Isolation and characterization of microsatellite markers from the threatened eastern indigo snake (Drymarchon couperi)

Shamblin, Brian M.; Alstad, Travis I.; Stevenson, Dirk J.; Macey, John N.; Snow, Frankie; Nairn, Campbell J.

doi:10.1007/s12686-010-9348-5

Cited by 4 publications

(5 citation statements)

References 10 publications

(8 reference statements)

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“…We extracted DNA using the Qiagen DNeasy blood and tissue extraction kit (Qiagen, Inc., Valencia, CA). We ran 17 microsatellite loci [ 52 ] within three multiplexed panels using the Qiagen Multiplex PCR kit ( S2 Table for details). Each reaction contained 1X Qiagen Multiplex PCR Master Mix, 0.2 μM multiplexed primer mix (each primer at equal concentrations), and 1 μl of DNA extract in a total volume of 7 μl.…”

Section: Methodsmentioning

confidence: 99%

“…Each reaction contained 1X Qiagen Multiplex PCR Master Mix, 0.2 μM multiplexed primer mix (each primer at equal concentrations), and 1 μl of DNA extract in a total volume of 7 μl. The PCR protocol was modified from Shamblin et al [ 52 ] for multiplex PCR and consisted of an initial denaturation of 95°C for 15 min, 20 touchdown cycles of 94°C for 30 s, 60°C minus 0.5°C per cycle for 90 s and 72°C for 1 min, followed by 30 cycles of 94°C for 30 s, 50°C for 90 s and 72°C for 1 min, and a final elongation step of 60°C for 30 min. Multiplexed PCR products were run on a 3130xl Applied Biosystems Genetic Analyzer at the University of Idaho’s Laboratory for Ecological, Evolutionary, and Conservation Genetics.…”

Section: Methodsmentioning

confidence: 99%

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Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi

et al. 2019

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Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6–8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi

et al. 2019

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“…We extracted DNA using the Qiagen DNeasy blood and tissue extraction kit. We genotyped individuals at 15 microsatellite loci (Shamblin et al, 2011) divided into three multiplexed panels run on an ABI 3130xl sequencer and scored using genemapper software (see Folt et al, 2019 for detail on PCR conditions and multiplex panels). We reran select samples to verify questionable genotypes and retained samples that amplified at ≥13 loci.…”

Section: Methodsmentioning

confidence: 99%

Multiscale assessment of functional connectivity: Landscape genetics of eastern indigo snakes in an anthropogenically fragmented landscape in central Florida

et al. 2021

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Landscape features can strongly influence gene flow and the strength and direction of these effects may vary across spatial scales. However, few studies have evaluated methodological approaches for selecting spatial scales in landscape genetics analyses, in part because of computational challenges associated with optimizing landscape resistance surfaces (LRS). We used the federally threatened eastern indigo snake (Drymarchon couperi) in central Florida as a case study with which to compare the importance of landscape features and their scales of effect in influencing gene flow. We used genetic algorithms (ResistanceGA) to empirically optimize LRS using categorical land cover surfaces, multiscale resource selection surfaces (RSS), and four combinations of landscape covariates measured at multiple spatial scales (multisurface multiscale LRS). We compared LRS where scale was selected using pseudo-and full optimization. Multisurface multiscale LRS received more empirical support than LRS optimized from categorical land cover surfaces or RSS. Multiscale LRS with scale selected using full optimization generally outperformed those with scale selected using pseudo-optimization. Multiscale LRS with large spatial scales (1200-1800 m) received the most empirical support. Our results highlight the importance of considering landscape features across multiple spatial scales in landscape genetic analyses, particularly broad scales relative to species movement potential. Different effects of scale on home range-level movements and dispersal could explain weak associations between habitat suitability and gene flow in other studies. Our results also demonstrate the importance of large tracts of undeveloped upland habitat with heterogenous vegetation communities and low urbanization for promoting indigo snake connectivity.

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“…We extracted DNA using the Qiagen DNeasy blood and tissue extraction kit (Qiagen, Inc., Valencia, CA). We ran 17 microsatellite loci [39] within three multiplexed panels using the Qiagen Multiplex PCR kit (S2 Table for details). Each reaction contained 1X Qiagen Multiplex PCR Master Mix, 0.2 μM multiplexed primer mix (each primer at equal concentrations), and 1 μl of DNA extract in a total volume of 7 μl.…”

Section: Methodsmentioning

confidence: 99%

“…Each reaction contained 1X Qiagen Multiplex PCR Master Mix, 0.2 μM multiplexed primer mix (each primer at equal concentrations), and 1 μl of DNA extract in a total volume of 7 μl. The PCR protocol was modified from Shamblin et al [39] for multiplex PCR and consisted of an initial denaturation of 95°C for 15 min, 20 touchdown cycles of 94°C for 30 s, 60°C minus 0.5°C per cycle for 90 s and 72°C for 1 min, followed by 30 cycles of 94°C for 30 s, 50°C for 90 s and 72°C for 1 min, and a final elongation step of 60°C for 30 min. Multiplexed PCR products were run on a 3130xl Applied Biosystems Genetic Analyzer at the University of Idaho’s Laboratory for Ecological, Evolutionary, and Conservation Genetics.…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic, population genetic, and morphological analyses reveal evidence for one species of Eastern Indigo Snake (Drymarchon couperi)

Folt

Bauder

Spear

et al. 2018

Preprint

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30Accurate species delimitation and description are necessary to guide effective conservation 31 management of imperiled species. The Eastern Indigo Snake (Drymarchon couperi) is a large 32 species in North America that is federally-protected as Threatened under the Endangered Species 33 Act. Recently, two associated studies hypothesized that Drymarchon couperi is two species. 34 Here, we use diverse approaches to test the two-species hypothesis for D. couperi. Our analyses 35 reveal that (1) phylogenetic reconstruction in previous studies was based entirely on variance of 36 mitochondrial DNA sequence data, (2) microsatellite data demonstrate significant population 37 admixture and nuclear gene flow between mitochondrial lineages, and (3) morphological 38 analyses recover a single diagnosable species. Our results are inconsistent with the two-species 39 hypothesis, thus we reject it and formally place Drymarchon kolpobasileus into synonymy with 40 D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA may be 41 driven by high dispersal of males relative to females. We caution against species delimitation 42 exercises when one or few loci are used without evaluation of contemporary gene flow, 43 particularly species with strong sex-biased dispersal (e.g., squamates) and/or when results have 44 implications for ongoing conservation efforts. 45 46 65 Mississippi, Alabama, and the Florida panhandle [13]. 66 Current conservation management plans for D. couperi were developed under the 67 hypothesis that D. couperi represents a single species. However, this hypothesis was recently 68 challenged by Krysko et al. [15], who used DNA sequence analyses to describe two genetic 69 lineages of D. couperi -an Atlantic lineage, including populations in southeastern Georgia and 70 eastern peninsular Florida, and a Gulf lineage of populations in western and southern peninsular 71Florida and the Florida panhandle. This phylogeographic study was followed by a second paper 72 [16] that analyzed morphological variation between the Atlantic and Gulf lineages and provided 73 4 an official description of the Gulf lineage as a purported novel species, the Gulf Coast Indigo 74 Snake (Drymarchon kolpobasileus). 75 Given the conservation status of D. couperi (sensu lato), these results have potentially 76 important consequences for the conservation of Eastern Indigo Snakes. First, division of D. 77 couperi (sensu lato) into two smaller-ranged species results in two species with substantially 78 smaller population sizes that are, therefore, at greater risk of extinction (sensu [2]; e.g., [17]). 79 Second, conservation and recovery of two rare species requires more time and funds than one, 80 and both resources are in short supply. Finally, as noted by Krysko et al. [15], active 81 conservation management plans for D. couperi (sensu lato) include population repatriation 82 projects in Alabama and the Florida panhandle, where populations attributed to the Gulf lineage 83 presumably were...

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Isolation and characterization of microsatellite markers from the threatened eastern indigo snake (Drymarchon couperi)

Cited by 4 publications

References 10 publications

Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi

Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi

Multiscale assessment of functional connectivity: Landscape genetics of eastern indigo snakes in an anthropogenically fragmented landscape in central Florida

Phylogenetic, population genetic, and morphological analyses reveal evidence for one species of Eastern Indigo Snake (Drymarchon couperi)

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