Dear Evoldir members, I listed the answers addressed to my query about clonality and definition of Multilocus Genotypes (MLG). To give some precision about the context of my study, I work on the assessment of the structuration of genetic diversity in several populations of Potamogeton pectinatus, an aquatic macrophyte reproducing by both clonal and sexual reproduction. I analysed genetic diversity and clonality with the help of 9 nuclear microsatellites. Some MLGs are very distinct from each other (i.e. differing by two or more alleles) but some are not so distinct (only one difference). My question was to have your opinion about the best way to treat those MLG. I am very grateful to each of you who spent time to answer to my question and I hope that the answer listed below could be useful for others! stef ___________ Original message : Dear EvolDir members, I have a methodological question that deals with the assessment of clonal diversity with microsatellite loci. Could we consider that each observed multilocus genotype (MLG) mirrors reality? Or is it more realistic to consider two or more MLG differing by only one allele at a locus (out of several) as a unique MLG? For example, could one consider the two following MLG to be different or identical? : (1) 0102 0202 0405 0405 0103 0203 0405 0405 (2) 0102 0202 0405 0405 0103 0202 0405 0405 Does someone know papers, methods or softwares that could help to take a decision by taking into account allelic frequencies or genotyping error rates? thank you very much for any help and advices! Stef sfenart@vub.ac.be ---------- ANSWERS: Hi Stef, I would consider those two examples of MLG different i.e. not identical. I do not know about software that would decide for you what is identical - I would say that such individuals must be the same on all loci, then they are likely to be a product of asexual (clonal) reproduction. For that you can just use excel file and function of finding identical individuals. However, identical can be also product of sexual reproduction (recombination) and to distinguish between those two - Software MLGsim (Stenberg et al. 2003, Molecular ecology notes, 3: 329-331) calculates probability that two and more identical (same on all loci) multilocus genotypes are the result of sexual reproduction and not asexual (clonal) reproduction. It works on microsatellite data, using allele frequencies and sample size. I'm uncertain, but I don't think it takes into account genotyping errors. Hope this helps a bit. It's very interesting question, and I would be interested in the responses you get. Can you please summarize them and forward to me? Good luck, Monika email: mzavodna@purdue.edu _________ Hi Stef, An addition to my previous email: I've heard from my colleage that Genalex software (Peakall and Smouse, 2006, Molecular Ecology notes, 6: 288-295) is also able to identify identical MLG (function 'matches' or so). You can probably check it out, I am uncertain about details. Monika ________ Calculating the probability of identity will help you determine whether you can answer this question. We did this for a wild rice species from texas in the attached article, using an Excel spreadsheet. You can always modify the PID formulas to add genotyping error, but testing whether the probability passes some kind of threshold is where it would get a bit complicated. Best, M. Antolin Richards, C. M., M. F. Antolin, A. Reilley, J. Poole, and C. Walter. 2006. Capturing genetic diversity of wild populations for ex situ conservation: Texas wild rice (Zizania texana) as a model. Genet Resour Crop Evol Published online: 7 December 2006 ________ Dear Stef, interesting question about the genotypes & clones... sorry that I cannot help a lot, but would be interested in reading the answers that you get, please post them! Just a thought: my hunch is that the inferences you make from your genotype data will largely depend on the method you use for genotyping, i.e. types of markers. Because, for instance microsatellites are well known for high mutation rates... so especially if your example of the two genotypes was based on microsats, these two would, in my view, clearly point towards not only common ancestry but actual possibility of grouping them together as a single "clonal lineage". Very often it is useful to group genotypes in such a way, simply because it provides more clarity in your dataset. e.g. if your two genotypes (1) 0102 0202 0405 0405 0103 0203 0405 0405 (2) 0102 0202 0405 0405 0103 0202 0405 0405 are compared with a third one: (3) 0101 0303 0405 0505 0101 0101 0202 0303 the number 3 is clearly very distant from the first two... hope this helps. good luck! Ruza -- ~~~~~~~~ Ruza Bruvo Department of Animal and Plant Sciences The University of Sheffield Western Bank Sheffield S10 2TN r.bruvo@sheffield.ac.uk Tel. +44 (0)114 22 20113 ________ Dear Stephane, depends on (i) the whole variability observed: 30 loci with many different alleles at each are not the same than 5 diallelic loci. (ii) the reproducibility of your experiments. (iii) the question under study and the goal of your research. I would say (if the technique is fine) that (1) and (2) are 2 very closely related clones, which most recent common ancestor lived recently. You may have a look at the paper in attachment, dealing with pathogens. Tibayrenc, M., and F. J. Ayala. 2002. The clonal theory of parasitic protozoa: 12 years on. Trends In Parasotology 18:405-410. Best regards, Michel Tibayrenc ________ Dear Stef, > > We are trying in our team to work on the issues of interpreting >those dataset in terms of clonality assessment, and I thought you >might be interested in the articles we wrote in our team during the >past years, and by our software GenClone (you will find the url on the >primer note in attachment). > > According to your question, the more relevant article will I think >be the one we published in Journal of Heredity, and one of the two >references that are in press, to be published in Journal of >Biogeography. In this one we tried in this one to address both the >issue of identical MLG possibly belonging to distinct clone, and the >issue of distinct MLG belonging to the same clone but distinct because >of somatic mutations or scoring errors. > > I hope those informations will be of help, don´t hesitate to >contact me for any further details. > > Best regards, > > Sophie Alberto F, Gouveia L, Arnaud-Haond S, et al. (2005) Spatial genetic structure, neighbourhood size and clonal subrange in seagrass (Cymodocea nodosa) populations. Molecular Ecology 14, 2669-2681. Arnaud-Haond S, Alberto F, Teixeira S, et al. (2005) Assessing genetic diversity in clonal organisms: low diversity or low resolution? Combining power and cost-efficiency in selecting markers. Journal of Heredity 96, 434-440. Arnaud-Haond S, Belkhir K (2007) GenClone 1.0: a new program to analyse genetics data on clonal organisms. Molecular Ecology Notes 7, 15-17. Arnaud-Haond S, Diaz Almela E, Teixeira S, et al. (2007) Vicariance patterns in the Mediterranean sea: East-West cleavage and low dispersal in the endemic seagrass Posidonia oceanica. Journal of Biogeography, in press. Diaz-Almela E, Arnaud-Haond S, van de Vliet MS, et al. (in press) Feed-backs between genetic structure and Perturbation-driven decline in seagrass (Posidonia oceanica) meadows. Conservation Genetics. ________ Dear Stef we have had experience with this situation in two separate contexts: (1) parthenogenetic aphids eg Sunnucks, P., De Barro, P. J., Lushai, G., Maclean, N. & Hales, D. F. (1997) Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis, differentiated lineages, and host specialization. Molecular Ecology 6: 1059-1073. Sunnucks, P., England, P.R., Taylor, A.C. & Hales, D.F. (1996) Microsatellite and chromosome evolution of Parthenogenetic Sitobion aphids in Australia. Genetics 144: 747-756. Depending on your organism and level of sampling, a special analysis may not be necessary. If the rare variant differing by only a single allele at a single locus is not a singleton, it should be a new, identifiable lineage. If it is a singleton (and you cannot get an progeny), it is harder to estimate if it is an additional clone or not. You could take the approach of assuming that singletons are all errors and that none are errors, and see if you get much difference. I would guess that in most reasonable-sized data sets, singleton variants differing by only one allele at one locus would be very rare, and would not contribute a lot to false inferences in a way that matters. If genotypes differ by more than one allele at one locus, they are vanishingly unlikely to be errors. Note that you can get an idea of how microsatellites mutate in clonal organisms, and that the types of changes might be predictable.... Wilson, A.C.C., Sunnucks, P. & Hales, D.F. (2003) Heritable genetic variation and potential for adaptive evolution in asexual aphids (Aphidoidea). Biological Journal of the Linnean Society, 79, 115-135. (2) Non-invasive typing. Many studies deal with the same issue eg in censusing populations of mammals from remotely-collected hair. If two genotypes differ by a small amount, are they really new or not? Software can interpolate roughly how many individuals really would mismatch by one allele at one locus, by using the data on how many mismatch at more than than. Banks, S.C., Hoyle, S.D., Horsup, A., Sunnucks, P., Wilton, A. & Taylor, A.C. (2003) Demographic monitoring of an entire species by genetic analysis of non-invasively collected material. Animal Conservation, 6, 101-108 You might also like to look at the work of Lisette Waits and Gordon Luikart on this subject. you get get a lot of our papers from my website at http://www.biolsci.monash.edu.au/staff/sunnucks.html if you need more Paul ________ Dear Stef Please try Geneclone available at: www.ualg.pt/ccmar/maree/software.php Arnaud-Haond S, Belkhir K (2007) GenClone 1.0: a new program to analyse genetics data on clonal organisms. Molecular Ecology Notes, 7: 15-17 A nice paper about this is Arnaud-Haond et al 2005 Filipe Alberto, PhD CCMAR-CIMAR University of Algarve campus de Gambelas 8005-139 Faro, Portugal http://www.ualg.pt/ccmar/maree/ ________ Dear Stef, The first person to deal with this issue in aphids was Paul Sunnucks: Sunnucks P, De Barro PJ, Lushai G, Maclean N, Hales DF (1997) Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis, differentiated lineages, and host specialization. Molecular Ecology 6: 1059-1073. For a paper where clonal diversity was very low and genotypes that are mutationally related to each other are described see: Wilson ACC, Sunnucks P, Hales DFH (1999) Microevolution, low clonal diversity and genetic affinities of parthenogenetic Sitobion aphids in New Zealand. Molecular Ecology 8: 1655-1666. The method of analyzing the data set so that it includes multiple clonal copies and does not include multiple copies of the same genotype is attributed to Sunnucks and has become standard in aphid population genetic analyses and has also been applied in other cyclically parthenogenetic systems (off the top of my head, see the work of Gomez). A review paper I published in 2003 may also be useful and of interest to you: Wilson ACC, Sunnucks P, Blackman RL, Hales D (2002) Microsatellite variation in cyclically parthenogenetic populations of Myzus persicae in south-eastern Australia. Heredity 88: 258-266. All the best for your study. Alex Wilson ________ Hoi Stef I do not think that there is a clearcut solution to your problem. How you should approach this is very much dependent on your research question, on your sampling, the used markers, but mostly on the reproductive biology of the system you are working on. If you work with an organism that is 100% strictly clonal, the differences between individuals is due only to mutation, and in that case there it makes no sense to distinguish between lineages on the basis of the number of mutational differences that you find. On the other hand, if there is a small amount of sex in your organism there will be both differences between individuals within clonal lineages that are due mutation and differences between different clonal lineages, that are mainly due to recombination. These two can be seperated if you have enough loci, as (depending on the mutation rate and the frequency of sex) genetic distances between individuals from the same clonal lineage will be very close or equal to zero, and genetic distances between individuals from different clonal lineages will be further removed from zero. This phenomenon is often very clearly visible when drawing a frequency histogram of the pairwise distances between all individuals. I wrote two programs to help in such analyses, one is called GenoType and will allow you to determine clonal lineages depending on a number of mutationsteps that is at max allowed within lineages. The second is called GenoDive and calculates clonal diversity. You can find them, and a manual, on: http://www.bentleydrummer.nl/software. I attached a paper that describes the software. Meirmans, P. G., and P. H. van Tienderen. 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organims. Mol Ecol Notes 4:792-794 Groetjes en zo, Patrick _________ Dear Stef, you can try Gimlet (free ware program) to calculate a probability of identity (Pid). This basically gives you the probability that you are mistakingly identify an individual as a clonemate when in fact it is not. We have used it for a study on clonal structure in corals (see attached). Iliana Baums, I. B., M. W. Miller, and M. E. Hellberg. 2006. Geographic variation in clonal structure in a reef-building caribbean coral, Acropora palmata. Ecol Monogr 76:503-519. _________ Stef, Here are some papers that I believe discuss the issue you've encountered. Meirmans, P. G., and P. H. van Tienderen. 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organims. Mol Ecol Notes 4:792-794 Douhovnikoff, V., and R. S. Dodd. 2003. Intra-clonal variation and a similiraity threshold for identification of clones: application to Salix exigua using AFLP molecular markers. Theor Appl Genet 106:1307-1315. Nick _________ I am treating one allele pairwise differences as separate genets in my studies, but I am including the number of such MLGs that differ by a single allele in a table where I report diversity statistics. The Program GenClone by Arnoud-Ahound and Belkhir lists such MLG pairs in its output. Another possibility is that they represnt somatic mutations in a ramet of a single genet. Of course my perspective is from plant genetics. If your organisms are animals incapable of asexual reproduction, I am not sure how to advise you. Essentially, you have to make up your mind how you want to treat these samples. If your sample is large enough, why not just drop those individuals? Alan Meerow _________ Dear Stephane Fénart, you can quantify the probabilities that 2 or more identical MLG come from sexual reproduction if you have some knowing the allele frequencies in your populations and by extension the probabilities that MLG you consider as clones are false. To my knowledge, there is no method that estimates, with a fixed mutation or a genotyping errors rates, the probabilities that 2 MLG that differs with only one or two alleles come from asexual reproduction from the same 'parent'. In fact, to take into account mutation, you need to have some ideas about the coalescence time and the age of the clones you are studying. Find joined one of my paper about asexual propagation in wild cherry trees, you will find there (in 'Occurrence of asexual reproduction' part of M&M) the literature related to clone identification using probability of identity. I made a software that calculates lower and upper limits of probabilities of identity as recommended by Waits et al. (2001), but it is not built into an *.exe to date. I'am studying the impacts of reproductive systems on structuration and evolution of genetic diversity (neutral and selected --> I have a paper in revision with Vincent Castric & Xavier Vekemans from GEPV)). I develop models and use field or experimental genetic data to progress. If you have some further questions about these topics, I will be pleased to help you. Hope that it will help you, Solenn _________ Hallo Stephane, I think the variation in Microsatellite markers in one way or the other mirrors reality especially for the neutral diversity. The alleles differing by a single basepair may be true alleles or just a stuttering effect. Looking at your peaks once again may help you solve the problem. I have used the software Micro-Checker to check my microsatellite data for the presence of null alleles and scoring errors (stuttering). The software is freely available over the internet. Looking at the following papers may also help: 1. Bonin et al. 2004. Molecular Ecology 13, 3261-3273. 2. Broquet and Petit 2004. Molecular Ecology 13, 3601-3608. Regards Joram Mwacharo mwacharo@gmail.com -- Stéphane Fénart Algemene Plantkunde en Natuurbeheer (Plant Biology and Nature Management) Room 7F416 Vrije Universiteit Brussel Pleinlaan, 2 B-1050 Brussels Belgium tel: + 32 2 629 3057 fax: + 32 2 629 3413 Visit our website: http://www.vub.ac.be/APNA/ Stéphane Fénart