Thanks from our lab to all those friendly responses to our query. Herewith again my original request and the promised summary of all the suggestions and tips (if you are interested in PDFs or protocolls or other links - please let me know, I won't attach it all here cause I don't want to hog all your mailboxes): our initial query: "we are busy with phyolgeographic studies of Scarabs (Coleoptera) and are struggling to get enough good loci in a microsatellite library (even using enrichment). Does anyone have experience with microsatellites in beetles and would be prepared to share this (especially encountered problems or special tricks or idiosyncracies)? Is it possible that Coleoptera (just as Lepidoptera) do not have that many microsatellite loci? Could you recommend any other nuclear marker for phylogeographic and conservation genetic studies in beetles?" ..and now ...the answers: 1) I would say that Coleoptera have plenty of microsatellites, but they are difficult to find. I suggest two things. 1- try and get access to the EST libraries of Volger and company, see attached paper. Scan these EST seqeunces for microsatellites. 2- move on and try and get nuclear gene markers that are variable with indels. I think that this is pretty easy to do. Again, get the ribosomal protein genes for Coleoptera, which are now in gene bank. Make primers at the 5' and 3' end of these genes and PCR them for several individuals. I imagine you will find some size variation in the PCR products. Just use this as your molecular marker. You could also try and do Restriction Enzyme digests on these PCR products that don't have any size variation- to get variation among individuals in what cuts/does not cut. See the second attached paper explaining that using nuclear genes can be just as informative as microsatellite loci. Given that you probably can't get the micorsatellite loci, you certainly can get the ribosomal proteins to PCR for your species. best of luck and let me know if you have any questions, Chris 2) Dear Ute, my husband was just forwarding me your mail, there are two things: first it looks as if some beetles really do not have many repetative sequences in the genome, but that is not easy to estimate. In the lab I used to work were two students which were isolating mircosats from beetles (Curculionoidea and Chrysomeloidea), and both had problems but at the end they got some. Here in Madrid they use a very fast PCR based method that seems to work well for the group of animals they are working with. If you want I can ask the detailed protocol. The other thing is that you could try ITS, this is easy to amplify and for population/subspecies studies not too bad. This might be worth to try. I have been using this for Lepidoptera and certainly can find the primer sequence but they should be available in the literature. Hope that helps a bit, in case you have some molecular questions please do not hesitate to ask, I am not very good with the beetles but the molecular work is fine. Greetings to Clarke, even though he might not remember me from the time in London, since I am not a beetle person. All the best, Ali -- Dr. Alexandra Cieslak Department Biodiversidad y Biologia Evolutiva Museo Nacional de Ciencias Naturales Jose Gutierrez Abascal, 2 28006 Madrid Spain 3) We have developed primer sets for about 65 gene regions (single- copy protein coding genes) IF you are interested in cDNA work (RT-PCR). If you are uncomfortable working with RNA, we have a few of the same genes that work with genomic DNA. bernie ball Duke University 4) Dear Ute, In response to your message regarding useful nuclear markers in Coleoptera: I am a phylogeneticist specializing in Auchenorrhyncha (Hemiptera), however, I worked on a molecular phylogenetic the Hawaiian Platynini (Carabidae; Jim Liebherr's group of interest) as a Post-doc. Attached is a .pdf of the paper resulting from my work with that group, in which I sequenced 28S and wingless from the nuclear genome. I know that Jim Liebherr has published a number of other papers on Platynini that deal more specifically with the group's phylogeography, as well. Best of luck! Jason Jason R. Cryan, Ph.D. Director, Laboratory for Conservation and Evolutionary Genetics New York State Museum 3140 Cultural Education Center Albany, NY 12230 Phone: (518) 486-2008 E-Mail: jcryan@mail.nysed.gov FAX: 518-486-2034 5) Hi Ute, I'm a PhD student working on beetles (Tenebrionidae) in fragmented systems. I too have found it very difficult to develop good microsatellite loci as well as other non-microsatelite nuclear markers for population genetics. The best I can offer is our paper (attached): Schmuki C, Blacket MJ, Sunnucks, P (2006) Anonymous scnDNA markers for two endemic log-dwelling beetles: Apasis puncticeps and Adelium calosomoides (Tenebrionidae: Lagriinae: Adeliini). Molecular Ecology Notes 6, 362-364. Unfortunately our exhaustive efforts yielded only 5 polymorphic markers in total for two species with number of alleles varying between 2 and 10, but the markers successfully detected effects of habitat fragmentation for the two species. Good luck with it, Christina Schmuki. 6) Hello Ute, we just were working on P. chalcographus (Scolytinae, Coleoptera) and had problems isolating loci. Already 1997 I worked on Ips typographus , another scolytid, without success, however not using enrichment. But since that time I met several people working on scolytids encountering similar problems. Wolfgang Arthofer did his PhD in my group and he spent some hard months and got only some loci which we are trying now to publish. I/He could send you a pdf of his PhD so that you know what he tried. Generally we oberved - only few loci per library -repaeted isolation of identical loci - often short and interrupted motifs. The ones published for scolytids by other groups are not really working well at least for Ips tpyographus - I can send you a paper in press in Biol J Linn Soc. Further there was a isolation in chrysomelids but the authors never published an "bigger" paper - for me indicating that the loci are not working that well.... It would be nice if you let us know which answers you receive and particularly your specific methods and problems - it seems there is a general problem with isolation from coleopterans - exception carabids. If you have questions don't hesitate to contact me or Wolfgang. Best wishes Christian Christian Stauffer, Department of Forest & Soil Sciences, Institute of Forest Entomology, Forest Pathology & Forest Protection; BOKU, University of Natural Resources & Applied Life Sciences, 1190 Vienna, Austria Tel: +43 1 3686352/25 Fax: /97 http://ifff.boku.ac.at email: christian.stauffer@boku.ac.at 7) Dear Ute, I have experience with microsatellite screening in Scolytidae, and also experienced troubles... We had to use an enrichment protocol, and even doing so the proportion of positive clones was very low. We even had to forget about microsatellites in one species (the few we found were monomorphic or impossible to interpret). I think the best strategy is to enrich once or twice (but then be aware that you'll find redundant clones in the positive clones you'll find) and clone as many colonies as possible because you'll find a low number of loci actually containing a microsat. The situation cannot be generalized to Coleoptera though. Carabid species seem to be OK (genera Carabus and Chrysocarabus were ok), as well as some Curculionids (We had no problem with the genus Pissodes). I am sorry to hear that Scarabeids have also low microsat contents. You could try AFLP, but you cannot answer all questions using those... Good luck, Carole -- Carole Kerdelhué INRA, UMR BioGeCo 69 Route d’Arcachon F-33612 Cestas Cedex, France. Tel : +33 5 57 12 28 58 Fax : +33 5 57 12 28 81 http://www.pierroton.inra.fr/biogeco/entomologie/personnel/Kerdelhue /Kerde lhueperso.html 8) Dear Ute, I am afraid I do not have any answers to your questions. However, as I work on species-level evolution and molecular genetics in beetles (mostly chrysomelid leaf beetles), I would be interested in what information you receive from your request. It would be great if you could post these responses on the evoldir as other authors have done. Although I have not yet worked with microsatellites, we have recently been having considerable success using AFLPs to study genome-level genetic differentiation. The technique has proved straightforward and the data are highly informative. Good luck with your work. Sincerely, Dan Daniel J. Funk Assistant Professor Dept. of Biological Sciences Vanderbilt University 9) Dear Ute, My lab has had experience getting microsatellite loci from water beetles (Dytiscidae) without too many problems that I am aware of. Michelle Guzik (Michelle.Guzik@adelaide.edu.au), a post doc with me, has been doing the lab work if you would like to get more details of her protocol (a microsatellite enrichment technique) and the types of loci she has isolated. I think there are a number of other beetle microsatellite loci cloned (see Molecular Ecology Notes). regards, Steve Dr Steve Cooper Evolutionary Biology Unit South Australian Museum 10) Dear Ute. I found short time ago this paper talking about the singularity of the microsats in Lepidoptera. I attach it, and hope can be interesting for you and your project. Best regards. Joaquin Munoz. -- Un buen lugar para descansar y disfrutar de la naturaleza y gastronomia: http://www.laposadademontellano.com/ Joaquin Munoz Garcia (PhD. Student) Laboratory of Molecular Ecology. Estacion Biologica Donana (CSIC). Pabellon del Peru. Avda. Maria Luisa, s/n. Apartado de Correos 1056. 41013-Sevilla. Spain. Phone: #34-95-4232340 (Office ext. 236; Lab. ext. 221) Fax: #34-95-4621125 http://www.ebd.csic.es http://www2.ebd.csic.es/lem/Personal/Quini.htm 11) Hi Ute, In reference to your post on Evoldir, we developed a panel of microsatellites in southern pine beetle (Dendroctonus frontalis) using a common enrichment protocol and had no difficulties. I know colleagues who have developed microsats in similar species with varying levels of success. Regards, Ed Heist Edward J. Heist, Ph.D. Southern Illinois University at Carbondale Fisheries and Illinois Aquaculture Center Life Sciences II, 1125 Lincoln Drive Carbondale, IL 62901-6511 Voice: (618) 453-4131 Fax: (618) 453-6095 email: edheist@siu.edu web: http://www.science.siu.edu/zoology/heist/index.html 12) Hello Ute, My name is Elena and I am working at the National History Museum of Madrid. I am actually writing you in behalf of Alexandra Cieslak, (see email below). Here I send you some protocols I've been using for the isolation of microsats. I hope they will help you. Please, feel free to ask me if you have any other question. Good luck! Cheers, Elena 13) Hi Ute, Well, i cant help with the microsats, but on the question of other nuclear markers, did you see the paper by Pons et al. in sys. biol. about the MP20 introns? They used Cicindela (adephagan Coleoptera), but the primers were designed from a broader sample of beetles. I was just playing with generating a matrix of insect MP20, to look at the broader evolution of the gene. There is plenty of data from this gene if you want to redesign primers that might be more specific to scarabs. The closest taxa to scarabs with data for this gene is Georissus (Staphylinid), but there is lots of comparative data from elateriform or cucujiform beetles. The paper on MP20 as a marker in adephaga is available here. http://72.14.203.104/search?q=cache:KYFvWyeZlM4J:hydrodictyon.e eb.uconn.ed u/courses/systematicsseminar/restricted/Pons_et_al_2004_SystBiol_ 53_554-57 0.pdf+MP20+cicindela&hl=en&gl=us&ct=clnk&cd=5 Let me know if you want to talk about available MP20 data and modifying the primers etc. Best of luck stuart Dr. S. Longhorn, MSc PhD PostDoctoral Researcher Mitogenomic Analysis of the Arachnid Orders Rm. 105 Graduate and faculty office, SB 1; Dept. of Biology Portland State university Portland, OREGON, 97207 Lab: TEl:001-503-725-3855 Email: sjl197@hotmail.com or stul@pdx.edu WORK CURRENT: http://web.pdx.edu/~smasta/ WORK PREVIOUS: http://www.bio.ic.ac.uk/research/apvogler/vogler.htm 14) Dear Ute, Someone has just forwarded me your enquiry about markers for beetles, I must somehow have missed your message. I developed microsatellite markers for ladybird beetles (primer note attached), so have some experience of this. Are you still in need of help or have you sorted your problems out? I'm not sure if there's anything I can do, but if there is then please do let me know. Best wishes Penny Haddrill so - once again, thank you all you wonderful co-scientists, and may the (nuclearDNA) force be with you!!! Ute Ute Kryger (PhD) Scarab Research Group Department of Zoology & Entomology University of Pretoria Pretoria 0002 - RSA Fax +27 12 362 5242 Phone +27 12 420 5343 Ute Kryger