Characterization of Microsatellite Loci for the European Species of Hermit Beetles (Osmoderma

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, KazimierzWielki University, Chodkiewicza 30, 85-064 Bydgoszcz, Poland

(b) Faculty of Sciences, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech Republic

*corresponding author: e-mail:

Abstract

Fourteen microsatellite loci are described for the eastern European hermit beetles, Osmodermabarnabita, 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 Osmodermaeremita.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 OsmodermaLep. 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 Osmodermaeremita(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 OsmodermabarnabitaMotsch., the species belonging to the eastern clade within O. eremitacomplex, and their cross-amplification in the western species O. eremitas. 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. barnabitafrom 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. eremitaonly 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. eremitas. 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 PavelŠebek for help with sample collection, JiříKošnar and EwaSztupecka for their laboratory work and Igor Chybicki for discussion of the results.

References

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