Department of Zoology and Monte L. Bean Museum, Brigham Young University
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Abstract |
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Introduction |
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Materials and Methods |
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Phylogeny Reconstruction
Sequences were aligned using CLUSTAL W (Thompson, Higgins, and Gibson 1994
). Some adjustments were made by eye. The sequences were then imported into PAUP* (Swofford 1999
) for phylogenetic analyses. When estimating phylogenetic relationships among sequences, one assumes a model of evolution regardless of the optimality criteria employed. We therefore employed minimum evolution rather than maximum parsimony, as this method can take into account more of the inherent characteristics of the data set (e.g., differences in nucleotide frequencies, rate heterogeneity, etc.). Determining which model to use given one's data is a statistical problem (Goldman 1993
). We used the approach outlined by Huelsenbeck and Crandall (1997)
to test alternative models of evolution, employing PAUP* and Modeltest (Posada and Crandall 1998
). A starting tree was obtained using neighbor joining. With this tree, likelihood scores were calculated for various models of evolution and then compared statistically using a chi-square test with degrees of freedom equal to the difference in free parameters between the models being tested. The null hypotheses tested in this way included the hypotheses that: (1) nucleotide frequencies are equal, (2) transition rates are equal to transversion rates, (3) transition rates are equal and transversion rates are equal, (4) there is rate homogeneity within the data set, and (5) there is no significant proportion of invariable sites. Once a model of evolution was chosen, it was used to estimate a tree using the minimum-evolution optimality criteria (Rzhetsky and Nei 1992
). Confidence in resulting nodes was assessed using the bootstrap technique (Felsenstein 1985
) with 1,000 replicates (fig. 2
).
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Results |
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To test for saturation by multiple substitutions within the ITS1 data set, observed pairwise proportions of transitions and transversions were plotted against uncorrected sequence divergence (fig. 3 ). There is a clear separation between the Orconectes sequences and the Procambarus sequences, with genetic divergences within genera being less than 5% but genetic divergence between them being more than 25%. This evidence and the difficulty in accurately aligning Orconectes sequences to those of Procambarus led us to analyze the Orconectes sequences without including those of Procambarus.
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Discussion |
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Phylogenetics
Phylogenetically, ITS1 sequences are not informative in separating the three populations of O. luteus, despite the fact that these populations are uniquely defined using amplified fragment length polymorphisms and 16S mtDNA (Fetzner and Crandall 1999
). This is due to the high level of intragenomic variation examined. This variation, although particularly prevalent in the microsatellite regions, is not confined to them. Removal of the microsatellites reduces bootstrap support for all species groups except O. punctimanus. However, with one exception, the clones of O. luteus from the Big Piney and Niangua rivers are then separated (62% bootstrap) from the two individuals from the Meramec River, indicating that it is these regions in particular that cause problems for phylogenetic construction at the population level.
At the generic level, at least between Orconectes and Procambarus, the ITS1 sequences are too divergent to be phylogenetically useful, but at the species level, ITS1 sequences are partially informative, with high bootstrap support for a clade of O. punctimanus, O. longidigitus, and O. virilis. This relationship is not altered by the presence or absence of the microsatellite regions. It is, however, different from the hypothesis of relationships derived from 16S mtDNA sequence data (Crandall and Fitzpatrick 1996
), which has been used to estimate a phylogeny of (((((O. punctimanus, O. virilis), O. luteus), O. longidigitus), O. neglectus), O. macrus), i.e., the positions of O. luteus and O. longidigitus are reversed. The topology derived from 16S sequence data is significantly different from the minimum-evolution tree estimated from the ITS1 data set using the Kishino and Hasegawa (1989)
likelihood ratio test (likelihood of minimum-evolution tree = -1,173, likelihood of tree constrained to topology derived from 16S sequence data = -1,197, SD = 10; T = 2.24; P = 0.025). This could be due to the limited sampling of species for the ITS data sets, but it does highlight the problems of using only a single gene phylogeny to estimate organismal relationships.
Despite the fact that such variation has been known to exist for some time (e.g., Vogler and DeSalle 1994
), very few of the recent phylogenetic analyses using ITS sequences take this into account by examining multiple sequences from single individuals. The impact of this kind of variation on phylogenetic studies is increased if many phylogenetically distinct groups show similar intragenomic variation. In all cases so far reported where intraindividual variation has been looked for within ITS1 and ITS2, it has been found to some degree. This includes studies on beetles (Vogler and DeSalle 1994
), crayfish (this study), yellow monkey flowers Mimulus (Ritland et al. 1993), maize Zea mays (Buckler and Holtsford 1996
), fungus Fusarium (O'Donnell and Cigelnik 1997), and coral Acropora (Odorico and Miller 1997). ITS sequence data are extremely widely used in plant systematics and population studies. However, for example, none of the papers using ITS sequence variation to examine phylogenies in Plant Systematics and Evolution in 1999 to date investigate this problem (Koch et al. 1999; Leskinen and Alstrom-Rapaport 1999; Susanna et al. 1999), despite analyses that show that major NOR loci (which contain rDNA arrays) may move within and among chromosomes and that this movement may occur via magnification of minor loci consisting of a few rDNA copies (Dubcovsky and Dvorak 1995
). Deletion of major rDNA sites and their replacement by minor, potentially paralogous, rDNA sites can lead to sudden stochastic fluctuations in the rDNA consensus sequence in an evolutionary lineage. Therefore, if paralogous copies are present, phylogenetic reconstruction should be treated extremely cautiously.
In some cases in which genomes contain considerable diversity of paralogous rDNA, the divergent paralogs are probably rDNA pseudogenes, since they have low predicted secondary structure stability (Buckler, Ippolito, and Holtsford 1997
). In their analysis of Zea relationships, Buckler and Holtsford (1996)
found four putative pseudogenes from Z. mays, which were basal to all other Z. mays ITS sequences. The pseudogenes had undergone many substitutions relative to the normal alleles. This was unlike the situation for Mimulus ITS types that were closely related to each other within types (Ritland et al. 1993). The crayfish ITS sequences do not appear to be pseudogenes, as the minimum-energy secondary structures (calculated using the program mFold [Zuker 1989]) were all very similar from the different ITS copies (approximately -266kcal/mol at 37°C for ITS1). Given that the different sequences are not pseudogenes, the level of intragenomic variation is much higher than those reported in other cases. This is almost certainly due to the presence of the microsatellites in both the ITS1 and the ITS2 sequences. Therefore, in studies which have microsatellites within their rDNA sequences, one should be particularly cautious about inferring relationships without investigating the levels of intragenomic variation.
Microsatellites
Known variation within microsatellite regions of ITS1 from single individuals has had little or no impact on microsatellite studiesfew of these report the positions of microsatellite loci within the genome, and the regions are routinely assumed to behave as independent codominant markers. This is probably due to the shortness of the microsatellites previously reported within multicopy genes. For example, Vogler and DeSalle (1994)
showed the presence of two microsatellites in their ITS1 data set, (TA)59 and (GA)48, while Koch et al. (1999), in their study of the Brassicaceae, reported a (TA)6 repeat in one species. The repeat units found in ITS2 sequences have also typically been short: Fritz et al. (1994)
reported a (GA)48 repeat in mosquito Anopheles populations. All of these repeat sequences are shorter than most new microstatellite loci that are published (e.g., 30 microsatellite loci reported for red drum, Sciaenops ocellatus, ranged from (TG)5 to (CA)22(GT)5 Turner et al. 1998). However, the microsatellite regions from this study are considerably longer, e.g., (CAG)58 in ITS2 and (TGC)36(TCC)36, (GA)312, and (CAG)58 in ITS1 (table 4
), and other long microsatellites have been reported, such as the (GA)1120 repeat found in the 5' half of human ITS clones (Gonzalez et al. 1990
). Although shorter than many reported microsatellite loci, they are within the range of those used in population studies. Given the hybridization techniques used to locate microsatellites (Strassmann et al. 1996
), microsatellites in multiple-copy regions like ITS1 and ITS2 will be preferentially found. The occurrence of microsatellites in such regions may explain why length variation of microsatellites has not always correlated with predicted phylogenies in previous studies, e.g., in the case of the horseshoe crab Limulus polyphemus (Orti, Pearse, and Avise 1997
). One indirect way of detecting this sort of variation would be through mismatches between known mother-offspring pairs or by significant deviation from Hardy-Weinberg equilibriathe same methods that are used to identify nonamplifying alleles (Pemberton et al. 1995
). A more direct method, sequencing multiple clones from a single individual, will show whether variation is prevalent, and this method should be used as a standard in the microsatellite technique to avoid spurious results.
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Acknowledgements |
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Footnotes |
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1 Present address: Unidade de Genética Animal e Conservação, Campus Agrário de Vairão, R. Monte-Crasto, Portugal.
2 Keywords: ITS1
ITS2
Orconectes,
Procambarus,
microsatellites
phylogeny
crayfish
rDNA
3 *Address for correspondence and reprints: Keith A. Crandall, Department of Zoology and Monte L. Bean Museum, Brigham Young University, 574 Widtsoe Building, Provo, Utah 84602-5255. E-mail: kac{at}email.byu.edu
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