Characterization of microsatellite loci for the European species of hermit beetles (Osmoderma
spp.) (Coleoptera: Cetoniidae)
A. Oleksaa, K. Meyzaa, L. Cizekb,c, L. Dragb,c
(a) Institute of Experimental Biology, Kazimierz Wielki University, Chodkiewicza 30, 85-064 Bydgoszcz,
Poland
(b) Faculty of Sciences, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech
Republic
(c) Institute of Entomology, Biology Centre CAS, v. v. i., Branisovska 31, 37005 Ceske Budejovice,
Czech Republic
*corresponding author: e-mail: olek@ukw.edu.pl
Abstract
Fourteen microsatellite loci are described for the eastern European hermit beetles, Osmoderma
barnabita, a vulnerable and internationally protected species associated with mature hollow trees.
Based on 45 individuals from Poland, 13 of 14 loci were polymorphic. The number of alleles per
polymorphic locus ranged from 2 to 13, and the observed and expected heterozygosity was 0 – 0.889
(mean = 0.231) and 0.033 – 0.868 (mean = 0.253), respectively. Three loci showed deviation from
Hardy–Weinberg equilibrium. The probability of null alleles was negligible for all but one locus. Seven
loci cross-amplified in the closely related Osmoderma eremita. The markers reported here can be
valuable tool for detecting genetic structure and gene flow in O. barnabita.
Keywords: saproxylic insects; 454-Pyrosequencing; SSR markers; conservation genetics
European species of hermit beetles (genus Osmoderma Lep. et Serv.) are closely related,
internationally protected representatives of the diverse and endangered fauna associated with
hollows in senescent trees (Ranius et al. 2005). All European hermit beetles were regarded as a single
species Osmoderma eremita (Scop.). Examination of COI gene of the mtDNA, however, indicated that
the taxon should be split into several species, origin of which result from long isolation in Pleistocene
refugia (Audisio et al. 2009). In this study, we report 14 microsatellite loci developed for Osmoderma
barnabita Motsch., the species belonging to the eastern clade within O. eremita complex, and their
cross-amplification in the western species O. eremita s. str. Both taxa are protected and red-listed in
many European countries and protected under the EU Habitats Directive. Globally, they are classified
as vulnerable. Highly informative, co-dominant markers are urgently needed for all species of hermit
beetles to provide efficient tools for clarifying their taxonomic status and detecting genetic structure
and gene flow at fine spatial scales (Oleksa et al. 2013).
The microsatellites were developed using SSR-enrichment and 454-pyrosequencing as described in
Drag et al. (2013). Genomic DNA was extracted from one individual collected in SE Czech Republic
using the Genomic DNA Mini Kit Tissue (Geneaid).
The final O. barnabita dataset comprised of 78,536 reads (average length = 517 bp). Combining
homologous reads, we obtained 18 unique SSR loci that were tested using M13-tailed assay on 12
individuals. Finally, 14 pairs of primers with consistent amplification were chosen, and their
variability was screened based on 45 individuals of O. barnabita from northern Poland and 45
individuals of O. eremita from southern Sweden. All loci were amplified using two multiplex PCR
reactions (Electronic Supplementary Material 1), each of them were performed in a reaction mixture
(final volume of 10μL) containing 5μL of 2× Multiplex PCR master mix (Qiagen), 10ng of DNA
template, 100 to 150nM of each fluorescently-labelled forward primer (6-FAM, NED, PET, or VIC
dyes—Applied Biosystems, ABI), and 100 to 150nM of each reverse primer. The PCR products were
genotyped using a 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) and the sizes
were scored using GeneMarker v2.6.3 Software (SoftGenetics LLC).
All tested loci except for Hermit_08 were polymorphic in O. barnabita, while out of 7 loci amplified in
O. eremita only two (Hermit_10 and Hermit_13) were polymorphic (Table 1). The number of alleles
per locus in polymorphic loci ranged from 2 to 13 in O. barnabita, and was equal to 2 in O. eremita.
Based on the analysis in INEST 2.0 (Chybicki and Burczyk 2009), observed and expected
heterozygosities (HO and HE) ranged from 0 to 0.889 (mean = 0.231) and 0 to 0.855 (mean = 0.253),
respectively. No evidences of linkage disequilibrium between loci were detected. Three loci in O.
barnabita (Hermit_02, Hermit_07 and Hermit_12) showed deviation from Hardy-Weinberg
equilibrium. The probability of the presence of null alleles was negligible (<0.03) for all loci except for
Hermit_12, in which it amounted to 0.19.
In conclusion, at least 12 of 14 markers presented here seem to be a suitable tool to explore the
genetic structure of the eastern hermit beetle O. barnabita. Although their usefulness for studies of
the western species O. eremita s. str. is much more limited, all loci could represent valuable tool for
investigating potential hybridisation between the two taxa. Also, their polymorphism might increase
in populations found further from the northern distribution limit of O.eremita.
Acknowledgments
This study was supported by the research grant from Polish Ministry of Science and Higher Education
(N N304 4175 33 to AO) and the Grant Agency of the University of South Bohemia (04-168/2013/P).
The authors would like to thank Robert Gawroński, Mattias C. Larsson, Glenn P. Svensson and Pavel
Šebek for help with sample collection, Jiří Košnar and Ewa Sztupecka for their laboratory work and
Igor Chybicki for discussion of the results.
References
Audisio P, Brustel H, Carpaneto GM, et al. (2009) Data on molecular taxonomy and genetic diversification
of the European Hermit beetles, a species complex of endangered insects (Coleoptera: Scarabaeidae,
Cetoniinae, Osmoderma ). J Zool Syst Evol Res 47:88–95. doi: 10.1111/j.1439-0469.2008.00475.x
Chybicki IJ, Burczyk J (2009) Simultaneous estimation of null alleles and inbreeding coefficients. J Hered
100:106–13. doi: 10.1093/jhered/esn088
Drag L, Kosnar J, Cizek L (2013) Development and characterization of ten polymorphic microsatellite loci
for the Great Capricorn beetle (Cerambyx cerdo) (Coleoptera: Cerambycidae). Conserv Genet Resour
5:907–909. doi: 10.1007/s12686-013-9930-8
Oleksa A, Chybicki IJ, Gawroński R, et al. (2013) Isolation by distance in saproxylic beetles may increase
with niche specialization. J Insect Conserv 17:219–233. doi: 10.1007/s10841-012-9499-7
Ranius T, Aguado L, Antonsson K, et al. (2005) Osmoderma eremita (Coleoptera, Scarabaeidae,
Cetoniinae) in Europe. Anim Biodivers Conserv 28:1–44.