Abstract
The Western Massasauga (Sistrurus tergeminus) is a small North American rattlesnake found west of the Mississippi River. Sistrurus tergeminus has previously been divided into two putative subspecies, Desert (S. t. edwardsii) and Prairie Massasaugas (S. t. tergeminus) based upon qualitative variation in morphology, coloration, and habitat. The Desert Massasauga subspecies has been formally petitioned for federal listing under the U.S. Endangered Species Act. Our overarching goal was to evaluate genetic structure and genomic differentiation between specimens of the two putative subspecies in an effort to inform ongoing conservation assessments. To that end, we generated whole genome sequence data for both putative taxa and then developed nearly 200 genetic markers from different fractions of the genome (~ 50 intergenic and ~ 50 genic markers from each of the two subspecies) to test for population structure across much of the Western Massasauga range. Mean genomic divergence between subspecies was 0.0041 ± 0.0080 (Kimura’s 2-parameter distance) for nuclear sequences and 0.0175 ± 0.0031 for mitochondrial sequences, both exceedingly low values which approach the minimum of zero. Admixture analyses and F-statistics both indicated that regardless of how the markers were partitioned, genetic structure was oriented far more along a geographic axis (isolation-by-distance) than a taxonomic axis (i.e., between putative subspecies). Overall, our analyses provide little support that formal protection of the purported Desert Massasauga is warranted based on the homogeneity of the collective Western Massasauga gene pool.
Similar content being viewed by others
Data availability
Sequencing reads and assemblies have been submitted to NCBI under BioProject accession PRJNA668351.
Code availability
Analyses were conducted in R or Unix environments and scripts are available in the Online Resources.
References
Anderson CD, Gibbs HL, Douglas ME, Holycross AT (2009) Conservation genetics of the Desert Massasauga rattlesnake (Sistrurus catenatus edwardsii). Copeia 2009:740–747. https://doi.org/10.1643/cg-08-152
Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc
Blair C, Sánchez-Ramírez S (2016) Diversity-dependent cladogenesis throughout western Mexico: evolutionary biogeography of rattlesnakes (Viperidae: Crotalinae: Crotalus and Sistrurus). Mol Phylogenet Evol 97:145–154. https://doi.org/10.1016/j.ympev.2015.12.020
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Broad Institute (2018) Picard Tools version 2.18.2. GitHub repository. Broad Institute. http://broadinstitute.github.io/picard/
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinform 10:1–9. https://doi.org/10.1186/1471-2105-10-421
Carranza S, Arnold EN, Wade E, Fahd S (2004) Phylogeography of the false smooth snakes, Macroprotodon (Serpentes, Colubridae): mitochondrial DNA sequences show European populations arrived recently from northwest Africa. Mol Phylogenet Evol 33:523–532. https://doi.org/10.1016/j.ympev.2004.07.009
Carroll EL, Bruford MW, DeWoody JA, Leroy G, Strand A, Waits L, Wang J (2018) Genetic and genomic monitoring with minimally invasive sampling methods. Evol Appl 11:1094–1119. https://doi.org/10.1111/eva.12600
Chikhi R, Medvedev P (2014) Informed and automated k-mer size selection for genome assembly. Bioinformatics 30(1):31–37. https://doi.org/10.1093/bioinformatics/btt310
Clark RW, Brown WS, Stechert R, Zamudio KR (2010) Roads, interrupted dispersal, and genetic diversity in timber rattlesnakes. Conserv Biol 24:1059–1069. https://doi.org/10.1111/j.1523-1739.2009.01439.x
Clark RW, Marchand MN, Clifford BJ, Stechert R, Stephens S (2011) Decline of an isolated timber rattlesnake (Crotalus horridus) population: interactions between climate change, disease, and loss of genetic diversity. Biol Conserv 144:886–891. https://doi.org/10.1016/j.biocon.2010.12.001
Colley M, Lougheed SC, Otterbein K, Litzgus JD (2017) Mitigation reduces road mortality of a threatened rattlesnake. Wildl Res 44:48–59. https://doi.org/10.1071/WR16130
Conant R, Collins JT (1991) A field guide to reptiles and amphibians. Eastern and central North America. Houghton Mifflin Co., Boston
Crother BI, Savage JM, Holycross AT (2011) Case 3571 Crotalinus catenatus Rafinesque, 1818 (currently Sistrurus catenatus) and Crotalus tergeminus Say in James, 1822 (currently Sistrurus tergeminus; Reptilia, Serpentes): proposed conservation of usage by designation of neotypes for both species. Bull Zool Nomencl 68:271–274. https://doi.org/10.21805/bzn.v68i4.a3
Crother BI, Savage JM, Holycross AT (2013) Crotalinus catenatus Rafinesque, 1818 (currently Sistrurus catenatus) and Crotalus tergeminus Say in James, 1822 (currently Sistrurus tergeminus; Reptilia, Serpentes): usage conserved by designation of neotypes for both species. Bull Zool Nomencl 70:271–274. https://doi.org/10.21805/bzn.v68i4.a3
Dixon JR, Werler JE (2005) Texas snakes: a field guide. University of Texas Press, Austin
Doyle JM, Katzner TE, Roemer GW, Cain JW III, Millsap BA, McIntyre CL, Sonsthagen SA, Fernandez NB, Wheeler M, Bulut Z, Bloom PH, DeWoody JA (2016) Genetic structure and viability selection in the golden eagle (Aquila chrysaetos), a vagile raptor with a Holarctic distribution. Conserv Genet 17:1307–1322. https://doi.org/10.1007/s10592-016-0863-0
Fernandes F, Pereira L, Freitas AT (2009) CSA: an efficient algorithm to improve circular DNA multiple alignment. BMC Bioinform 10:230. https://doi.org/10.1186/1471-2105-10-230
Fetzner JW Jr (1999) Extracting high-quality DNA from shed reptile skins: a simplified method. Biotechniques 26:1052–1054. https://doi.org/10.2144/99266bm09
Fitzgerald LA, Painter CW (2000) Rattlesnake commercialization: long-term trends, issues, and implications for conservation. Wildl Soc Bull 28:235–253
Frichot E, François O (2015) LEA: an R package for landscape and ecological association studies. Methods Ecol Evol 6(8):925–929. https://doi.org/10.1111/2041-210X.12382
Frichot E, Mathieu F, Trouillon T, Bouchard G, François O (2014) Fast and efficient estimation of individual ancestry coefficients. Genetics 196(4):973–983. https://doi.org/10.1534/genetics.113.160572
Gibbs HL, Murphy M, Chiucchi JE (2011) Genetic identity of endangered Massasauga rattlesnakes (Sistrurus sp.) in Missouri. Conserv Genet 12:433–439. https://doi.org/10.1007/s10592-010-0151-3
Gloyd H (1955) A review of the Massasauga, Sistrurus catenatus, of the southwestern United States (Serpentes: Crotalidae). Bull Chin Acad Sci 10:83–98
Gregory TR (2020) Animal genome size database. http://www.genomesize.com. Accessed 22 Sep 2020
Guicking D, Joger U, Wink M (2008) Molecular phylogeography of the viperine snake Natrix maura (Serpentes: Colubridae): evidence for strong intraspecific differentiation. Org Divers Evol 8:130–145. https://doi.org/10.1016/j.ode.2007.05.001
Hahn C, Bachmann L, Chevreux B (2013) Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Res 41(13):129. https://doi.org/10.1093/nar/gkt371
Hillis DM (2020) The detection and naming of geographic variation within species. Herpetol Rev 51:52–56
Holycross AT, Mackessy SP (2002) Variation in the diet of Sistrurus catenatus (Massasauga), with emphasis on Sistrurus catenatus edwardsii (Desert Massasauga). J Herpetol 36:454–464. https://doi.org/10.1670/0022-1511(2002)036[0454:VITDOS]2.0.CO;2
Holycross AT, Anton TG, Douglas ME, Frost DR (2008) The type localities of Sistrurus catenatus and Crotalus viridis (Serpentes: Viperidae), with the unraveling of a most unfortunate tangle of names. Copeia 2008:421–424. https://doi.org/10.1643/ch-07-095
Jackman SD, Vandervalk BP, Mohamadi H, Chu J, Yeo S, Hammond SA, Jahesh G, Khan H, Coombe L, Warren RL, Birol I (2017) ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter. Genome Res 27(5):768–777. https://doi.org/10.1101/gr.214346.116
Jombart T (2008) Adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24:1403–1405. https://doi.org/10.1093/bioinformatics/btn129
Letunic I, Bork P (2021) Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49(W1):W293–W296
Kelly CMR, Barker NP, Villet MH, Broadley DG, Branch WR (2008) The snake family Psammophiidae (Reptilia: Serpentes): phylogenetics and species delimitation in the African sand snakes (Psammophis Boie, 1825) and allied genera. Mol Phylogenet Evol 47:1045–1060. https://doi.org/10.1016/j.ympev.2008.03.025
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581
Klauber LM (1982) Rattlesnakes: their habits, life histories, and influence on mankind. University of California Press, Berkeley
Knight A, Styer D, Campbell JA, Pelikan S, Densmore LD, Mindell DP (1993) Choosing among hypotheses of rattlesnake phylogeny a best fit rate test for DNA sequence data. Syst Biol 42:356–367. https://doi.org/10.1093/sysbio/42.3.356
Kubatko LS, Gibbs HL, Bloomquist EW (2011) Inferring species-level phylogenies and taxonomic distinctiveness using multilocus data in Sistrurus rattlesnakes. Syst Biol 60:393–409. https://doi.org/10.1093/sysbio/syr011
Kumar S, Stecher G, Suleski M, Hedges SB (2017) TimeTree: a resource for timelines, timetrees, and divergence times. Mol Biol Evol 34:1812–1819. https://doi.org/10.1093/molbev/msx116
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Li H (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. ArXiv. http://arxiv.org/abs/1303.3997
Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience 1(1):2047-217X-1–18. https://doi.org/10.1186/2047-217X-1-18
Mackessy SP, Keinath DA, Mcgee M, Mcdonald D, Ise T (2005) Desert Massasauga rattlesnake (Sistrurus catenatus edwardsii): a technical conservation assessment. https://www.fs.usda.gov/Internet/FSE_DOCUMENTS/stelprdb5182073.pdf. Accessed 22 Sep 2020
McCluskey EM, Bender D (2015) Genetic structure of Western Massasauga rattlesnakes (Sistrurus catenatus tergeminus). J Herpetol 49:343–348. https://doi.org/10.1670/14-016
Murphy RW, Fu J, Lathrop A, Feltham JV, Kovac V (2002) Phylogeny of the rattlesnakes (Crotalus and Sistrurus) inferred from sequences of five mitochondrial DNA genes. In: Schuett GW, Hoggren M, Douglas ME, Greene HW (eds) Biology of the vipers. Eagle Mountain Publishing LC, Eagle Mountain, pp 66–92
O’Brien SJ, Mayr E (1991) Bureaucratic mischief: recognizing endangered species and subspecies. Science 251:1187–1188. https://doi.org/10.1126/science.251.4998.1187
Paradis E, Claude J, Strimmer K (2004) APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20:289–290. https://doi.org/10.1093/bioinformatics/btg412
Pasquesi GIM, Adams RH, Card DC, Schield DR, Corbin AB, Perry BW, Reyes-Velasco J, Ruggiero RP, Vandewege MW, Shortt JA, Castoe TA (2018) Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals. Nat Commun 9:2774. https://doi.org/10.1038/s41467-018-05279-1
Reyes-Velasco J, Adams RH, Boissinot S, Parkinson CL, Campbell JA, Castoe TA, Smith EN (2020) Genome-wide SNPs clarify lineage diversity confused by coloration in coralsnakes of the Micrurus diastema species complex (Serpentes: Elapidae). Mol Phylogenet Evol 147:106770. https://doi.org/10.1016/j.ympev.2020.106770
Rousset F (2008) GENEPOP’007: a complete re-implementation of the GENEPOP software for Windows and Linux. Mol Ecol Resour 8:103–106. https://doi.org/10.1111/j.1471-8286.2007.01931.x
Ryberg WA, Harvey JA, Blick A, Hibbitts TJ, Voelker G (2015) Genetic structure is inconsistent with subspecies designations in the Western Massasauga Sistrurus tergeminus. J Fish Wildl Manag 6:350–359. https://doi.org/10.3996/122014-JFWM-093
Schield DR, Card DC, Hales NR, Perry BW, Pasquesi GM, Blackmon H, Adams RH, Corbin AB, Smith CF, Ramesh B, Demuth JP, Bertrán E, Tollis M, Meik JM, Mackessy SP, Castoe TA (2019) The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes. Genome Res 29:590–601. https://doi.org/10.1101/gr.240952.118
Stebbins RC (1980) A field guide to western reptiles and amphibians. Houghton Mifflin Company, Boston
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Torstrom SM, Pangle KL, Swanson BJ (2014) Shedding subspecies: the influence of genetics on reptile subspecies taxonomy. Mol Phylogenet Evol 76:134–143
Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella KV, Altshuler D, Gabriel S, DePristo MA (2013) From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr Protoc Bioinform 43:11.10.1-11.10.33. https://doi.org/10.1002/0471250953.bi1110s43
von Thaden A, Nowak C, Tiesmeyer A, Reiners TE, Alves PC, Lyons LA, Mattucci F, Randi E, Cragnolini M, Galián J, Hegylei Z, Kitchener AC, Lambinet C, Lucas JM, Mölich T, Ramos L, Schockert V, Cocchiararo B (2020) Applying genomic data in wildlife monitoring: development guidelines for genotyping degraded samples with reduced single nucleotide polymorphism (SNP) panels. Mol Ecol Resour 20:662–680. https://doi.org/10.1111/1755-0998.13136
Waples RS (2020) Serendipity and me. ICES J Mar Sci 77:1658–1665
Waples RS, Allendorf F (2015) Testing for Hardy-Weinberg proportions: have we lost the plot? J Hered 106:1–19. https://doi.org/10.1093/jhered/esu062
Wickham H (2016) ggplot2: elegant graphics for data analysis. Springer, New York
Wild Earth Guardians (2010) Petition to list the Desert Massasauga (Sistrurus catenatus edwardsii) under the U.S. Endangered Species Act. https://pdf.wildearthguardians.org/site/DocServer/listing_petition_desert_massasauga.pdf?docID=685&AddInterest=1059. Accessed 22 Sep 2020
Acknowledgements
We thank Andrew Holycross, Lisle Gibbs, Stephen Mackessy, Paul Crump, Jason Jones, Doug Burkett, Ian Latella, Sean Graham, Nathan Rains, and Dan Walker for providing samples of Sistrurus tergeminus and Sistrurus catenatus. We also acquired tissue samples from the Texas Natural History Collection (Travis LaDuc), Gladys Porter Zoo (Thomas deMaar), Museum of Southwestern Biology (Tom Giermakowski), Sternberg Museum of Natural History (Curtis Schmidt), Sam Noble Oklahoma Museum of Natural History (Jessa Watters), and Texas State University (Michael R. J. Forstner). We thank Lisle Gibbs, Alex Ochoa, and Scott Martin for their generosity in sharing the Sistrurus catenatus nuclear and mitochondrial genome assemblies. We thank Drew R. Schield for his help in obtaining and navigating the Crotalus viridis genome assembly. We also thank Drs. Lisle Gibbs, Anna Brüniche-Olsen, Phillip San Miguel, Janna Willoughby, Alex Ochoa and members of the DeWoody Lab for their invaluable feedback and advice at many different parts of the process. This research was supported in part by the National Institute for Food and Agriculture, National Fish and Wildlife Foundation, U.S. Fish and Wildlife Service, and Texas Parks and Wildlife Department. Field work was conducted under the authority of Permits issued by the Texas Parks and Wildlife Department (SPR-0506-662) and the New Mexico Department of Game and Fish (NMDGF Sci# 3742) to Toby J. Hibbitts. This study was approved by the Texas A&M University Institutional Animal Care and Use Committee (AUP 2016-178). This is publication number 1657 of the Biodiversity Research and Teaching Collections at Texas A&M University.
Funding
This research was supported in part by the National Institute for Food and Agriculture, National Fish and Wildlife Foundation, U.S. Fish and Wildlife Service, and Texas Parks and Wildlife Department.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors claim no competing interests.
Ethical approval
Field work was conducted under the authority of Permits issued by the Texas Parks and Wildlife Department (SPR-0506-662) and the New Mexico Department of Game and Fish (NMDGF Sci# 3742) to Toby J. Hibbitts. This study was approved by the Texas A&M University Institutional Animal Care and Use Committee (AUP 2016-178).
Informed consent
Not applicable.
Consent for publication
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Bylsma, R., Walkup, D.K., Hibbitts, T.J. et al. Population genetic and genomic analyses of Western Massasauga (Sistrurus tergeminus ssp.): implications for subspecies delimitation and conservation. Conserv Genet 23, 271–283 (2022). https://doi.org/10.1007/s10592-021-01420-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10592-021-01420-8