Ants are predestined for studies of the genetic consequences of habitat fragmentation due to the combination of sessile colonies, which can be recorded with ...
Springer 2005
Conservation Genetics (2005) 6:859–861 DOI 10.1007/s10592-005-9033-5
Isolation of polymorphic microsatellite loci for the study of habitat fragmentation in the harvester ant Messor structor W. Arthofer1,*, , B. C. Schlick-Steiner1,2, , F. M. Steiner1,2, , H. Konrad1, X. Espadaler3 & C. Stauffer1 1
Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection; 2Department of Integrative Biology, Institute of Zoology, BOKU, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Str. 33, Vienna, Wien, A-1180, Austria; 3C.R.E.A.F., Universitat Auto`noma de Barcelona, E-08193, Bellaterra, Spain (*Corresponding author: Phone: +43-13686352-25; Fax: +43-1-3686352-97; E-mail: wolfgang.arthofer@ boku.ac.at) Received 12 October 2004; accepted 21 December 2004
Key words: conservation, habitat fragmentation, Messor structor, microsatellites, social insects
Ants are predestined for studies of the genetic consequences of habitat fragmentation due to the combination of sessile colonies, which can be recorded with topographic exactness (Steiner and Schlick-Steiner 2002), and of vagile alate sexuals, which ensure gene flow between moderately isolated habitat islands. The social organisation allows conclusions about supra-organismal colony characters like queen number and dispersal strategy. Further, the male-haploid genetic system facilitates genetic mapping (Crozier et al. 1997) and the early recognition of inbreeding effects (Chapman and Bourke 2001). The genetic effects of habitat fragmentation on ants have only been addressed once (Gyllenstrand and Seppa¨ 2003). Harvesting ants such as Messor structor (Latreille, 1798) are keystone species. M. structor in Central Europe is a stenotopic inhabitant of natural xerothermous grassland (Schlick-Steiner et al. 2003). Genomic DNA of 25 degastrated M. structor workers from two Austrian populations was extracted using the Sigma GenElute kit. According to the FIASCO protocol (Fast Isolation by AFLP of Sequences COntaining repeats) of Zane et al. (2002), a one-step reaction for MseI digestion and W. Arthofer, B. C. Schlick-Steiner and F. M. Steiner contributed equally to this work.
adaptor ligation followed by recovery PCR with adaptor primers was performed. PCR products were denatured and annealed to 100 pM 5¢-biotinylated oligoprobes (ac)8 and (ag)8 in the presence of 4.2 SSC and 0.07% SDS for 15 min at room temperature. Agarose bound avidin D particles (Vectrex) suspended in TBT (100 mM Tris, 0.1% Tween20, pH 7.5) were added to the hybrids and incubated at 50 C under constant agitation for 60 min. Captured products were separated from the buffer by centrifugation. After repeated stringent washes (0.2 SSC / 0.1% SDS) the remaining products were eluted into a buffer containing 10 mM Tris and 0.1 mM EDTA. Another recovery PCR was performed as described above. PCR products were purified with the QIAquick PCR purification kit (Quiagen), cloned into the pGEMT vector (Promega) and used for transformation of JM109 competent E. coli cells. About 276 white colonies were inoculated onto masterplates and transferred to Nylon membranes (Roche 1 699 075) as suggested by the manufacturer. After overnight hybridization at 60 C with 5¢ biotinylated SSR oligoprobes and subsequent washing steps screening was performed with the Biotin Luminescent Detection Kit (Roche). 19 positive colonies were transferred to liquid culture and plasmid DNA was extracted by alkaline lysis (Sambrook et al. 1989) and sequenced for deter-
TGATACGAGCGAGTGGAAC TCCGTTTTTGTAGTGCGTC GATGCGAGCGACTCCAATA CTCGAGCAGTTTTCCAGAC TTGGCTTTTTGCAGTTTCG TCTACTGAGCTCGGCGAC CGCTCGTTAGTGGTCGATAC TTCTCACCGTTCTCTCAGC CACGTAGGACGAACGTTG TAGAAATGGGTAGGCGTTCG CGTGCTTTGAGGAAGGGAT AGCCTCTCTGTCTTGTTCTC CGGCACGGAGACAATACTTC GCTGTTCGGCGAAAACTATC
Ms1A
(TC)4(TC)25(TC)4
(AG)11
(AG)20
(CA)17(CG)5
(CG)4(TG)13(TA)6
(TC)17
(AG)10(AG)8
Repeat motif
164–236
152–178
172 211
158–221
149–195
173–210
174–202
141–186
Allele size range (bp)
194
194
197
193
159
Clone allele (bp)
21
5
17
8
11
10
4
5
4
8
6
7
3
5
1
–
–
–
–
2
1
a
2
1
1
2
–
1
2
M. mar. n=2 i=4
0.59/0.83c
0.20/0.59c
0.57/0.68c
0.3/0.72c
0.76/0.85c
0.60/0.68
0.57/0.63
M. str. from Retz n=34 i=54
M. bou. n=4 i=4
M. str. n=44 i=102
M. bar. n=6 i=8
Heterozygosityb
Number of allelesa
Nucleotide sequences of the microsatellite clones are registered in the GenBank under the accession numbers AY735104–AY735110. Number of different alleles found (n = number of sampled nests, i = number of sampled workers). b Observed (Ho) and expected (He) heterozygosity in the format: Ho/He (n = number of sampled nests, i = number of sampled workers). c Significant deviations from Hardy-Weinberg expectations after Bonferroni corrections.
Ms2D
Ms2C
Ms2A
Ms1F
Ms1E
Ms1B
Primer sequences (5¢–3¢)
Locus
Table 1. Primer sequences and characteristics of seven M. structor = M. str. microsatellite loci with indication of cross species amplification in M. barbarus = M. bar., M. bouvieri = M. bou., M. maroccanus = M. mar
860
861 mination of repeat regions. Eight out of thirteen sequences containing microsatellites were selected for subsequent primer design. Amplifications for population screening were performed in 15 ll reaction volume containing 1 reaction buffer, 0.2 mM dNTPs, 0.2 lM of each primer, 0.6 U of Taq polymerase (Biotherm), 60 ng DNA and 11.58 ll H2Odd. Cycling conditions were 94 C for 30 sec, 60 C for 1 min, 72 C for 45 sec for 32 cycles with an initial denaturation step at 94 C for 5 min and a final extension step at 68 C for 20 min. The primers for loci Ms1A, Ms1B, Ms1E, Ms2A and Ms1F, Ms2C, Ms2D, respectively (Table 1) were designed for simultaneous migration of their PCR products in a single run each of an ABI PRISM 310 genetic analyzer (Applied Biosystems). The forward primers were 5¢-end labelled with a fluorescent dye, either 6-FAM, HEX or TET. GeneScan 500-TAMRA was used as internal size standard. GENESCAN and GENOTYPER software (Applied Biosystems) was used for fragment analysis data processing. Fifty-four workers of M. structor from 34 nests of 34 habitat islands from a 60 km2 region near Retz, Austria were analysed. Ten additional populations were screened (Austria, Croatia, Germany). The seven loci revealed four to 21 alleles (Table 1). The Retz population had expected heterozygosity values of 0.59–0.85 per locus. Calculations with GENEPOP (Raymond and Rousset 1995) revealed significant deviations from Hardy–Weinberg equilibrium (HWE) for all loci except locus Ms1A and Ms1B (after Bonferroni correction for multiple comparisons). When three subpopulations (22, 16 and 22 samples, respectively) were considered, the number of loci deviating from HWE were reduced to 2–4 per subpopulation, with changing identity of the deviating loci (data not shown). Linkage disequilibrium tested for with GENEPOP (Raymond and Rousset 1995) was detected for the following seven out of the total of 21 pairs of loci: Ms1E with each of Ms1B, Ms1F, Ms2D, Ms2C; Ms2D with Ms1B and Ms1F; Ms1B with Ms2A after Bonferroni correction. When three subpopulations (22, 16 and 22 samples, respectively) were considered, the number of pairs of loci with linkage disequilibrium was 0, 0 and 5 in the single subpopulations (data
not shown). Deviations from HWE and linkage disequilibrium might be partially due to habitat fragmentation. Cross species amplification was performed using two Spanish populations of M. barbarus and M. bouvieri and one Spanish population of M. marocanus. The number of sampled individuals per population and allele data are given in Table 1. The microsatellite loci described provide the tools to use M. structor as model organism for the study of the influence of habitat fragmentation on social structure and population structure of social insects. The resulting estimates will help to define adequate conservation units.
Acknowledgements To B. Becker, A. Buschinger, E. Christian, R. Crozier, K. Go´mez, G. Heller, R. Huertas, K. Moder, K. Sefc, B. Seifert, M. Stachowitsch. This work was funded by the Austrian Science Foundation P16794-B06. References Chapman R, Bourke AFG (2001) The influence of sociality on the conservation biology of social insects. Ecol. Lett., 4, 650– 662. Crozier RH, Oldroyd BP, Tay WT, Kaufmann BE, Johnson RN, Carew ME, Jennings KM (1997) Molecular advances in understanding social insect population structure. Electrophoresis, 18, 1672–1675. Gyllenstrand N, Seppa¨ P (2003) Conservation genetics of the wood ant, Formica lugubris, in a fragmented landscape. Mol. Ecol., 12, 2931–2940. Raymond M, Rousset F (1995) GENEPOP Version 1.2: Population genetics software for exact tests and ecumenism. J. Heredity, 86, 248–249. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York. Schlick-Steiner BC, Steiner FM, Scho¨dl S (2003) Rote Listen ausgewa¨hlter Tiergruppen Niedero¨sterreichs – Ameisen (Hymenoptera: Formicidae), 1. Fassung 2002, Amt der NO¨ Landesregierung, Abteilung Naturschutz, St. Po¨lten. Steiner FM, Schlick-Steiner BC (2002) Einsatz von Ameisen in der naturschutzfachlichen Praxis. Begru¨ndungen ihrer vielfa¨ltigen Eignung im Vergleich zu anderen Tiergruppen. Naturschutz und Landschaftsplanung, 34, 5–13. Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation: a review. Mol. Ecol., 11, 1–16.
