Failure of Microsatellite's Cross-Species Amplification In

Baltic J. Coleopterol. 11(1) 2011 ISSN 1407 - 8619 Failure of microsatellite’s cross-species amplification in common ground beetle Pterostichus melanarius (Illiger) Robert Rutkowski, Anna Szczuka, Marcin Zalewski, Julita Korczyńska, Grzegorz Gryziak Rutkowski R., Szczuka A., Zalewski M., Korczyńska J., Gryziak G. 2011. Failure of microsatellite’s cross-species amplification in common ground beetle Pterostichus melanarius (Illiger). Baltic J. Coleopterol., 11(1): 17 - 24. In the present paper, we describe results of cross-species amplification of microsatellite loci in wing dimorphic carabid Pterostichus melanarius - model species for diverse ecological studies. In particular wing dimorphism determined genetically, with simple Mendelian fashion and discovery of brachyptery gene provides unique opportunities to study gene flow within metapopulation. One hundred individuals originating form nine island populations of Mamry Lake archipelago were studied. Using PCR primers from five different species of Carabidae, especially including closely related species – Pterostichus oblongopunctatus we failed to establish firm ground of population genetic tool for the beetle. We review works on cross- species amplification in invertebrates and show that success of cross-species strategy among coleopteran species from different genus is rather highly doubtful. We present procedures and results that that might be of importance for ecologists tempted by scientific opportunities given by P. melanarius. Key words: Pterostichus melanarius, Coleoptera, Carabidae, cross-species amplification. Robert Rutkowski. Museum and Institute of Zoology PAS, Wilcza 64, 00-679 Warsaw, Poland; E-mail: [email protected] Anna Szczuka, Julita Korczyńska. The Nencki Institute of Experimental Biology PAS, Pasteur 3, 02-093 Warsaw, Poland Marcin Zalewski*. Center for Ecological Research PAS, M. Konopnickiej 1, 05-092 Łomianki, Poland Grzegorz Gryziak. Plant Breeding and Acclimatization Institute – National Research Institute, Radzików, 05-870 Błonie, Poland * corresponding author INTRODUCTION Pterostichus melanarius is another example. There are tens of works inspecting different There are many good “model species”, that pose aspects of its biology (Ericson 1977, David 2000, unique features, which help to explain particular Symondson et al. 2000, Winder et al. 2001). scientific questions. For example laboratory mice, Particularly, wing dimorphism make this animal Drosophila, or Daphnia proved their importance perfect object in studies on invasions and in biological studies. Common ground beetle 1 7 Rutkowski R., Szczuka A., Zalewski M., Korczyńska J., Gryziak G. dispersal (Niemelä & Spence 1991, 1999, Zalewski identification of the marker is expensive and 2004). Briefly, populations of this common ground labour-consuming, moreover requires cloning beetle contain long winged (macropterous), as and sequencing. To overcome these well as wingless (brachypterous) individuals. The disadvantages, researchers often adapt trait is determined genetically, with simple information about microsatellite markers, Mendelian fashion of inheritance and originally developed for one (the source) species, brachyptery gene being a dominant allele for use in other, usually closely related species (Aukema et al. 1996). As consequence of wing (the target). This strategy, which is based on type inheritance and emigration fraction of using PCR primers described for one species to macropterous individuals in population gives amplify homologous microsatellite in other good idea about population age (Den Boer et al. species was called cross-species microsatellite 1980, Zalewski 2004) – the characteristic rarely amplification and become widely applied, studied in natural populations. Additionaly frequently with success (Moore et al. 1991, brachyptery gene was discovered (Ober 2002). Gemmell et al. 1997, Primmer & Ellegren 1998, Thus, P. melanarius provides opportunity to Gibbs et al. 2000). However, application of cross- study genetic processes in new versus old species microsatellite amplification has been populations and genetic structure of the species shown to have many limitations. First, the formed by two different phenotypic forms: strategy works preferably for species belonging dispersive and non-dispersive, as well as gene to the same genus or to recently separated genera flow in metapopulations. All of them, are presently (Scribner & Pearce 2000). Second, in many cases important questions in population biology a given microsatellite may fail to amplify or may (Hanski et al. 2004, Ovaskainen et al. 2008) and be less or even non-polymorphic in target species are reasons for conducting this study. (Rubinsztein et al. 1995, Morin et al. 1998). Thus, application of cross-species strategy requires Simultaneously, resolving of such complex pilot study, which would assess the amplification population’s questions requires utilization of success of particular markers and their level of molecular markers, which present high polymorphisms in target species, as only polymorphism on the species level. polymorphic microsatellites could be Microsatellites are one of the most popular successfully used in population genetic studies genetic marker of this type. They are defined as (Frankham et al. 2003). tandem repeats of short (from two to six nucleotides) DNA motif, forming more or less In the present paper, we describe results of cross- uniform tracts up to 100 nucleotides long species amplification of microsatellite loci in (Chambers & MacAvoy 2000). Microsatellites are Pterostichus melanarius, using PCR primers from found in just about every organisms and several different species of Carabidae, especially organelle and are relatively evenly spaced including closely related species – Pterostichus throughout the genomes (Edwards et al. 1991, oblongopunctatus. Nevertheless our broad Stallings et al. 1991, Chambers & MacAvoy cross-species research failed to establish firm 2000). High level of polymorphisms interlinked ground of population genetic tool for the beetle, with the power that they provide to solve we present procedures and results that might be biological problems, as well as the possibility of of importance for ecologists using scientific analysis using fast and effective technique of opportunities given by P. melanarius. Our basic PCR cause the microsatellites useful genetic scientific question was about genetic diversity marker for wide range of genetic investigation, of young vs. old populations and importance of ranging from identification of individuals to macropterous vs. brachypterous individuals for studies on population level (e.g. Sloane et al. gene flow in metapopulation. However, as we fail 2000, Girman et al. 2001, Lee et al. 2001, Roeder to study these interesting issues due to problems et al. 2001). One of the factors limiting even with amplification they remain to be solved in broader use of microsatellites is that initial 1 8 Failure of microsatellite’s cross-species amplification in common ground beetle Pterostichus melanarius (Illiger) future when PCR primers for Pterostichus As no product was obtained amplification of each melanarius will be developed. microsatellite was repeated under different PCR conditions, using gradient of annealing temperatures ranging from 44ºC to 60ºC. MATERIAL AND METHODS The length of amplified fragments was estimated Beetles were captured alive on islands and using CEQ8000 Beckman Coulter automatic mainland sites on the archipelago and shores of sequencer. Data were analyzed using Beckman Mamry Lake (coordinates 21’30°–21’52° E, Coulter Fragment Analysis Software. 54’00°–54’10° N). Collected specimens were preserved in 70% ethanol and stored frozen. In total 500 beetles were collected. RESULTS Genomie DNA was isolated from 100 individuals originating from 9 different sites. Extraction was Satisfying results, meaning electropherograms, performed applying DNeasy® Tissue Kit which showed characteristic microsatellite’s (Qiagen). Fragments of tissues form abdomens structure of a peak after analysis of allele’s were crushed and suspended in 180 μl of ATL fragment length in sequencing machine – the buffer and incubate at 56°C over night with 20 μl “stutters bands” and +A peaks were obtained of proteinase K (20μg/ml). Following incubation, only for Pob10 marker amplified in annealing extraction of DNA followed standard protocol temperature 55ºC. In all tested individuals (both with the DNA being eluted in 100 μl of elution macropterous and wingless specimens) amplified buffer. Results of DNA isolation were checked fragment had exactly the same length (126 base using electrophoresis technique in 1.5% agaroze pairs) indicating severe fixation of an allele. gels. No clear amplification products were visible for As no microsatellite markers for P. melanarius other tested loci. Either, complete lack of amplified had been described, we applied cross-species fragments or multiple “peaks” were obtained in a amplification strategy. In total we have tested 11 wide range of annealing temperatures. markers. Following primers, amplifying microsatellite sequences in five other Carabidae species were used: Pob1; Pob3–Pob5; Pob10 and DISCUSSION Pob14 (source species – Pterostichus oblongopunctatus; Lagisz & Wolff 2004), Cins11 Our study on cross-species amplification of and Cins33 (source species – Carabus microsatellite between Pterostichus insulicola; Takami & Katada 2001), Cn11/152 oblongopunctatus (source species) and P. (source species – Carabus nemoralis,

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