to pollinators that are attracted by these traits [Disjunct populations of widespread species are often exposed to varying environmental conditions that can lead to population differentiation and local adaptation. Indeed, geographic mosaics of interacting species may generate different selection regimes among populations  that cane traits . AlthougGiven that venoms are typically comprised of direct gene products that operate directly at the interface of predation , venomouCone snails are trophic specialists that show strong interspecific differences in diet ,22,23,24Conus miliaris is a widely distributed cone snail inhabiting tropical and subtropical waters from the eastern shores of Africa in the Indian Ocean to Easter Island in the southern Pacific [C. miliaris co-occurs with up to 36 congeners [C. miliaris has undergone ecological release and feeds on a much more diverse assemblage of prey, including additional species of eunicids and members of nine other polychaete families [C. miliaris at Easter Island offers an excellent opportunity to evaluate the impacts of different feeding ecologies on the evolution and differentiation of venoms and to identify the molecular mechanisms contributing to this differentiation.  Pacific . Throughongeners  and is aongeners . Howeverfamilies . The expC. miliaris in the Indo-West Pacific, based on analyses of sequences of regions of the mitochondrial cytochrome oxidase I gene and two O-superfamily conotoxin loci, illustrate that most populations exhibit low levels of genetic differentiation [Previous investigations of patterns of genetic differentiation of populations of ntiation ,28. The ntiation . Nonethentiation ,28.C. miliaris, we characterized venom duct transcriptomes of individuals of this species from three locations in the Indo-West Pacific . After assembling and annotating transcripts, we categorized transcripts into two classes: those that show homology to genes encoding peptides that are known venom components (toxin-related) and all other transcripts (non-toxin-related) that presumably represent mRNAs of “house-keeping” genes. We evaluated these transcript sets to determine if populations exhibit more differentiation at transcripts encoding venom components than at other transcripts. In addition, while past work has shown that the population at Easter Island has a wider dietary breadth than populations elsewhere, we characterized diets of individuals from these three locations using a DNA-based approach to enable quantitative measures of dietary breadth and differences in prey utilization among populations and to determine if individuals at Easter Island are specialists for particular prey species. Moreover, we present diet data for populations at Guam and American Samoa as these were previously unavailable. We specifically sought to determine how dietary differences are associated with structural and regulatory differentiation at toxin-related and non-toxin-related transcripts among populations. Although we assume that many of the transcripts encoding venom components are utilized in venoms for capturing prey, several recent studies have shown that some cone snail species utilize their venom to deter predation and that offensive venoms differ considerably from defensive ones [C. miliaris at these sites. Hence, patterns of variation observed for transcriptomes may be due to other factors and not necessarily result from differences in diet.To determine patterns of genetic variation and patterns of variation in gene expression among populations of ive ones ,31. MoreC. miliaris from Guam, American Samoa, and Easter Island. These included 59 sequences from feces of 58 specimens of C. miliaris at Guam , 21 sequences from 21 specimens at American Samoa, and 90 sequences from 68 specimens at Easter Island  (GenBank accession numbers MH634087-MH634256). In gene trees, recovered sequences occurred in 15 tip clades that were interpreted to represent 15 species of polychaetes , were unique to single locations. Estimates of dietary overlap  among populations ranged from 0.023 to 0.083 and were less than all values calculated from resampled data  . The Shannon diversity index calculated for the Easter Island population (1.77) was nearly twice as large as those for populations from American Samoa (0.93) and Guam (0.98) . Although most individuals at American Samoa utilized this taxon (~60%), it was less frequently recovered at Guam (~8% of prey items consumed) and Easter Island (~2%). All other inferred prey species, including the most commonly detected prey species at Guam  and Easter Island (m (0.98) . In addim (0.98) .C. miliaris individual cDNA libraries in a single flow cell lane averaging 15,876,996  reads per individual  and Pinctada fucata . Howeve  of the transcripts from the C. miliaris reference transcriptome had similarity  with known proteins or conotoxins in the reference databases  had confident genotypes for all 22 individuals. The multiplexing of 22 individuals in a single HiSeq lane likely accounts for the low percentage of confidently genotyped SNPs; the most highly expressed genes  were likely sequenced to a sufficient depth to be confidently genotyped for all individuals. Examination of the levels of diversity exhibited at these SNPs revealed that while non-toxin-related SNPs show similar levels of diversity among the three populations, the population at Easter Island shows greater transcript diversity at toxin-related SNPs than do the other populations .ST values for all 26,920 SNPs were lower than, but comparable to those reported for the mitochondrial cytochrome oxidase I gene [ST values based on the 24,557 non-toxin-related SNPs indicated little genetic differentiation between populations, the 2363 toxin-related SNPs exhibited strong genetic differentiation, especially between the Easter Island population and the American Samoa and Guam populations  are available from Dryad as a VCF file under accession number: 10.5061/dryad.t74q4.Raw NGS sequence data for each individual are available from NCBI SRA under accession numbers PRJNA257931 and SRP045405. Assembled transcripts are available from Dryad for C. miliaris exhibit substantial differences in diets in terms of the particular species preyed upon and dietary breadth at different locations  generalist individuals with similarly broad niche breadths , (ii) sets of specialist individuals that each utilize a subset of available resources , or (iii) sets of generalist and specialist individuals that differ in niche width  [C. miliaris individuals  ,35,36,37ividuals , individThe feeding ecologies of the three populations examined are all distinct with very little overlap in resource use . While iC. miliaris based on an analysis of mitochondrial cytochrome oxidase I sequences suggested that the population at Easter Island is genetically isolated from populations elsewhere in the Indo-West Pacific and that other populations in this region were not genetically structured [C. miliaris utilizes its venom defensively nor how predation pressures on this species differ among locations, it is possible that these or other factors are responsible for the patterns of variation that we observed. Future studies aimed at understanding patterns of variation in predation pressures and identifying defensive and offensive components of venoms of C. miliaris would help to address this issue.Previous analyses of patterns of variation exhibited at two polymorphic conotoxin loci revealed significant differences in allelic frequencies at Easter Island, while populations at Guam and American Samoa showed no evidence of differentiation . In addiructured . A much ructured ,31. AlthPopulations with expanded niche breadths are proposed to exhibit greater phenotypic variation than populations with narrow niche breadths when individuals exhibit specializations for subsets of resources ,39,40,41C. miliaris at Easter Island is distinct from other populations in terms of structural and regulatory divergence of toxin-related transcripts as well as its wider dietary breadth and lack of genetic connectivity with other populations in the Indo-West Pacific. Populations at Guam and American Samoa differ in diet, but have similar dietary breadths, show similar toxin-related transcript expression patterns, and apparently experience high levels of gene flow with each other. Although populations at Guam and American Samoa show greater differentiation at toxin-related than at non-toxin-related transcripts, the level of differentiation is much less than found for comparisons involving Easter Island. Differences in diet are associated with the genetic differentiation of conotoxin loci among the three population comparisons. Nonetheless, the extent of this differentiation is greatest when populations show large differences in dietary breadth and low levels of genetic connectivity. Because populations at Guam and American Samoa show similar conotoxin gene expression patterns despite large differences in diet, differences in conotoxin expression patterns appear to only be associated with differences in dietary breadth and lack of genetic connectivity of populations , but not differences in diet. A similar association between the extent of differentiation of conotoxin genes and dietary breadth was also observed for C. ebraeus, another worm-eating species that exhibits geographical variation in feeding ecology [The population of  ecology . Given tC. miliaris. While the three populations showed very little overlap in prey utilization, the population at Easter Island exhibited a much broader dietary breadth than populations at American Samoa and Guam. Contrary to previous studies, no associations between particular conotoxin gene superfamilies or total conotoxin diversity and prey type were observed, supporting the hypothesis that differences in prey do not correlate to changes in gene superfamily size or total conotoxin diversity [To elucidate the contributions of predator-prey interactions to intraspecific genetic and phenotypic divergence, we characterized diets and applied a comparative transcriptomic approach to three populations of the cone snail iversity ,44. Howeiversity ,47, a phiversity ,49.C. miliaris were collected at locations in Guam, American Samoa, and Easter Island and placed in small containers with seawater within one to two hours after collection. Fecal or regurgitated materials were recovered and preserved in 95% ethanol. A mark-recapture study was performed at Easter Island, allowing for the recovery of feces from multiple feeding bouts from a subset of individuals. Feces were examined via microscopy to guide the DNA-based prey determinations. In particular, presence of multiple pairs of jaws, jaw parts, and either types of setae or acicula were used to infer the number of prey taxa present in feces. Worm fragments or small sections of regurgitated worms were removed from feces (one fragment from feces that appeared to contain a single prey item and multiple fragments (up to five) for feces that appeared to contain more than one prey item) and used for genetic identification. Specimens of Total genomic DNA was extracted from collected material using the EZNA Mollusc DNA extraction kit  following the manufacturer’s recommendations. A combination of previously reported annelid specific primers , a newlSuccessfully amplified products were diluted 1:5 in deionized water and submitted for sequencing at the University of Michigan DNA Sequencing Core facilities. Ambiguities in chromatograms were corrected by comparison with the complementary DNA strand in Sequencher v5.0 . Sequences were manually aligned using Se-Al v2.0a11  with sevI values [Levels of dietary overlap among populations were evaluated by estimating proportional similarity indices  from spI values  and averC. miliaris were collected from Easter Island, Guam, and American Samoa, respectively. Total RNA was extracted from 22 dissected venom ducts and prepared for sequencing in a single flowcell lane on an Illumina HiSeq 2000 at the University of Michigan DNA Sequencing Core.Detailed descriptions of sample collection, library preparation and sequencing, as well as transcriptome assembly and annotation are described previously in Genomic Resources Development Consortium . BrieflyC. miliaris reference transcriptome, reads from all 22 individuals were combined and digitally normalized [Quality scores, nucleotide distributions, and the overall quality of the 100 bp paired-end reads were examined using the FASTX Toolkit . Given trmalized  prior tormalized . For thee-value < 10−5. Transcripts that failed to match any toxins in our conotoxin database were then compared to local databases comprised of: (1) annotated proteins from the two mollusc species available on the NCBI Unigene database, Aplysia californica, and Lottia gigantea; (2) all Conus related proteins downloaded from NCBI’s nr database; and (3) the UniProt protein database using BLASTx with an e-value cut off of 1 × 10−5. The assemblies were then divided into two sets: transcripts that were toxin-related, which are those transcripts that had homology with proteins in our conotoxin database and those that were annotated but had no homology to proteins in our conotoxin database . Toxin-related transcripts were assigned to superfamilies based on their best hits in the BLASTx search against the conotoxin database.To annotate the de novo assemblies for each dataset (whole species and three geographic populations), transcripts were first searched against a locally curated databases of non-redundant conotoxin and conotoxin signal sequences downloaded from the Conoserver database  using BLC. miliaris assembly were used as the reference transcriptome for the evaluation of expression values and SNP detection. Prior to mapping, reads were quality trimmed based on a phred-scale quality score cut-off of 20 with reads shorter than 20 bases being discarded using the FASTX Toolkit. Trimmed, single-end reads for each individual were then individually mapped against the reference transcriptome with Bowtie v1.0.1 [C. miliaris annotated transcripts, (2) C. miliaris transcripts not related to toxins, and (3) toxin-related C. miliaris transcripts following the methods outlined by Hass et al. [http://bioconductor.org). For visualization, transcripts that showed more than fourfold differences in expression with a false discovery-corrected statistical significance of <0.001 were hierarchically clustered using the heatmaps2 package in R. To evaluate patterns of variation in expression of toxin-related transcripts of the three populations, we calculated the coefficients of variation of expression values and determined if more loci show higher coefficients of variation at Easter Island than do the other populations. Annotated transcripts from the reference e v1.0.1  using the v1.0.1  was useds et al. . For eacs et al. . The difs et al.  in BiocoC. miliaris reference transcriptome using Bowtie2 v2.1.0 [For SNP detection, trimmed paired-end reads for each individual were mapped to the 2 v2.1.0  with the2 v2.1.0 , SNPs we2 v2.1.0 ,69 follo2 v2.1.0 .ST values for all SNPs, non-toxin-related SNPs, and toxin-related SNPs were calculated in GenePop [A principal component analysis (PCA) was conducted on high quality SNPs (Q > 20) using the EignSoft package  to inves GenePop .