Cordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has become a common invader of both fresh and brackish water ecosystems of North America and Europe. We describe 11 polymorphic microsatellite loci for this species. Preliminary analyses indicate that population substructure may contribute to departures from Hardy–Weinberg equilibrium. In addition, new loci failed to consistently amplify Cordylophora samples known to be genetically distant from those utilized in this study, indicating the presence of cryptic diversity within the taxon

Darlings, John A.

Drake, Carrie A.

Folino-Rorem, Nadine C.

Kuenzi, Ashley M.

Schable, Nancy A.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Environmental Protection Agency Papers U.S. Environmental Protection Agency 2008 Microsatellite loci for the invasive colonial hydrozoan Cordylophora caspia Nancy A. Schable U.S. Environmental Protection Agency, schable.nancy@epa.gov Ashley M. Kuenzi U.S. Environmental Protection Agency Carrie A. Drake U.S. Environmental Protection Agency Nadine C. Folino-Rorem Wheaton College - Wheaton John A. Darlings U.S. Environmental Protection Agency Follow this and additional works at: https://digitalcommons.unl.edu/usepapapers Schable, Nancy A.; Kuenzi, Ashley M.; Drake, Carrie A.; Folino-Rorem, Nadine C.; and Darlings, John A., "Microsatellite loci for the invasive colonial hydrozoan Cordylophora caspia" (2008). U.S. Environmental Protection Agency Papers. 151. https://digitalcommons.unl.edu/usepapapers/151 This Article is brought to you for free and open access by the U.S. Environmental Protection Agency at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Environmental Protection Agency Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Molecular Ecology Resources (2008) 8, 968–970 doi: 10.1111/j.1755-0998.2008.02109.x© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing LtdBlackwell Publishing LtdPERMANENT GENETIC RESOURCESMicrosatellite loci for the invasive colonial hydrozoan Cordylophora caspiaNANCY A.  SCHABLE,* ASHLEY M.  KUENZI ,† CARRIE A.  DRAKE,* NADINE C.  FOLINO-ROREM‡ and JOHN A.  DARLING§*Dynamac Corporation, c/o US EPA, Cincinnati, OH 45268, USA; †Independent Contractor to US EPA, Cincinnati, OH 45248, USA; ‡Biology Department, Wheaton College, Wheaton, IL 60187-5593, USA; §National Exposure Research Laboratory, Molecular Ecology Research Branch, US EPA, Cincinnati, OH 45268, USAAbstractCordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has becomea common invader of both fresh and brackish water ecosystems of North America andEurope. We describe 11 polymorphic microsatellite loci for this species. Preliminary analysesindicate that population substructure may contribute to departures from Hardy–Weinbergequilibrium. In addition, new loci failed to consistently amplify Cordylophora samplesknown to be genetically distant from those utilized in this study, indicating the presenceof cryptic diversity within the taxon.Keywords: Cordylophora caspia, cryptic diversity, hydrozoan, invasive species, microsatellite, primerdevelopmentReceived 6 November 2007; revision accepted 12 December 2007The Ponto-Caspian hydrozoan Cordylophora caspia recentlyhas established with increasing frequency throughoutNorth America and Western Europe (Folino 2000). Inaddition to potential impacts on native biological com-munities, Cordylophora frequently fouls intake pipes andfilters of power plants, impinging on plant efficiency andproduction (Folino 2000). Cordylophora possesses broadenvironmental tolerance, can reproduce asexually throughbudding, and has the ability to form a dormant menontstage capable of regenerating under favourable conditions(Folino 2000). These characteristics make it ideally suitedfor translocation through ballast water or ship fouling, andpotentially contribute to complex colonization scenarios.The high resolution markers described here will help toadequately assess invasion dynamics for this species.For microsatellite development, Cordylophora DNA wasisolated from fresh tissue of a single large Lake Michigancolony using DNAzol (Invitrogen), digested using BstU Ior EcoRV (New England Biolabs, NEB) and modified bylinker ligation as originally described by Hamilton et al.(1999). SuperSNX linker-ligated fragments were enrichedfor microsatellites and cloned following procedures descr-ibed by Glenn & Schable (2005). Specifically, fragmentswere hybridized with biotinylated microsatellite probes(TG12, AG12, AAG8, ATC8, AAC8, AAT12, ACT12, AAAC6,AAAG6, AATC6, AATG6, ACCT6, ACAG6, ACTC6, ACTG6,AAAT8, AACT8, AAGT8, ACAT8 and AGAT8), capturedusing streptavidin magnetic beads (NEB), recovered byamplification using SuperSNX24 forward primer, andcloned using the TOPO TA Cloning Kit (Invitrogen). Positivecolonies were amplified using a 25-μL polymerase chainreaction (PCR) containing 0.5 U Jumpstart Taq DNAPolymerase (Sigma), 1× PCR buffer, 0.4 μm each M13forward and reverse primers, 0.6 mm dNTPs, 2.0 mmMgCl2, 25 μg/mL bovine serum albumin (BSA) and 1.5 μLEscherichia coli grown overnight in Luria-Bertani broth.PCR products were cleaned using Antarctic Phosphataseand Exonuclease I (NEB) and sequenced using BigDyeTerminator version 3.1 Cycle Sequencing Reagent (AppliedBiosystems, ABI). Sequence reactions were purified usingDyeEx 96 (QIAGEN) and run on an ABI 3730xl DNAAnalyser. Sequence data were assembled and edited usingsequencher 4.6 (Gene Codes) and searched for micro-satellite repeats using msatcommander (Faircloth 2007).Thirty-two microsatellite primer pairs were designed witholigo 3.0 using a three-primer system (cf. Boutin-GanacheCorrespondence: Nancy Schable, Fax: 513-569-7554; E-mail: schable.nancy@epa.govP E R M A N E N T  G E N E T I C  R E S O U R C E S 969© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltdet al. 2001). New primer pairs were initially examined foramplification success and polymorphism using 11 indi-viduals. Each 15-μL PCR contained 0.375 U Jumpstart TaqDNA polymerase (Sigma), 1× PCR buffer, 1 μm untaggedprimer, 0.1 μm tagged primer, 0.9 μm FAM-labelled CAG orM13 universal primer, 1 mm dNTPs, 1.5 mm MgCl2, 25 μg/mL BSA and 10–100 ng of DNA template. All primers weretested using a 60 °C touchdown thermal cycling programme(Don et al. 1991). Cycling parameters were: 95 °C for 2 : 30;20 cycles of 95 °C for 20 s, 60 °C (–0.5 °C per cycle) for 20 s,72 °C for 30 s; 15 cycles of 95 °C for 20 s, 50 °C for 20 s, 72 °Cfor 30 s; 72 °C for 10 min. Samples were sized on an ABI3730xl DNA Analyser using GeneScan-500 LIZ size standard(ABI) and analysed using genemarker 1.60. Primer pairsthat failed to amplify, were difficult to score, or that appearedmonomorphic were not used for additional polymorphismtesting.Eleven polymorphic primer pairs were direct-labelledfor additional polymorphism testing. Most loci wereamplified with one of two touchdown PCR programmes.Touchdown cycling parameters were: 95 °C for 1 min; 12cycles of 95 °C for 30 s, 64 °C (or 59 °C) for 30 s (–0.8 °C percycle), 72 °C for 45 s; 23 cycles of 95 °C for 30 s, 54 °C (or49 °C) for 30 s, 72 °C for 45 s; 72 °C for 15 min. Each 15-μLPCR contained 0.5 U Taq DNA polymerase (QIAGEN),1× PCR buffer, 1 μm each forward and reverse primer,1 mm dNTPs, 1.6 mm MgCl2, and 10–100 ng of DNA tem-plate. Locus CC11 was amplified using a single annealingtemperature with cycling parameters: 94 °C for 5 min; 35cycles of 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min;72 °C for 15 min.A total of 128 individual Cordylophora colonies fromthree sites in Lake Michigan were tested for microsatellitepolymorphism. Loci were tested for linkage and Hardy–Weinberg equilibrium (HWE) using genepop 3.4 (Raymond& Rousset 1995); observed and expected heterozygositieswere determined using Arlequin (Schneider et al. 2000)(Table 1). No significant linkage disequilibrium was obser-ved, but five loci deviated significantly from HWE whendata were analysed as a single population, and three ofthese results remained significant after Bonferroni correction.When subpopulations were analysed individually, nosingle locus consistently deviated from HWE for all popu-lations. While this suggests the likelihood of underlyingpopulation structure, the possibility of null alleles cannotbe ruled out. Although the presence of clonal genotypeswas generally rare, several clones were observed in neigh-bouring colonies from the same sampling site (not shown).Clonal genotypes were removed from the data set for equi-librium analyses.Table 1 Characterization of microsatellite loci for Cordylophora caspia Locus Primer sequence Ta (°C) Repeat motif Size range (bp) NA n HE HO Np GenBankCC02 F: 5′-ACCCTTAGCACCTTACATA-3′ 64–54 TD (TACA)4(ACAT)10 218–310 21 122 0.91972 0.68033* 2 EU196241R: 5′-PET-CGCAACAACACCTAAAT-3′CC08 F: 5′-NED-ATCTGCCAATAAATAAGAAT-3′ 59–49 TD (AAC)19(ACG)10 205–250 12 121 0.75488 0.7438 1 EU196246R: 5′-TATCGACGCCATCTACT-3′CC10 F: 5′-VIC-ATTATCTCAAGCGAACATC-3′ 64–54 TD (ACAG)21 203–285 18 127 0.59923 0.55906 0 EU196247R: 5′-TGTCTTTCAATACCCAATG-3′CC11 F: 5′-PET-ACCGAAATCACTCTACACA-3′ 56 (AAC)9 188–208 5 115 0.55508 0.49565 0 EU196248R: 5′-GGTATTTCTTTCGTTCCTTA-3′CC12 F: 5′-PET-GTGTGCCATTGAGAAGTT-3′ 59–49 TD (AAAT)5 286–347 5 110 0.58813 0.53636 0 EU196249R: 5′-TGTATGTACTGAAACGACTATC-3′CC16 F: 5′-AATCGTTATCCTTATACATATT-3′ 59–49 TD (ACT)21 263–350 17 122 0.72509 0.70492 0 EU196250R: 5′-VIC-CTTTAATTTGCGATCTCT-3′CC22 F: 5′-FAM-CTCTTTTGGCTTCTTCTAC-3′ 64–54 TD (ACAT)8 151–236 24 119 0.9093 0.77311 1 EU196251R: 5′-TGAAACCGCATTACTAAA-3′CC29 F: 5′-NED-CATCGTCTGGTAGAGAGAA-3′ 59–49 TD (ACTC)6 170–182 3 125 0.55595 0.44* 1 EU196242R: 5′-ACCAAACCCGTATTCTAAT-3′CC30 F: 5′-FAM-GTTTTGCAGAATTGGTACA-3′ 59–49 TD (TTG)19 311–346 14 118 0.78485 0.72034 0 EU196243R: 5′-GTCGTCTCGTCGTTCA-3′CC31 F: 5′-CCATACGGCCTTACA-3′ 64–54 TD (AATC)23 189–269 22 123 0.86872 0.87805 0 EU196244R: 5′-FAM-TTTCTCTTTCTCCCCATA-3′CC32 F: 5′-NED-TAGACGTAACGCTTGC-3′ 64–54 TD (ACTC)17 255–393 39 117 0.89645 0.64103* 2 EU196245R: 5′-CACTTTTCTATGATACCTGC-3′Ta, annealing temperature; TD, touchdown PCR programme; NA, number of alleles; n, number of individuals tested; HE, expected heterozygosity; HO, observed heterozygosity; * denotes loci that deviate significantly from HWE after Bonferroni correction for multiple tests; Np, number of subpopulations (out of three total) exhibiting significant deviation from HWE (after correction) when populations are tested independently. Fluorescent labels (ABI) are shown in bold.970 P E R M A N E N T  G E N E T I C  R E S O U R C E S© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing LtdGiven the taxonomic uncertainty surrounding the genusCordylophora (Folino 2000), primers were tested on samplesthat, although nominally conspecific, are genetically distinctenough to represent cryptic sister species to the LakeMichigan samples (N.C. Folino-Rorem, C. b’Ausilio, andJ.A. Darling, unpublished data). Failure to consistentlyamplify these samples (Table 2) indicates that these primersare not useful for all specimens currently recognized asCordylophora caspia and further indicates the need fortaxonomic re-evaluation of the genus.AcknowledgementsWe thank Suzanne Jackson for helpful discussions. The US Envi-ronmental Protection Agency through its Office of Research andDevelopment funded and collaborated in the research describedhere under Contract EPD06096 to Dynamac Corporation. It hasbeen subjected to Agency review and approved for publication.Mention of trade names or commercial products does not constituteendorsement or recommendation for use.ReferencesBoutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001)M13-tailed primers improve the readability and usability ofmicrosatellite analyses performed with two different allele-sizingmethods. BioTechniques, 31, 24–28.Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)‘Touchdown’ PCR to circumvent spurious priming during geneamplification. Nucleic Acids Research, 19, 4008.Faircloth BC (2007) msatcommander: detection of microsatelliterepeat arrays and automated, locus-specific primer design.Molecular Ecology Notes. doi: 10.1111/j.1471–8286.2007.01884.x.Folino NC (2000) The freshwater expansion and classifica-tion of the colonial hydroid Cordylophora (Phylum Cni-daria, Class Hydrozoa). In: Marine Bioinvasions: Proceedings ofthe First National Conference January 24–27, 1999 (ed. Pederson J),pp. 139–144. Massachusetts Institute of Technology SeaGrant College Program, Cambridge, Massachusetts.Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. In:Methods in Enzymology 395, Molecular Evolution: Producing theBiochemical Data, Part B (eds Zimmer EA, Roalson EH), pp. 202–222. Academic Press, San Diego, California.Hamilton MB, Pincus EL, Di Fiore A, Flesher RC (1999) Universallinker and ligation procedures for construction of genomicDNA libraries enriched for microsatellites. BioTechniques, 27,500–507.Raymond M, Rousset F (1995) genepop (version 1.2): populationgenetics software for exact tests and ecumenicism. Journal ofHeredity, 86, 248–249.Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN ver. 2.000: ASoftware for Population Genetics Data Analysis. Genetics andBiometry Laboratory. University of Geneva, Switzerland.Table 2 Allele sizes (in base pairs) observed in Cordylophora samples genetically distant from those used for polymorphism testing. Geneticdistance (p) is based on uncorrected p-distance (percentage) from the Lake Michigan sample at the mitochondrial COI locus. GenBankaccession numbers for COI sequences are indicated; five samples from Petaluma River with the same COI haplotype were all tested forpolymorphism. ‘−’ denotes amplification failureSample location GenBank p CC02 CC08 CC10 CC11 CC12 CC16 CC22 CC29 CC30 CC31 CC32Lake Balaton, Hungary EF540787 8.02 — 274/319 — — 339/339 — 169/229 — — — —Petaluma River, California EF540792 12.6 — 229/229 — — — — — — — — —Petaluma River, California EF540792 12.6 — 229/229 — — — — — — — — —Petaluma River, California EF540792 12.6 — 229/236 — — — — — — — — —Petaluma River, California EF540792 12.6 — — — — — — — — — — —Petaluma River, California EF540792 12.6 — — — — — — — — — — —Napa River, California EF540790 12.6 — — — — — — — — — — —Exeter, New Hampshire EF540782 14.6 — — — — — — — — — — —Ryck River, Germany EF540784 14.6 — — — — — — — — — — —

Microsatellite loci for the invasive colonial hydrozoan
\u3ci\u3eCordylophora caspia\u3c/i\u3e

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
U.S. Environmental Protection Agency Papers U.S. Environmental Protection Agency 
2008 
Microsatellite loci for the invasive colonial hydrozoan 
Cordylophora caspia 
Nancy A. Schable 
U.S. Environmental Protection Agency, schable.nancy@epa.gov 
Ashley M. Kuenzi 
U.S. Environmental Protection Agency 
Carrie A. Drake 
U.S. Environmental Protection Agency 
Nadine C. Folino-Rorem 
Wheaton College - Wheaton 
John A. Darlings 
U.S. Environmental Protection Agency 
Follow this and additional works at: https://digitalcommons.unl.edu/usepapapers 
Schable, Nancy A.; Kuenzi, Ashley M.; Drake, Carrie A.; Folino-Rorem, Nadine C.; and Darlings, John A., 
"Microsatellite loci for the invasive colonial hydrozoan Cordylophora caspia" (2008). U.S. Environmental 
Protection Agency Papers. 151. 
https://digitalcommons.unl.edu/usepapapers/151 
This Article is brought to you for free and open access by the U.S. Environmental Protection Agency at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Environmental 
Protection Agency Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Molecular Ecology Resources (2008) 8, 968–970 doi: 10.1111/j.1755-0998.2008.02109.x
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
Blackwell Publishing LtdPERMANENT GENETIC RESOURCES
Microsatellite loci for the invasive colonial hydrozoan 
Cordylophora caspia
NANCY A.  SCHABLE,* ASHLEY M.  KUENZI ,† CARRIE A.  DRAKE,* NADINE C.  FOLINO-ROREM‡ 
and JOHN A.  DARLING§
*Dynamac Corporation, c/o US EPA, Cincinnati, OH 45268, USA; †Independent Contractor to US EPA, Cincinnati, OH 45248, USA; 
‡Biology Department, Wheaton College, Wheaton, IL 60187-5593, USA; §National Exposure Research Laboratory, Molecular Ecology 
Research Branch, US EPA, Cincinnati, OH 45268, USA
Abstract
Cordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has become
a common invader of both fresh and brackish water ecosystems of North America and
Europe. We describe 11 polymorphic microsatellite loci for this species. Preliminary analyses
indicate that population substructure may contribute to departures from Hardy–Weinberg
equilibrium. In addition, new loci failed to consistently amplify Cordylophora samples
known to be genetically distant from those utilized in this study, indicating the presence
of cryptic diversity within the taxon.
Keywords: Cordylophora caspia, cryptic diversity, hydrozoan, invasive species, microsatellite, primer
development
Received 6 November 2007; revision accepted 12 December 2007
The Ponto-Caspian hydrozoan Cordylophora caspia recently
has established with increasing frequency throughout
North America and Western Europe (Folino 2000). In
addition to potential impacts on native biological com-
munities, Cordylophora frequently fouls intake pipes and
filters of power plants, impinging on plant efficiency and
production (Folino 2000). Cordylophora possesses broad
environmental tolerance, can reproduce asexually through
budding, and has the ability to form a dormant menont
stage capable of regenerating under favourable conditions
(Folino 2000). These characteristics make it ideally suited
for translocation through ballast water or ship fouling, and
potentially contribute to complex colonization scenarios.
The high resolution markers described here will help to
adequately assess invasion dynamics for this species.
For microsatellite development, Cordylophora DNA was
isolated from fresh tissue of a single large Lake Michigan
colony using DNAzol (Invitrogen), digested using BstU I
or EcoRV (New England Biolabs, NEB) and modified by
linker ligation as originally described by Hamilton et al.
(1999). SuperSNX linker-ligated fragments were enriched
for microsatellites and cloned following procedures descr-
ibed by Glenn & Schable (2005). Specifically, fragments
were hybridized with biotinylated microsatellite probes
(TG12, AG12, AAG8, ATC8, AAC8, AAT12, ACT12, AAAC6,
AAAG6, AATC6, AATG6, ACCT6, ACAG6, ACTC6, ACTG6,
AAAT8, AACT8, AAGT8, ACAT8 and AGAT8), captured
using streptavidin magnetic beads (NEB), recovered by
amplification using SuperSNX24 forward primer, and
cloned using the TOPO TA Cloning Kit (Invitrogen). Positive
colonies were amplified using a 25-μL polymerase chain
reaction (PCR) containing 0.5 U Jumpstart Taq DNA
Polymerase (Sigma), 1× PCR buffer, 0.4 μm each M13
forward and reverse primers, 0.6 mm dNTPs, 2.0 mm
MgCl2, 25 μg/mL bovine serum albumin (BSA) and 1.5 μL
Escherichia coli grown overnight in Luria-Bertani broth.
PCR products were cleaned using Antarctic Phosphatase
and Exonuclease I (NEB) and sequenced using BigDye
Terminator version 3.1 Cycle Sequencing Reagent (Applied
Biosystems, ABI). Sequence reactions were purified using
DyeEx 96 (QIAGEN) and run on an ABI 3730xl DNA
Analyser. Sequence data were assembled and edited using
sequencher 4.6 (Gene Codes) and searched for micro-
satellite repeats using msatcommander (Faircloth 2007).
Thirty-two microsatellite primer pairs were designed with
oligo 3.0 using a three-primer system (cf. Boutin-Ganache
Correspondence: Nancy Schable, Fax: 513-569-7554; 
E-mail: schable.nancy@epa.gov
P E R M A N E N T  G E N E T I C  R E S O U R C E S 969
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
et al. 2001). New primer pairs were initially examined for
amplification success and polymorphism using 11 indi-
viduals. Each 15-μL PCR contained 0.375 U Jumpstart Taq
DNA polymerase (Sigma), 1× PCR buffer, 1 μm untagged
primer, 0.1 μm tagged primer, 0.9 μm FAM-labelled CAG or
M13 universal primer, 1 mm dNTPs, 1.5 mm MgCl2, 25 μg/
mL BSA and 10–100 ng of DNA template. All primers were
tested using a 60 °C touchdown thermal cycling programme
(Don et al. 1991). Cycling parameters were: 95 °C for 2 : 30;
20 cycles of 95 °C for 20 s, 60 °C (–0.5 °C per cycle) for 20 s,
72 °C for 30 s; 15 cycles of 95 °C for 20 s, 50 °C for 20 s, 72 °C
for 30 s; 72 °C for 10 min. Samples were sized on an ABI
3730xl DNA Analyser using GeneScan-500 LIZ size standard
(ABI) and analysed using genemarker 1.60. Primer pairs
that failed to amplify, were difficult to score, or that appeared
monomorphic were not used for additional polymorphism
testing.
Eleven polymorphic primer pairs were direct-labelled
for additional polymorphism testing. Most loci were
amplified with one of two touchdown PCR programmes.
Touchdown cycling parameters were: 95 °C for 1 min; 12
cycles of 95 °C for 30 s, 64 °C (or 59 °C) for 30 s (–0.8 °C per
cycle), 72 °C for 45 s; 23 cycles of 95 °C for 30 s, 54 °C (or
49 °C) for 30 s, 72 °C for 45 s; 72 °C for 15 min. Each 15-μL
PCR contained 0.5 U Taq DNA polymerase (QIAGEN),
1× PCR buffer, 1 μm each forward and reverse primer,
1 mm dNTPs, 1.6 mm MgCl2, and 10–100 ng of DNA tem-
plate. Locus CC11 was amplified using a single annealing
temperature with cycling parameters: 94 °C for 5 min; 35
cycles of 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min;
72 °C for 15 min.
A total of 128 individual Cordylophora colonies from
three sites in Lake Michigan were tested for microsatellite
polymorphism. Loci were tested for linkage and Hardy–
Weinberg equilibrium (HWE) using genepop 3.4 (Raymond
& Rousset 1995); observed and expected heterozygosities
were determined using Arlequin (Schneider et al. 2000)
(Table 1). No significant linkage disequilibrium was obser-
ved, but five loci deviated significantly from HWE when
data were analysed as a single population, and three of
these results remained significant after Bonferroni correction.
When subpopulations were analysed individually, no
single locus consistently deviated from HWE for all popu-
lations. While this suggests the likelihood of underlying
population structure, the possibility of null alleles cannot
be ruled out. Although the presence of clonal genotypes
was generally rare, several clones were observed in neigh-
bouring colonies from the same sampling site (not shown).
Clonal genotypes were removed from the data set for equi-
librium analyses.
Table 1 Characterization of microsatellite loci for Cordylophora caspia 
Locus Primer sequence Ta (°C) Repeat motif Size range (bp) NA n HE HO Np GenBank
CC02 F: 5′-ACCCTTAGCACCTTACATA-3′ 64–54 TD (TACA)4(ACAT)10 218–310 21 122 0.91972 0.68033* 2 EU196241
R: 5′-PET-CGCAACAACACCTAAAT-3′
CC08 F: 5′-NED-ATCTGCCAATAAATAAGAAT-3′ 59–49 TD (AAC)19(ACG)10 205–250 12 121 0.75488 0.7438 1 EU196246
R: 5′-TATCGACGCCATCTACT-3′
CC10 F: 5′-VIC-ATTATCTCAAGCGAACATC-3′ 64–54 TD (ACAG)21 203–285 18 127 0.59923 0.55906 0 EU196247
R: 5′-TGTCTTTCAATACCCAATG-3′
CC11 F: 5′-PET-ACCGAAATCACTCTACACA-3′ 56 (AAC)9 188–208 5 115 0.55508 0.49565 0 EU196248
R: 5′-GGTATTTCTTTCGTTCCTTA-3′
CC12 F: 5′-PET-GTGTGCCATTGAGAAGTT-3′ 59–49 TD (AAAT)5 286–347 5 110 0.58813 0.53636 0 EU196249
R: 5′-TGTATGTACTGAAACGACTATC-3′
CC16 F: 5′-AATCGTTATCCTTATACATATT-3′ 59–49 TD (ACT)21 263–350 17 122 0.72509 0.70492 0 EU196250
R: 5′-VIC-CTTTAATTTGCGATCTCT-3′
CC22 F: 5′-FAM-CTCTTTTGGCTTCTTCTAC-3′ 64–54 TD (ACAT)8 151–236 24 119 0.9093 0.77311 1 EU196251
R: 5′-TGAAACCGCATTACTAAA-3′
CC29 F: 5′-NED-CATCGTCTGGTAGAGAGAA-3′ 59–49 TD (ACTC)6 170–182 3 125 0.55595 0.44* 1 EU196242
R: 5′-ACCAAACCCGTATTCTAAT-3′
CC30 F: 5′-FAM-GTTTTGCAGAATTGGTACA-3′ 59–49 TD (TTG)19 311–346 14 118 0.78485 0.72034 0 EU196243
R: 5′-GTCGTCTCGTCGTTCA-3′
CC31 F: 5′-CCATACGGCCTTACA-3′ 64–54 TD (AATC)23 189–269 22 123 0.86872 0.87805 0 EU196244
R: 5′-FAM-TTTCTCTTTCTCCCCATA-3′
CC32 F: 5′-NED-TAGACGTAACGCTTGC-3′ 64–54 TD (ACTC)17 255–393 39 117 0.89645 0.64103* 2 EU196245
R: 5′-CACTTTTCTATGATACCTGC-3′
Ta, annealing temperature; TD, touchdown PCR programme; NA, number of alleles; n, number of individuals tested; HE, expected 
heterozygosity; HO, observed heterozygosity; * denotes loci that deviate significantly from HWE after Bonferroni correction for multiple 
tests; Np, number of subpopulations (out of three total) exhibiting significant deviation from HWE (after correction) when populations are 
tested independently. Fluorescent labels (ABI) are shown in bold.
970 P E R M A N E N T  G E N E T I C  R E S O U R C E S
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
Given the taxonomic uncertainty surrounding the genus
Cordylophora (Folino 2000), primers were tested on samples
that, although nominally conspecific, are genetically distinct
enough to represent cryptic sister species to the Lake
Michigan samples (N.C. Folino-Rorem, C. b’Ausilio, and
J.A. Darling, unpublished data). Failure to consistently
amplify these samples (Table 2) indicates that these primers
are not useful for all specimens currently recognized as
Cordylophora caspia and further indicates the need for
taxonomic re-evaluation of the genus.
Acknowledgements
We thank Suzanne Jackson for helpful discussions. The US Envi-
ronmental Protection Agency through its Office of Research and
Development funded and collaborated in the research described
here under Contract EPD06096 to Dynamac Corporation. It has
been subjected to Agency review and approved for publication.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
References
Boutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001)
M13-tailed primers improve the readability and usability of
microsatellite analyses performed with two different allele-sizing
methods. BioTechniques, 31, 24–28.
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)
‘Touchdown’ PCR to circumvent spurious priming during gene
amplification. Nucleic Acids Research, 19, 4008.
Faircloth BC (2007) msatcommander: detection of microsatellite
repeat arrays and automated, locus-specific primer design.
Molecular Ecology Notes. doi: 10.1111/j.1471–8286.2007.01884.x.
Folino NC (2000) The freshwater expansion and classifica-
tion of the colonial hydroid Cordylophora (Phylum Cni-
daria, Class Hydrozoa). In: Marine Bioinvasions: Proceedings of
the First National Conference January 24–27, 1999 (ed. Pederson J),
pp. 139–144. Massachusetts Institute of Technology Sea
Grant College Program, Cambridge, Massachusetts.
Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. In:
Methods in Enzymology 395, Molecular Evolution: Producing the
Biochemical Data, Part B (eds Zimmer EA, Roalson EH), pp. 202–
222. Academic Press, San Diego, California.
Hamilton MB, Pincus EL, Di Fiore A, Flesher RC (1999) Universal
linker and ligation procedures for construction of genomic
DNA libraries enriched for microsatellites. BioTechniques, 27,
500–507.
Raymond M, Rousset F (1995) genepop (version 1.2): population
genetics software for exact tests and ecumenicism. Journal of
Heredity, 86, 248–249.
Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN ver. 2.000: A
Software for Population Genetics Data Analysis. Genetics and
Biometry Laboratory. University of Geneva, Switzerland.
Table 2 Allele sizes (in base pairs) observed in Cordylophora samples genetically distant from those used for polymorphism testing. Genetic
distance (p) is based on uncorrected p-distance (percentage) from the Lake Michigan sample at the mitochondrial COI locus. GenBank
accession numbers for COI sequences are indicated; five samples from Petaluma River with the same COI haplotype were all tested for
polymorphism. ‘−’ denotes amplification failure
Sample location GenBank p CC02 CC08 CC10 CC11 CC12 CC16 CC22 CC29 CC30 CC31 CC32
Lake Balaton, Hungary EF540787 8.02 — 274/319 — — 339/339 — 169/229 — — — —
Petaluma River, California EF540792 12.6 — 229/229 — — — — — — — — —
Petaluma River, California EF540792 12.6 — 229/229 — — — — — — — — —
Petaluma River, California EF540792 12.6 — 229/236 — — — — — — — — —
Petaluma River, California EF540792 12.6 — — — — — — — — — — —
Petaluma River, California EF540792 12.6 — — — — — — — — — — —
Napa River, California EF540790 12.6 — — — — — — — — — — —
Exeter, New Hampshire EF540782 14.6 — — — — — — — — — — —
Ryck River, Germany EF540784 14.6 — — — — — — — — — — —


https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1151&amp;context=usepapapers

Microsatellite loci for the invasive colonial hydrozoan \u3ci\u3eCordylophora caspia\u3c/i\u3e

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