New evidence contradicts the rapid spread of invasive genes into a threatened native species

New evidence contradicts the rapid spread of invasive genes into a threatened native species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article New evidence contradicts the rapid spread of invasive genes into a threatened native species Benjamin Minault Fitzpatrick, Evan McCartney-Melstad, Jarrett Johnson, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3924969/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Single nucleotide polymorphism (SNP) genotyping has become the default strategy for genetic analyses of natural populations. However, because of their simplicity, SNPs can sometimes be misleading. We used a seemingly well-curated panel of diagnostic SNPs to evaluate patterns of hybridization between introduced and native tiger salamanders ( Ambystoma ) across California. We found evidence of three genes with non-native alleles at high frequencies in otherwise pure native populations far from the previously recognized hybrid zone. While both fascinating and important for conservation, these ‘superinvasive’ alleles also challenged our perception of salamander movement abilities. Here, we further tested our initial interpretation, first by isolating DNA from two specimens of native California tiger salamanders collected several decades before the introduction of barred tiger salamanders. Both specimens had the putative invasive SNPs, suggesting that they are not diagnostic of nonnative ancestry. We followed up with a novel genealogical analysis of DNA sequences of the loci containing the questionable SNPs, and showed that the genotypes formerly interpreted as “superinvasive” are better explained as native variants that share a SNP with the introduced species. These results indicate that the hybrid invasion, while still enormous in extent, remains limited to areas near the original introduction sites. Our study demonstrates how mistakes in DNA-based analyses of invasions can be recognized and corrected using genealogical analysis of DNA sequences (tree-based haplotype inference) rather than SNPs, which are more subject to ascertainment bias. It also demonstrates the value of revisiting previous inferences, especially when important conservation targets are at stake. hybridization gene flow tiger salamander ascertainment bias Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction When introduced and native species interbreed, biologists and conservation managers are faced with the problem of tracking and understanding the impacts of non-native genes in native populations and communities (Allendorf et al. 2001 ; Draper et al. 2021 ; Fitzpatrick et al. 2015 ; Rhymer and Simberloff 1996 ; Todesco et al. 2016 ; Wayne and Shaffer 2016 ). Of particular interest is the potential for rapid spread of invasive alleles owing to strong selection arising from ecological or sexual advantages (Chatfield et al. 2010 ; Lipshutz et al. 2019 ). Previously, we presented evidence in this journal of differential introgression of three genes from introduced barred tiger salamanders, Ambystoma mavortium , deep into the native range of the California tiger salamander, A. californiense (Fitzpatrick et al. 2010 ). Because A. californiense is listed as Threatened under both the US and California Endangered Species Acts, the legal status of hybrid and introgressed populations can have major repercussions for land use and recovery efforts. However, ascertaining the ancestry of alleles in hybrid populations is often tricky, and at a minimum depends on sample data from reference populations (Fitzpatrick 2012 ; Pereira et al. 2020 ). Ideally, these should reflect a complete inventory of genetic variation in both species to ensure that ancestral polymorphisms shared between species are not misinterpreted as invasive alleles that have introgressed into the native species’ range. In our case study, the epicenter of the original introduction is well-established; it occurred in the central coast of California in Monterey County, with many populations reaching levels of non-native allele frequencies exceeding 50% across the genome. Because and we did not have genetic samples of A. californiense from before the historically documented introductions of A. mavortium in the 1950’s (Riley et al. 2003 ), we avoided using samples from the known area of hybridization in our reference panel to establish CTS ancestry, since they potentially carried non-native and native alleles at all loci. Instead, we used native reference populations from those parts of the native range that were geographically isolated from the hybrid swarm, and could therefore be defined as pure native A. californiense genotypes. While this opened the possibility that we could miss native variants that were geographically restricted to the region dominated by the invasion, it also safeguarded us against ignoring potentially informative diagnostic markers by erroneously misidentifying A. mavortium SNP alleles from within the hybrid swarm as ancestral polymorphisms segregating in A. californiense . Given our otherwise comprehensive sampling across the range of the native species (we only excluded the geographically isolated, and independently managed population in Santa Barbara County), the only real concern was the potential presence of native variation where one SNP allele was largely restricted to the central coast region, with the rest of the range fixed or nearly fixed for the alternative allele. We viewed this as an unlikely possibility. Here we provide two new lines of evidence that strongly support a reinterpretation of our earlier results. First, we were able to sequence A. californiense samples collected from the central coast decades before the known introduction of A. mavortium , and show that the three SNPs that we had interpreted as ‘superinvasive’ were naturally already present in A. californiense . Following that observation, we used new DNA sequencing capabilities to analyze the flanking genomic regions containing those SNPs to more accurately recover the ancestry information in DNA data from hybrid populations, even without perfect knowledge of all haplotype variation in the reference species. Our conclusion is that we erroneously interpreted naturally occurring, geographically restricted SNPs as extraordinarily mobile non-native A. mavortium alleles, and the hybrid invasion is still largely restricted to the central coast part of the A. californiense range. Methods We obtained two ethanol-preserved samples of A. californiense from the California Academy of Sciences collected near Stanford University in 1909 and 1921 (California Academy of Sciences specimen numbers CAS SUA-3466 and CAS 50182, respectively). These samples predate the introduction of barred tiger salamanders to California in the 1950’s (Riley et al. 2003 ), and were collected in the central coast region near the epicentre of deliberate introductions in the Salinas Valley. We included these samples in a large exon capture analysis (McCartney-Melstad et al. 2016 ) with 1624 previously collected hybrid and reference samples. For consistency with the previous work, we used the same reference samples listed in ref. (Fitzpatrick et al. 2009 ), except for one that was listed there in error ( Supporting Information ). We also included one representative of the closely related model species, the Mexican axolotl ( A. mexicanum ), which is more closely related to A. mavortium than A. californiense , to help contextualize estimated gene trees (Everson et al. 2021 ; Fitzpatrick et al. 2010 ; McCartney-Melstad et al. 2016 ). We extracted DNA from tissue samples using a salt-based extraction protocol (Sambrook and Russell 2001 ). Extractions were normalized to 100 ng/uL and sheared to approximately 300-500bp on a Bioruptor NGS (Diagenode). We prepared libraries using KAPA LTP library preparation kit half reactions (KAPA Biosystems, Wilmington, MA) and universal stubs, adding dual 8-bp adapter index sequences via a 6-cycle PCR reaction (Glenn et al. 2019 ). We pooled libraries into groups of 8 with 500 ng of input library each and enriched for a set of 5,237 exons using biotinylated RNA probes (Arbor Biosciences, Ann Arbor, MI) in the presence of 30,000 ng of ambystomatid c 0 t-1 sequence blocker (McCartney-Melstad et al. 2016 ). We amplified enriched libraries with 14 cycles of PCR and combined 19 enrichments at an equimolar ratio to create pools of 152 samples for sequencing on Illumina HiSeq 4000 150 bp paired-end lanes. We used skewer v0.2.2 (Jiang et al. 2014 ) to trim reads for adapter contamination, discarding trimmed reads shorter than 40bp. We used the Genome Analysis Toolkit (GATK) version 3.8-1 to call SNPs and genotypes (McKenna et al. 2010 ; Van der Auwera et al. 2013 ). We ran HaplotypeCaller on individual samples across the target regions (McCartney-Melstad et al. 2016 ), and GenotypeGVCFs to jointly call and genotype SNPs and short indels. We filtered SNPs with hard filters as follows: QD < 2.0, MQ 60.0, MQRankSum ReadPosRankSum > 8.0, SOR > 5.0, and QUAL < 30. We filtered indels with the following hard filters: QD 10.0, FS > 60.0, -8.0 > ReadPosRankSum > 8.0, and QUAL < 30. Genotype calls that had a depth lower than 8 or a GQ score lower than 20 were set to missing data. We removed samples with more than 75% missing data. Then, we removed indels and SNPs with more than 25% missing data across the remaining samples. We extracted data mapping to the loci containing the putative “superinvasive” SNPs (E6E11, E12C11, and E23C6) with vcftools (Danecek et al. 2011 ), and phased genotypes within each target region using BEAGLE without imputing missing data (Browning et al. 2021 ). We extracted target-level SNP haplotypes for each sample (a maximum of two haplotypes per sample). The final dataset included 1399 individuals ( Supporting Information ). To better evaluate the ancestry information in the SNP-containing loci, we used the inferred DNA sequences of the full phased haplotypes ( Supporting Information ) to estimate gene trees in MrBayes (Ronquist et al. 2012 ) for each of the three genomic regions that contained the previously identified superinvasive SNP. We used the HKY + I + G substitution model for 2x10 6 generations, 2 independent runs of 4 chains, and 25% burnin. Results For the three previously identified putatively ‘superinvasive’ SNPs, we recovered the same geographic pattern as published in 2010 using the older single base-pair SNP technology: Alleles previously identified as characteristic of A. mavortium were common throughout much of the range of California tiger salamanders (Fig. 1 ). However, both of the pre-introduction A. californiense samples from Stanford also had the SNP alleles characteristic of introduced A. mavortium. Therefore, these SNPs cannot be diagnostic of introduced ancestry, as previously thought. When we used the entire DNA sequence flanking the SNPs to estimate gene trees for each locus, the haplotypes carried by the pre-introduction samples grouped with other native haplotypes ( Supporting Information ). The trees for loci E6E11 and E12C11 clearly show several haplotypes clustering with native A. californiense despite having the SNP formerly characterized as invasive (Fig. 2 ), further confirming that the SNP is an ancestral polymorphism shared by both the native and introduced species. Two ambiguous haplotypes of E6E11 that fell on the long internal branch of the gene tree (Fig. 2 ) were observed only in the Salinas Valley; otherwise, all haplotypes were clearly clustering with one of the two species. The tree for the third locus, E23C6, is not resolvable because it has few ancestry-informative sites. This lack of clear genealogical separation between native and non-native reference haplotypes indicates that E23C6 is not an appropriate marker for differentiating CTS/BTS tiger salamander ancestry. Moreover, when we used BLAST to align E23C6 sequences to the axolotl genome, it mapped to two different chromosomes, making its interpretation even more ambiguous. We used the gene trees for E6E11 and E12C11 to score haplotypes (rather than just single SNPs contained within those haplotypes) as native or invasive based on their genealogical affinities illustrated in Fig. 2 . The geographic distribution of inferred invasive haplotypes matches the known distribution of the hybrid swarm (Fitzpatrick and Shaffer 2007b ), with no evidence of differential introgression (Fig. 3 ). Outside of the Salinas Valley in Monterey County, we found non-native A. mavortium genotypes of the loci E6E11 and E12C11 in samples previously identified as independent introductions outside of the range of native A. californiense in Lake, Siskiyou, Mono, Kern, and San Diego counties (Johnson et al. 2011 ). A non-native population was also sampled in the Lompoc Valley (Santa Barbara County) near pure native populations of the Santa Barbara Distinct Population Segment of A. californiense . In addition, we found previously unpublished non-native genotypes in Sonoma County (the northernmost non-native alleles within the native range shown in Fig. 4 ), Great Valley Grasslands State Park (Merced County), and Stanford (Santa Clara County). The Stanford samples are of undocumented specific origin and may have been collected in the Salinas Valley. Discussion Accurately identifying hybrid genotypes is central to evolutionary, population, and conservation genetics. Detection of introgression rests on the assumption that the ancestry of each allele can be inferred with an acceptable level of certainty. Unfortunately, homoplasy or shared ancestral variation can lead to false inferences of introgression when a putative recipient population is naturally segregating for an allele that is characteristic of a putative source population. False inference can be particularly problematic in biallelic SNPs because of their extreme simplicity. Only two possible allelic states are generally allowed at a single nucleotide position, which means that SNPs in isolation are particularly sensitive to the potential misinterpretation of ancestral polymorphism as the presence of hybrid genotypes when they are not actually present in a sample. This is a special case of the broader problem of SNP ascertainment bias, wherein population genetic inferences can be biased owing to the systematic choice of nucleotide variants with particular characteristics in a reference sample rather than a truly random sample of sites from the genome (Clark et al. 2005 ; Dokan et al. 2021 ; Rogers and Jorde 1996 ). Figure 2 illustrates how inadequate sampling of a structured population can lead to the inaccurate assignment of a diagnostic SNP. For example, locus E6E11 includes a clade of haplotypes that share several unique SNPs, including the one used in our original study. Our reference sample included only individuals carrying those haplotypes, leading us to attribute alleles from the other native haplotype clade to introgression from A. mavortium based on a single SNP. This limitation can be overcome by using the more expansive haplotype sequence and its associated gene tree to assign ancestry to haplotypes that were not observed in the reference sample. Doing so is a natural extension of the current practice of identifying diagnostic SNPs. However, by analyzing sequences, each containing multiple SNPs, the likelihood of mistinterpreting ancestral polymorphism is dramatically reduced. We believe that our previous inference of rapid spread of invasive alleles was incorrect, and instead represented our misinterpretation of ancestral polymorphism present within the California tiger salamander. Our incorrect inference stemmed from several aspects of our earlier study. First, by using biallelic SNPs defined as diagnostic from a systematic, but incomplete sampling of the range of the species, we assumed that the CTS allele present in the rest of the range also characterized the central coast (Fitzpatrick et al. 2009 ). For three loci and the individuals we happened to sample, that was not the case. Second, in that detailed analysis of the hybridization dynamics of California and barred tiger salamanders, we analyzed only a transect extending north from the hybrid swarm into pure CTS. While both reasonable and comprehensive in scope, this obscured the widespread occurrence of the putative invasive allele, especially at lower frequencies to the east (e.g., Fig. 1 ). And third, our relatively modest sampling of our reference populations, with a single individual from each of nine widely dispersed sites from outside of the known hybrid zone, means that we could, and did, miss an allele if it was at a low or moderate frequency, as happened for markers E6E11 and E12C11 in the southeastern populations of the southern San Joaquin Valley. For two of the three loci, tree-based haplotype scoring alone would have averted the false inferences. Even in the absence of comprehensive reference population data for the central coast, the haplotypes are sufficiently rich in variation that genealogical affinities of most observed haplotypes can be inferred (Fig. 2 ). In all three cases, the new data from the two pre-introduction specimens confirm that the SNPs formerly scored as non-native were present in A. californiense decades before the known introductions of A. mavortium in the 1950s. Our updated assessment of the geographic distribution of introduced A. mavortium in California is as follows (Fig. 4 ): Relatively isolated pure introduced populations are found outside of the range of A. californiense in Lake, Siskiyou, Mono, Kern, and San Diego counties (Johnson et al. 2011 ). Hybrid populations are widespread in the Salinas Valley (Monterey, San Benito, and southern Santa Clara counties), and have been detected, but are rare, in Great Valley Grasslands State Park (Merced County), the Santa Rosa Plain (Sonoma County), Lompoc Valley (Santa Barbara County), and possibly Stanford (Santa Clara County). Many of these sites are known to have been targets of deliberate introductions to establish harvestable populations for fishing bait (Fitzpatrick and Shaffer 2007b ; Riley et al. 2003 ). Contrary to our previous inference of extensive introgression of three genes, there is no evidence that the hybrid swarm centered in the Salinas Valley has spread much farther than 12km from specific known or suspected introduction sites in the last seven decades (Fitzpatrick and Shaffer 2007b ). However, hybrids do appear occasionally in places far from the Salinas Valley hybrid swarm. The presence of extralimital hybrids and pure A. mavortium suggest that humans have repeatedly translocated non-native tiger salamanders in California, and demonstrates the need for continued monitoring of native populations for signs of admixture and introgression. Barred tiger salamanders and hybrids exhibit significant ecological differences from native California tiger salamanders, including negative impacts on other native amphibians and endangered vernal pool invertebrates (Ryan et al. 2009 ; Searcy et al. 2016 ). While the results presented here are more optimistic than our previous geographical assessment of the invasion progression (Fitzpatrick et al. 2010 ), hybrid larvae have now been shown to have greater fitness than native CTS (Cooper and Shaffer 2023 ; Fitzpatrick and Shaffer 2007a ) and negative ecological consequences for vernal pool ecosystems (Ryan et al. 2009 ; Searcy et al. 2016 ). Therefore, the spread of hybrid genotypes remains a risk that must be both monitored, and ideally, reversed before it spreads further. Declarations Acknowledgements This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562, and the Stampede2 cluster at the University of Texas through allocation TG-DEB180005, as well as the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH Instrumentation Grant S10 OD018174. We thank the Department of Herpetology, California Academy of Sciences for tissue sample loans of the two specimens that predate the introduction of barred tiger salamanders to California. Funding: Funding was provided by the National Science Foundation, the Central Valley Project Conservation Program, and the California Department of Transportation to HBS. Competing Interests : The authors have no relevant financial or non-financial interests to disclose. Author Contributions: All authors contributed to the study conception, design, and field sampling. Lab work and bioinformatics were performed by Evan McCartney-Melstad. Data analysis was performed by Ben Fitzpatrick. The first draft of the manuscript was written by Ben Fitzpatrick and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Allendorf FW, Leary RF, Spruell P, Wenburg JK (2001) The problems with hybrids: setting conservation guidelines. Trends in Ecology and Evolution 16:613-622 Browning BL, Tian X, Zhou Y, Browning SR (2021) Fast two-stage phasing of large-scale sequence data. Am J Hum Genet 108:1880-1890 Chatfield MWH, Kozak KH, Fitzpatrick BM, Tucker PK (2010) Patterns of differential introgression in a salamander hybrid zone: inferences from genetic data and ecological niche modelling. Molecular Ecology 19:4265-4282 Clark AG, Hubisz MJ, Bustamante CD, et al. (2005) Ascertainment bias in studies of human genome-wide polymorphism. Genome research 15:1496-502 Cooper RD, Shaffer HB (2023) Managing invasive hybrids with pond hydroperiod manipulation in an endangered salamander system. Cons. Biol. 2023:e14167. Danecek P, Auton A, Abecasis G, et al. (2011) The variant call format and VCFtools. Bioinformatics 27:2156–2158 Dokan K, Kawamura S, Teshima KM (2021) Effects of single nucleotide polymorphism ascertainment on population structure inferences. G3 (Bethesda) 11 Draper D, Laguna E, Marques. I (2021) Demystifying Negative Connotations of Hybridization for Less Biased Conservation Policies. Frontiers in Ecology and Evolution 9 Everson KM, Gray LN, Jones AG, et al. (2021) Geography is more important than life history in the recent diversification of the tiger salamander complex. Proc Natl Acad Sci U S A 118 Fitzpatrick BM (2012) Estimating ancestry and heterozygosity of hybrids using molecular markers. BMC Evolutionary Biology 12 Fitzpatrick BM, Johnson JR, Kump DK, et al. (2009) Rapid fixation of non-native alleles revealed by genome-wide SNP analysis of hybrid tiger salamanders. BMC Evolutionary Biology 9:176 Fitzpatrick BM, Johnson JR, Kump DK, et al. (2010) Rapid spread of invasive genes into a threatened native species. Proceedings of the National Academy of Sciences, USA 107:3606-3610 Fitzpatrick BM, Ryan ME, Johnson JR, et al. (2015) Hybridization and the species problem in conservation. Current Zoology 61:206-216 Fitzpatrick BM, Shaffer HB (2007a) Hybrid vigor between native and introduced salamanders raises new challenges for conservation. Proceedings of the National Academy of Sciences, USA 104:15793-15798 Fitzpatrick BM, Shaffer HB (2007b) Introduction history and habitat variation explain the landscape genetics of hybrid tiger salamanders. Ecological Applications 17:598-608 Glenn TC, Nilsen RA, Kieran TJ, et al. (2019) Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext). PeerJ 7:e7755 Jiang H, Lei R, Ding S-W, Zhu. S (2014) Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15 Johnson JR, Thomson RC, Micheletti SJ, Shaffer HB (2011) The origin of tiger salamander ( Ambystoma tigrinum ) populations in California, Oregon, and Nevada: Introductions or relicts. Conservation Genetics 12:355-370 Lipshutz SE, Meier JI, Derryberry GE, et al. (2019) Differential introgression of a female competitive trait in a hybrid zone between sex-role reversed species. Evolution 73:188-201 McCartney-Melstad E, Mount GG, Shaffer. HB (2016) Exon capture optimization in amphibians with large genomes. Mol. Ecol. Resour 16:1084–1094 McKenna A, Hanna M, Banks E, et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome research 20:1297–1303 Pereira V, Santangelo R, Børsting C, et al. (2020) Evaluation of the Precision of Ancestry Inferences in South American Admixed Populations. Frontiers in genetics 11 Rhymer JM, Simberloff D (1996) Extinction by hybridization and introgression. Annual Review of Ecology and Systematics 27:83-109 Riley SPD, Shaffer HB, Voss SR, Fitzpatrick BM (2003) Hybridization between a rare, native tiger salamander ( Ambystoma californiense ) and its introduced congener. Ecological Applications 13:1263-1275 Rogers AR, Jorde LB (1996) Ascertainment Bias in Estimates of Average Heterozygosity. Am J Hum Genet 58:1033–1041 Ronquist F, Teslenko M, van der Mark P, et al. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61:539-42 Ryan ME, Johnson JR, Fitzpatrick BM (2009) Invasive hybrid tiger salamander genotypes impact native amphibians. Proceedings of the National Academy of Sciences, USA 106:11166-11171 Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor, New York. Searcy CA, Rollins HB, Shaffer HB (2016) Ecological equivalency as a tool for endangered species management. Ecol. Appl. 26:94–103 Todesco M, Pascual MA, Owens GL, et al. (2016) Hybridization and extinction. Evolutionary applications 9:892-908 Van der Auwera GA, Carneiro MO, Hartl C, et al. (2013) From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics 43:11.10.1-33. Wayne RK, Shaffer HB (2016) Hybridization and endangered species protection in the molecular era. Mol Ecol 25:2680-9 Supplementary Files E12C11hap.nex.txt E23C6hap.nex.txt E6E11hap.nex.txt SImetadataHBS.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 Feb, 2024 Reviewers invited by journal 04 Feb, 2024 Editor assigned by journal 02 Feb, 2024 First submitted to journal 01 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3924969","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271041779,"identity":"ca4404bf-d839-4355-9c88-954caeba326a","order_by":0,"name":"Benjamin Minault Fitzpatrick","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIie3OIQ+CUBDA8WNsj4Jmk36F92ZgTD/MUaBgJbJnIWGHb8FHgLGRHJkGdgOR4uZjJoPj0Qz3367db3cAFPWHcTUjAuwBGHxGgxgZIhyBrSKA4Elt4li3Sj6mOCj6BGGM6mXipq0nEdmlaFhpZK0G4V3oDIi2IpY0N4kO6Z9CXdkFfCYvLdLZM+HI1WOmoUPcNFTER5E3PlZpGywTx7qL63SOD9u6EcMUnZbJV+XKfYqiKOpXbwV2PHGU3gHyAAAAAElFTkSuQmCC","orcid":"","institution":"The University of Tennessee Knoxville","correspondingAuthor":true,"prefix":"","firstName":"Benjamin","middleName":"Minault","lastName":"Fitzpatrick","suffix":""},{"id":271041780,"identity":"07a32de9-746b-4298-8c93-a8fc49cf1d51","order_by":1,"name":"Evan McCartney-Melstad","email":"","orcid":"","institution":"UCLA: University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Evan","middleName":"","lastName":"McCartney-Melstad","suffix":""},{"id":271041781,"identity":"b745144f-fc86-4f25-886b-e26151037cf3","order_by":2,"name":"Jarrett Johnson","email":"","orcid":"","institution":"Western Kentucky University","correspondingAuthor":false,"prefix":"","firstName":"Jarrett","middleName":"","lastName":"Johnson","suffix":""},{"id":271041782,"identity":"5553c26a-ef33-4807-887e-962e54322f4e","order_by":3,"name":"H Bradley Shaffer","email":"","orcid":"","institution":"UCLA: University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"H","middleName":"Bradley","lastName":"Shaffer","suffix":""}],"badges":[],"createdAt":"2024-02-03 18:30:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3924969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3924969/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50727959,"identity":"19f0712d-a252-4f70-94fb-034f368969dc","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253731,"visible":true,"origin":"","legend":"\u003cp\u003eApproximate native range of the California tiger salamander (\u003cem\u003eAmbystoma californiense\u003c/em\u003e) and geographic distribution (blue) of SNP alleles characteristic of native reference populations, and (red) SNP alleles formerly thought to represent “superinvasive” alleles from non-native barred tiger salamanders (\u003cem\u003eA. mavortium\u003c/em\u003e). E6E11, E12C11, and E23C6 are loci mapped to separate chromosomes in the \u003cem\u003eAmbystoma\u003c/em\u003e genome (E23C6 actually maps to two chromosomes). The red alleles are concentrated in the central coast region where true non-native alleles dominate, and were rare or absent in the rest of the range.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/624b4dbf110e802b6b0b280d.png"},{"id":50727960,"identity":"195bae2f-08cb-44e4-bb31-e73f8459aa79","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":200550,"visible":true,"origin":"","legend":"\u003cp\u003eUnrooted gene trees estimated for each locus with mrBayes. Haplotypes with red branches contain the SNP alleles formerly assumed to be diagnostic of non-native ancestry. Our tree-based ancestry inference assigns as native all those haplotypes unambiguously clustering with \u003cem\u003eAmbystoma californiense\u003c/em\u003e reference samples (blue dots), and as introduced all those haplotypes unambiguously clustering with \u003cem\u003eA. mavortium\u003c/em\u003e reference samples (red dots). Haplotypes falling along the internal branch connecting these clusters are ambiguous and cannot be assigned with confidence to either species.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/77f468a188e9ed6d05789f75.png"},{"id":50727965,"identity":"5f2f4564-b82f-4c1d-b37b-9a8368ba3fc0","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182404,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distributions of haplotypes scored as native (blue and violet) and introduced (red) based on genealogical affinities (Fig. 2). Violet indicates native haplotypes containing the SNP formerly assumed to be diagnostic of non-native ancestry.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/2c3f1336cb19a7ecca6efdc3.png"},{"id":50727958,"identity":"a84fe72e-408d-4914-8dc9-c189ea2df428","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17200,"visible":true,"origin":"","legend":"\u003cp\u003eUpdated map of California, USA, showing the geographic distribution of the native California tiger salamander (\u003cem\u003eAmbystoma californiense\u003c/em\u003e) in black (870 unique locations from BerkeleyMapper and our sample sites) and localities with introduced barred tiger salamander (\u003cem\u003eA. mavortium\u003c/em\u003e) alleles in red.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/4a6934dca9baaef9821ca40a.png"},{"id":50728705,"identity":"69faa1d1-7bd0-4e82-a2a7-46439b994782","added_by":"auto","created_at":"2024-02-06 12:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":792876,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/8ab3bf1b-5481-4110-8b90-9b771978b24c.pdf"},{"id":50727966,"identity":"d356472b-8ffb-4ac4-aed0-9faa58c42203","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"txt","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":24252,"visible":true,"origin":"","legend":"","description":"","filename":"E12C11hap.nex.txt","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/6ddc742a3fec28e582f65ca8.txt"},{"id":50728101,"identity":"fd542e9d-7943-4e2b-9b62-fe1c7059f03d","added_by":"auto","created_at":"2024-02-06 12:06:58","extension":"txt","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14560,"visible":true,"origin":"","legend":"","description":"","filename":"E23C6hap.nex.txt","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/5a181f93d953dd324e9ba1ff.txt"},{"id":50727964,"identity":"355b6f99-5abb-4843-80e5-9d999e7675b2","added_by":"auto","created_at":"2024-02-06 11:58:58","extension":"txt","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":29723,"visible":true,"origin":"","legend":"","description":"","filename":"E6E11hap.nex.txt","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/423b851d248ae7e4a5401720.txt"},{"id":50728100,"identity":"6891be3f-1c9a-4d39-a260-363d91dadc33","added_by":"auto","created_at":"2024-02-06 12:06:58","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":134492,"visible":true,"origin":"","legend":"","description":"","filename":"SImetadataHBS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3924969/v1/00c18a78381126610e73b6b2.xlsx"}],"financialInterests":"","formattedTitle":"New evidence contradicts the rapid spread of invasive genes into a threatened native species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWhen introduced and native species interbreed, biologists and conservation managers are faced with the problem of tracking and understanding the impacts of non-native genes in native populations and communities (Allendorf et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Draper et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fitzpatrick et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rhymer and Simberloff \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Todesco et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wayne and Shaffer \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Of particular interest is the potential for rapid spread of invasive alleles owing to strong selection arising from ecological or sexual advantages (Chatfield et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lipshutz et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previously, we presented evidence in this journal of differential introgression of three genes from introduced barred tiger salamanders, \u003cem\u003eAmbystoma mavortium\u003c/em\u003e, deep into the native range of the California tiger salamander, \u003cem\u003eA. californiense\u003c/em\u003e (Fitzpatrick et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Because \u003cem\u003eA. californiense\u003c/em\u003e is listed as Threatened under both the US and California Endangered Species Acts, the legal status of hybrid and introgressed populations can have major repercussions for land use and recovery efforts. However, ascertaining the ancestry of alleles in hybrid populations is often tricky, and at a minimum depends on sample data from reference populations (Fitzpatrick \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ideally, these should reflect a complete inventory of genetic variation in both species to ensure that ancestral polymorphisms shared between species are not misinterpreted as invasive alleles that have introgressed into the native species\u0026rsquo; range.\u003c/p\u003e \u003cp\u003eIn our case study, the epicenter of the original introduction is well-established; it occurred in the central coast of California in Monterey County, with many populations reaching levels of non-native allele frequencies exceeding 50% across the genome. Because and we did not have genetic samples of \u003cem\u003eA. californiense\u003c/em\u003e from before the historically documented introductions of \u003cem\u003eA. mavortium\u003c/em\u003e in the 1950\u0026rsquo;s (Riley et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), we avoided using samples from the known area of hybridization in our reference panel to establish CTS ancestry, since they potentially carried non-native and native alleles at all loci. Instead, we used native reference populations from those parts of the native range that were geographically isolated from the hybrid swarm, and could therefore be defined as pure native \u003cem\u003eA. californiense\u003c/em\u003e genotypes. While this opened the possibility that we could miss native variants that were geographically restricted to the region dominated by the invasion, it also safeguarded us against ignoring potentially informative diagnostic markers by erroneously misidentifying \u003cem\u003eA. mavortium\u003c/em\u003e SNP alleles from within the hybrid swarm as ancestral polymorphisms segregating in \u003cem\u003eA. californiense\u003c/em\u003e. Given our otherwise comprehensive sampling across the range of the native species (we only excluded the geographically isolated, and independently managed population in Santa Barbara County), the only real concern was the potential presence of native variation where one SNP allele was largely restricted to the central coast region, with the rest of the range fixed or nearly fixed for the alternative allele. We viewed this as an unlikely possibility.\u003c/p\u003e \u003cp\u003eHere we provide two new lines of evidence that strongly support a reinterpretation of our earlier results. First, we were able to sequence \u003cem\u003eA. californiense\u003c/em\u003e samples collected from the central coast decades before the known introduction of \u003cem\u003eA. mavortium\u003c/em\u003e, and show that the three SNPs that we had interpreted as \u0026lsquo;superinvasive\u0026rsquo; were naturally already present in \u003cem\u003eA. californiense\u003c/em\u003e. Following that observation, we used new DNA sequencing capabilities to analyze the flanking genomic regions containing those SNPs to more accurately recover the ancestry information in DNA data from hybrid populations, even without perfect knowledge of all haplotype variation in the reference species. Our conclusion is that we erroneously interpreted naturally occurring, geographically restricted SNPs as extraordinarily mobile non-native \u003cem\u003eA. mavortium\u003c/em\u003e alleles, and the hybrid invasion is still largely restricted to the central coast part of the \u003cem\u003eA. californiense\u003c/em\u003e range.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eWe obtained two ethanol-preserved samples of \u003cem\u003eA. californiense\u003c/em\u003e from the California Academy of Sciences collected near Stanford University in 1909 and 1921 (California Academy of Sciences specimen numbers CAS SUA-3466 and CAS 50182, respectively). These samples predate the introduction of barred tiger salamanders to California in the 1950\u0026rsquo;s (Riley et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and were collected in the central coast region near the epicentre of deliberate introductions in the Salinas Valley. We included these samples in a large exon capture analysis (McCartney-Melstad et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with 1624 previously collected hybrid and reference samples. For consistency with the previous work, we used the same reference samples listed in ref. (Fitzpatrick et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), except for one that was listed there in error (\u003cem\u003eSupporting Information\u003c/em\u003e). We also included one representative of the closely related model species, the Mexican axolotl (\u003cem\u003eA. mexicanum\u003c/em\u003e), which is more closely related to \u003cem\u003eA. mavortium\u003c/em\u003e than \u003cem\u003eA. californiense\u003c/em\u003e, to help contextualize estimated gene trees (Everson et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fitzpatrick et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; McCartney-Melstad et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe extracted DNA from tissue samples using a salt-based extraction protocol (Sambrook and Russell \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Extractions were normalized to 100 ng/uL and sheared to approximately 300-500bp on a Bioruptor NGS (Diagenode). We prepared libraries using KAPA LTP library preparation kit half reactions (KAPA Biosystems, Wilmington, MA) and universal stubs, adding dual 8-bp adapter index sequences via a 6-cycle PCR reaction (Glenn et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We pooled libraries into groups of 8 with 500 ng of input library each and enriched for a set of 5,237 exons using biotinylated RNA probes (Arbor Biosciences, Ann Arbor, MI) in the presence of 30,000 ng of ambystomatid c\u003csub\u003e0\u003c/sub\u003et-1 sequence blocker (McCartney-Melstad et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We amplified enriched libraries with 14 cycles of PCR and combined 19 enrichments at an equimolar ratio to create pools of 152 samples for sequencing on Illumina HiSeq 4000 150 bp paired-end lanes.\u003c/p\u003e \u003cp\u003eWe used skewer v0.2.2 (Jiang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) to trim reads for adapter contamination, discarding trimmed reads shorter than 40bp. We used the Genome Analysis Toolkit (GATK) version 3.8-1 to call SNPs and genotypes (McKenna et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Van der Auwera et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We ran HaplotypeCaller on individual samples across the target regions (McCartney-Melstad et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and GenotypeGVCFs to jointly call and genotype SNPs and short indels.\u003c/p\u003e \u003cp\u003eWe filtered SNPs with hard filters as follows: QD\u0026thinsp;\u0026lt;\u0026thinsp;2.0, MQ\u0026thinsp;\u0026lt;\u0026thinsp;20.0, FS\u0026thinsp;\u0026gt;\u0026thinsp;60.0, MQRankSum \u0026lt; -12.5, -8.0\u0026thinsp;\u0026gt;\u0026thinsp;ReadPosRankSum\u0026thinsp;\u0026gt;\u0026thinsp;8.0, SOR\u0026thinsp;\u0026gt;\u0026thinsp;5.0, and QUAL\u0026thinsp;\u0026lt;\u0026thinsp;30. We filtered indels with the following hard filters: QD\u0026thinsp;\u0026lt;\u0026thinsp;2.0, SOR\u0026thinsp;\u0026gt;\u0026thinsp;10.0, FS\u0026thinsp;\u0026gt;\u0026thinsp;60.0, -8.0\u0026thinsp;\u0026gt;\u0026thinsp;ReadPosRankSum\u0026thinsp;\u0026gt;\u0026thinsp;8.0, and QUAL\u0026thinsp;\u0026lt;\u0026thinsp;30. Genotype calls that had a depth lower than 8 or a GQ score lower than 20 were set to missing data. We removed samples with more than 75% missing data. Then, we removed indels and SNPs with more than 25% missing data across the remaining samples.\u003c/p\u003e \u003cp\u003eWe extracted data mapping to the loci containing the putative \u0026ldquo;superinvasive\u0026rdquo; SNPs (E6E11, E12C11, and E23C6) with vcftools (Danecek et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and phased genotypes within each target region using BEAGLE without imputing missing data (Browning et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We extracted target-level SNP haplotypes for each sample (a maximum of two haplotypes per sample). The final dataset included 1399 individuals (\u003cem\u003eSupporting Information\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eTo better evaluate the ancestry information in the SNP-containing loci, we used the inferred DNA sequences of the full phased haplotypes (\u003cem\u003eSupporting Information\u003c/em\u003e) to estimate gene trees in MrBayes (Ronquist et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) for each of the three genomic regions that contained the previously identified superinvasive SNP. We used the HKY\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G substitution model for 2x10\u003csup\u003e6\u003c/sup\u003e generations, 2 independent runs of 4 chains, and 25% burnin.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFor the three previously identified putatively \u0026lsquo;superinvasive\u0026rsquo; SNPs, we recovered the same geographic pattern as published in 2010 using the older single base-pair SNP technology: Alleles previously identified as characteristic of \u003cem\u003eA. mavortium\u003c/em\u003e were common throughout much of the range of California tiger salamanders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, both of the pre-introduction \u003cem\u003eA. californiense\u003c/em\u003e samples from Stanford also had the SNP alleles characteristic of introduced \u003cem\u003eA. mavortium.\u003c/em\u003e Therefore, these SNPs cannot be diagnostic of introduced ancestry, as previously thought.\u003c/p\u003e \u003cp\u003eWhen we used the entire DNA sequence flanking the SNPs to estimate gene trees for each locus, the haplotypes carried by the pre-introduction samples grouped with other native haplotypes (\u003cem\u003eSupporting Information\u003c/em\u003e). The trees for loci E6E11 and E12C11 clearly show several haplotypes clustering with native \u003cem\u003eA. californiense\u003c/em\u003e despite having the SNP formerly characterized as invasive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), further confirming that the SNP is an ancestral polymorphism shared by both the native and introduced species. Two ambiguous haplotypes of E6E11 that fell on the long internal branch of the gene tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were observed only in the Salinas Valley; otherwise, all haplotypes were clearly clustering with one of the two species. The tree for the third locus, E23C6, is not resolvable because it has few ancestry-informative sites. This lack of clear genealogical separation between native and non-native reference haplotypes indicates that E23C6 is not an appropriate marker for differentiating CTS/BTS tiger salamander ancestry. Moreover, when we used BLAST to align E23C6 sequences to the axolotl genome, it mapped to two different chromosomes, making its interpretation even more ambiguous.\u003c/p\u003e \u003cp\u003eWe used the gene trees for E6E11 and E12C11 to score haplotypes (rather than just single SNPs contained within those haplotypes) as native or invasive based on their genealogical affinities illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The geographic distribution of inferred invasive haplotypes matches the known distribution of the hybrid swarm (Fitzpatrick and Shaffer \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e), with no evidence of differential introgression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOutside of the Salinas Valley in Monterey County, we found non-native \u003cem\u003eA. mavortium\u003c/em\u003e genotypes of the loci E6E11 and E12C11 in samples previously identified as independent introductions outside of the range of native \u003cem\u003eA. californiense\u003c/em\u003e in Lake, Siskiyou, Mono, Kern, and San Diego counties (Johnson et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A non-native population was also sampled in the Lompoc Valley (Santa Barbara County) near pure native populations of the Santa Barbara Distinct Population Segment of \u003cem\u003eA. californiense\u003c/em\u003e. In addition, we found previously unpublished non-native genotypes in Sonoma County (the northernmost non-native alleles within the native range shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), Great Valley Grasslands State Park (Merced County), and Stanford (Santa Clara County). The Stanford samples are of undocumented specific origin and may have been collected in the Salinas Valley.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccurately identifying hybrid genotypes is central to evolutionary, population, and conservation genetics. Detection of introgression rests on the assumption that the ancestry of each allele can be inferred with an acceptable level of certainty. Unfortunately, homoplasy or shared ancestral variation can lead to false inferences of introgression when a putative recipient population is naturally segregating for an allele that is characteristic of a putative source population. False inference can be particularly problematic in biallelic SNPs because of their extreme simplicity. Only two possible allelic states are generally allowed at a single nucleotide position, which means that SNPs in isolation are particularly sensitive to the potential misinterpretation of ancestral polymorphism as the presence of hybrid genotypes when they are not actually present in a sample. This is a special case of the broader problem of SNP ascertainment bias, wherein population genetic inferences can be biased owing to the systematic choice of nucleotide variants with particular characteristics in a reference sample rather than a truly random sample of sites from the genome (Clark et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Dokan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rogers and Jorde \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates how inadequate sampling of a structured population can lead to the inaccurate assignment of a diagnostic SNP. For example, locus E6E11 includes a clade of haplotypes that share several unique SNPs, including the one used in our original study. Our reference sample included only individuals carrying those haplotypes, leading us to attribute alleles from the other native haplotype clade to introgression from \u003cem\u003eA. mavortium\u003c/em\u003e based on a single SNP. This limitation can be overcome by using the more expansive haplotype sequence and its associated gene tree to assign ancestry to haplotypes that were not observed in the reference sample. Doing so is a natural extension of the current practice of identifying diagnostic SNPs. However, by analyzing sequences, each containing multiple SNPs, the likelihood of mistinterpreting ancestral polymorphism is dramatically reduced.\u003c/p\u003e \u003cp\u003eWe believe that our previous inference of rapid spread of invasive alleles was incorrect, and instead represented our misinterpretation of ancestral polymorphism present within the California tiger salamander. Our incorrect inference stemmed from several aspects of our earlier study. First, by using biallelic SNPs defined as diagnostic from a systematic, but incomplete sampling of the range of the species, we assumed that the CTS allele present in the rest of the range also characterized the central coast (Fitzpatrick et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For three loci and the individuals we happened to sample, that was not the case. Second, in that detailed analysis of the hybridization dynamics of California and barred tiger salamanders, we analyzed only a transect extending north from the hybrid swarm into pure CTS. While both reasonable and comprehensive in scope, this obscured the widespread occurrence of the putative invasive allele, especially at lower frequencies to the east (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). And third, our relatively modest sampling of our reference populations, with a single individual from each of nine widely dispersed sites from outside of the known hybrid zone, means that we could, and did, miss an allele if it was at a low or moderate frequency, as happened for markers E6E11 and E12C11 in the southeastern populations of the southern San Joaquin Valley. For two of the three loci, tree-based haplotype scoring alone would have averted the false inferences. Even in the absence of comprehensive reference population data for the central coast, the haplotypes are sufficiently rich in variation that genealogical affinities of most observed haplotypes can be inferred (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In all three cases, the new data from the two pre-introduction specimens confirm that the SNPs formerly scored as non-native were present in \u003cem\u003eA. californiense\u003c/em\u003e decades before the known introductions of \u003cem\u003eA. mavortium\u003c/em\u003e in the 1950s.\u003c/p\u003e \u003cp\u003eOur updated assessment of the geographic distribution of introduced \u003cem\u003eA. mavortium\u003c/em\u003e in California is as follows (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e): Relatively isolated pure introduced populations are found outside of the range of \u003cem\u003eA. californiense\u003c/em\u003e in Lake, Siskiyou, Mono, Kern, and San Diego counties (Johnson et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hybrid populations are widespread in the Salinas Valley (Monterey, San Benito, and southern Santa Clara counties), and have been detected, but are rare, in Great Valley Grasslands State Park (Merced County), the Santa Rosa Plain (Sonoma County), Lompoc Valley (Santa Barbara County), and possibly Stanford (Santa Clara County). Many of these sites are known to have been targets of deliberate introductions to establish harvestable populations for fishing bait (Fitzpatrick and Shaffer \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Contrary to our previous inference of extensive introgression of three genes, there is no evidence that the hybrid swarm centered in the Salinas Valley has spread much farther than 12km from specific known or suspected introduction sites in the last seven decades (Fitzpatrick and Shaffer \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, hybrids do appear occasionally in places far from the Salinas Valley hybrid swarm. The presence of extralimital hybrids and pure \u003cem\u003eA. mavortium\u003c/em\u003e suggest that humans have repeatedly translocated non-native tiger salamanders in California, and demonstrates the need for continued monitoring of native populations for signs of admixture and introgression. Barred tiger salamanders and hybrids exhibit significant ecological differences from native California tiger salamanders, including negative impacts on other native amphibians and endangered vernal pool invertebrates (Ryan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Searcy et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While the results presented here are more optimistic than our previous geographical assessment of the invasion progression (Fitzpatrick et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), hybrid larvae have now been shown to have greater fitness than native CTS (Cooper and Shaffer \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fitzpatrick and Shaffer \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007a\u003c/span\u003e) and negative ecological consequences for vernal pool ecosystems (Ryan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Searcy et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the spread of hybrid genotypes remains a risk that must be both monitored, and ideally, reversed before it spreads further.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562, and the Stampede2 cluster at the University of Texas through allocation TG-DEB180005, as well as the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH Instrumentation Grant S10 OD018174. We thank the Department of Herpetology, California Academy of Sciences for tissue sample loans of the two specimens that predate the introduction of barred tiger salamanders to California.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eFunding was provided by the National Science Foundation, the Central Valley Project Conservation Program, and the California Department of Transportation to HBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e All authors contributed to the study conception, design, and field sampling. Lab work and bioinformatics were performed by Evan McCartney-Melstad. Data analysis was performed by Ben Fitzpatrick. The first draft of the manuscript was written by Ben Fitzpatrick and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllendorf FW, Leary RF, Spruell P, Wenburg JK (2001) The problems with hybrids: setting conservation guidelines. Trends in Ecology and Evolution 16:613-622\u003c/li\u003e\n\u003cli\u003eBrowning BL, Tian X, Zhou Y, Browning SR (2021) Fast two-stage phasing of large-scale sequence data. Am J Hum Genet 108:1880-1890\u003c/li\u003e\n\u003cli\u003eChatfield MWH, Kozak KH, Fitzpatrick BM, Tucker PK (2010) Patterns of differential introgression in a salamander hybrid zone: inferences from genetic data and ecological niche modelling. Molecular Ecology 19:4265-4282\u003c/li\u003e\n\u003cli\u003eClark AG, Hubisz MJ, Bustamante CD, et al. (2005) Ascertainment bias in studies of human genome-wide polymorphism. Genome research 15:1496-502\u003c/li\u003e\n\u003cli\u003eCooper RD, Shaffer HB (2023) Managing invasive hybrids with pond hydroperiod manipulation in an endangered salamander system. Cons. Biol. 2023:e14167.\u003c/li\u003e\n\u003cli\u003eDanecek P, Auton A, Abecasis G, et al. (2011) The variant call format and VCFtools. Bioinformatics 27:2156\u0026ndash;2158\u003c/li\u003e\n\u003cli\u003eDokan K, Kawamura S, Teshima KM (2021) Effects of single nucleotide polymorphism ascertainment on population structure inferences. G3 (Bethesda) 11\u003c/li\u003e\n\u003cli\u003eDraper D, Laguna E, Marques. I (2021) Demystifying Negative Connotations of Hybridization for Less Biased Conservation Policies. Frontiers in Ecology and Evolution 9\u003c/li\u003e\n\u003cli\u003eEverson KM, Gray LN, Jones AG, et al. (2021) Geography is more important than life history in the recent diversification of the tiger salamander complex. Proc Natl Acad Sci U S A 118\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM (2012) Estimating ancestry and heterozygosity of hybrids using molecular markers. BMC Evolutionary Biology 12\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM, Johnson JR, Kump DK, et al. (2009) Rapid fixation of non-native alleles revealed by genome-wide SNP analysis of hybrid tiger salamanders. BMC Evolutionary Biology 9:176\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM, Johnson JR, Kump DK, et al. (2010) Rapid spread of invasive genes into a threatened native species. Proceedings of the National Academy of Sciences, USA 107:3606-3610\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM, Ryan ME, Johnson JR, et al. (2015) Hybridization and the species problem in conservation. Current Zoology 61:206-216\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM, Shaffer HB (2007a) Hybrid vigor between native and introduced salamanders raises new challenges for conservation. Proceedings of the National Academy of Sciences, USA 104:15793-15798\u003c/li\u003e\n\u003cli\u003eFitzpatrick BM, Shaffer HB (2007b) Introduction history and habitat variation explain the landscape genetics of hybrid tiger salamanders. Ecological Applications 17:598-608\u003c/li\u003e\n\u003cli\u003eGlenn TC, Nilsen RA, Kieran TJ, et al. (2019) Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru \u0026amp; iNext). PeerJ 7:e7755\u003c/li\u003e\n\u003cli\u003eJiang H, Lei R, Ding S-W, Zhu. S (2014) Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15\u003c/li\u003e\n\u003cli\u003eJohnson JR, Thomson RC, Micheletti SJ, Shaffer HB (2011) The origin of tiger salamander (\u003cem\u003eAmbystoma tigrinum\u003c/em\u003e) populations in California, Oregon, and Nevada: Introductions or relicts. Conservation Genetics 12:355-370\u003c/li\u003e\n\u003cli\u003eLipshutz SE, Meier JI, Derryberry GE, et al. (2019) Differential introgression of a female competitive trait in a hybrid zone between sex-role reversed species. Evolution 73:188-201\u003c/li\u003e\n\u003cli\u003eMcCartney-Melstad E, Mount GG, Shaffer. HB (2016) Exon capture optimization in amphibians with large genomes. Mol. Ecol. Resour 16:1084\u0026ndash;1094\u003c/li\u003e\n\u003cli\u003eMcKenna A, Hanna M, Banks E, et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome research 20:1297\u0026ndash;1303\u003c/li\u003e\n\u003cli\u003ePereira V, Santangelo R, B\u0026oslash;rsting C, et al. (2020) Evaluation of the Precision of Ancestry Inferences in South American Admixed Populations. Frontiers in genetics 11\u003c/li\u003e\n\u003cli\u003eRhymer JM, Simberloff D (1996) Extinction by hybridization and introgression. Annual Review of Ecology and Systematics 27:83-109\u003c/li\u003e\n\u003cli\u003eRiley SPD, Shaffer HB, Voss SR, Fitzpatrick BM (2003) Hybridization between a rare, native tiger salamander (\u003cem\u003eAmbystoma californiense\u003c/em\u003e) and its introduced congener. Ecological Applications 13:1263-1275\u003c/li\u003e\n\u003cli\u003eRogers AR, Jorde LB (1996) Ascertainment Bias in Estimates of Average Heterozygosity. Am J Hum Genet 58:1033\u0026ndash;1041\u003c/li\u003e\n\u003cli\u003eRonquist F, Teslenko M, van der Mark P, et al. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61:539-42\u003c/li\u003e\n\u003cli\u003eRyan ME, Johnson JR, Fitzpatrick BM (2009) Invasive hybrid tiger salamander genotypes impact native amphibians. Proceedings of the National Academy of Sciences, USA 106:11166-11171\u003c/li\u003e\n\u003cli\u003eSambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor, New York.\u003c/li\u003e\n\u003cli\u003eSearcy CA, Rollins HB, Shaffer HB (2016) Ecological equivalency as a tool for endangered species management. Ecol. Appl. 26:94\u0026ndash;103\u003c/li\u003e\n\u003cli\u003eTodesco M, Pascual MA, Owens GL, et al. (2016) Hybridization and extinction. Evolutionary applications 9:892-908\u003c/li\u003e\n\u003cli\u003eVan der Auwera GA, Carneiro MO, Hartl C, et al. (2013) From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics 43:11.10.1-33.\u003c/li\u003e\n\u003cli\u003eWayne RK, Shaffer HB (2016) Hybridization and endangered species protection in the molecular era. Mol Ecol 25:2680-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hybridization, gene flow, tiger salamander, ascertainment bias","lastPublishedDoi":"10.21203/rs.3.rs-3924969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3924969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSingle nucleotide polymorphism (SNP) genotyping has become the default strategy for genetic analyses of natural populations. However, because of their simplicity, SNPs can sometimes be misleading. We used a seemingly well-curated panel of diagnostic SNPs to evaluate patterns of hybridization between introduced and native tiger salamanders (\u003cem\u003eAmbystoma\u003c/em\u003e) across California. We found evidence of three genes with non-native alleles at high frequencies in otherwise pure native populations far from the previously recognized hybrid zone. While both fascinating and important for conservation, these \u0026lsquo;superinvasive\u0026rsquo; alleles also challenged our perception of salamander movement abilities. Here, we further tested our initial interpretation, first by isolating DNA from two specimens of native California tiger salamanders collected several decades before the introduction of barred tiger salamanders. Both specimens had the putative invasive SNPs, suggesting that they are not diagnostic of nonnative ancestry. We followed up with a novel genealogical analysis of DNA sequences of the loci containing the questionable SNPs, and showed that the genotypes formerly interpreted as \u0026ldquo;superinvasive\u0026rdquo; are better explained as native variants that share a SNP with the introduced species. These results indicate that the hybrid invasion, while still enormous in extent, remains limited to areas near the original introduction sites. Our study demonstrates how mistakes in DNA-based analyses of invasions can be recognized and corrected using genealogical analysis of DNA sequences (tree-based haplotype inference) rather than SNPs, which are more subject to ascertainment bias. It also demonstrates the value of revisiting previous inferences, especially when important conservation targets are at stake.\u003c/p\u003e","manuscriptTitle":"New evidence contradicts the rapid spread of invasive genes into a threatened native species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 11:58:53","doi":"10.21203/rs.3.rs-3924969/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-02-04T16:24:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-04T15:51:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-02T14:25:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Invasions","date":"2024-02-01T11:23:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"51fb35ae-3d81-438a-9644-e156c569d556","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T23:11:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-06 11:58:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3924969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3924969","identity":"rs-3924969","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}