Use of the nuclear gene glyceraldehyde 3-phosphate dehydrogenase for phylogeny reconstruction of recently diverged lineages in Mitthyridium (Musci: Calymperaceae)

A portion of the nuclear gene glyceraldehyde 3-phosphate dehydrogenase (gpd) was sequenced in 26 representatives of the paleotropical moss, Mitthyridium, and a group of 20 outgroup taxa to assess its utility for phylogenetic reconstruction compared with the better understood chloroplast markers, rps4 and trnL. Primers based on plant and fungal sequences were designed to amplify gpd in plants universally with the exclusion of fungal contaminants. The piece amplified spanned 4 introns and 3 of 9 exons, based on comparisons with complete sequence from Arabidopsis. Size variation in gpd ranged from 891 to 1007 bp, in part attributable to 6 indels of variable length found within the introns. Intron 6 contributed most of the length variation and contained a variable purine-repeat motif of possible use as a microsatellite. Phylogenetic analyses of the full gpd amplicon yielded well-resolved trees that were in nearly full accord with the trees derived from the cpDNA partitions for analyses of both the ingroup and ingroup + outgroup taxon sets. Pairwise nucleotide substitution rates of gpd were as much as 2.2 times higher than those in rps4 and 2.8 times higher than in trnL. Excision of the introns left suitable numbers of parsimony informative characters and demonstrated that the full gpd amplicon could be compartmentalized to provide resolution for both shallow and deep phylogenetic branches. Exons of gpd were found to behave in a clock-like fashion for the 26 ingroup taxa and select outgroups. In general, gpd was found to hold great promise not only for improving resolution of chloroplast-derived phylogenies, but also for phylogenetic reconstruction of recent, diversifying lineages.     

Phylogenetics of wigeons and allies (Anatidae: Anas): the importance of sampling multiple loci and multiple individuals

Species-level DNA phylogenies frequently suffer from two shortcomings--gene trees usually are constructed from a single locus, and often species are represented by only one individual. To evaluate the effect of these two shortcomings, we tested phylogenetic hypotheses within the wigeons and allies, a clade of Anas ducks (Anatidae) composed of five species. We sequenced two nuclear introns from the Z-chromosome-linked chromo-helicase binding protein gene (CHD1Zb and CHD1Za) and the mitochondrial DNA (mtDNA) control region for multiple individuals sampled from widespread geographic locations. We compared these phylogenies to previously published phylogenies constructed from morphology and protein coding regions of mtDNA. Relative to other nuclear introns, CHD showed remarkable phylogenetic utility. Of the 26 CHD1Zb alleles identified, only one was shared between two species, and the combined CHD datasets revealed that four of the five species were consistent with monophyly. Several species shared mtDNA haplotypes, which probably was a result of interspecific hybridization. Overall, the nuclear CHD tree and the mtDNA tree were more congruent with coding regions of mtDNA than they were with morphology.     

Examination of five nuclear markers for phylogenetic study of Hologalegina (Leguminosae)

Most legume phylogenies have relied heavily on plastid gene datasets, with or without nuclear ribosomal DNA ITS data, but the sequences of nuclear genes and gene-spanning regions offer certain advantages. We tested the phylogenetic utility of five nuclear loci across the species-rich legume clade Hologalegina: PGDH, TRPT, HRIP, RNAR, and CNGC4 (CNGC4-like protein). Our objective was to determine whether any of these nuclear loci could be beneficial at resolving lower-level phylogenetic relationships in this clade, with a particular interest in finding markers that might work at the species level. While the phylogenetic utility of these nuclear loci is unknown outside of Hologalegina, we determined that two of the loci, PGDH and TRPT, are useful for phylogenetic analyses within Hologalegina, depending upon the desired scale of resolution.     

Introns outperform exons in analyses of basal avian phylogeny using clathrin heavy chain genes

Neoaves is the most diverse major avian clade, containing ~95% of avian species, and it underwent an ancient but rapid diversification that has made resolution of relationships at the base of the clade difficult. In fact, Neoaves has been suggested to be a "hard" polytomy that cannot be resolved with any amount of data. However, this conclusion was based on slowly evolving coding sequences and ribosomal RNAs and some recent studies using more rapidly evolving intron sequences have suggested some resolution at the base of Neoaves. To further examine the utility of introns and exons for phylogenetics, we sequenced parts of two unlinked clathrin heavy chain genes (CLTC and CLTCL1). Comparisons of phylogenetic trees based upon individual partitions (i.e. introns and exons), the combined dataset, and published phylogenies using Robinson-Foulds distances (a metric of topological differences) revealed more similarity than expected by chance, suggesting there is structure at the base of Neoaves. We found that introns provided more informative sites, were subject to less homoplasy, and provided better support for well-accepted clades, suggesting that intron evolution is better suited to determining closely-spaced branching events like the base of Neoaves. Furthermore, phylogenetic power analyses indicated that existing molecular datasets for birds are unlikely to provide sufficient phylogenetic information to resolve relationships at the base of Neoaves, especially when comprised of exon or other slowly evolving regions. Although relationships among the orders in Neoaves cannot be definitively established using available data, the base of Neoaves does not appear to represent a hard polytomy. Our analyses suggest that large intron datasets have the best potential to resolve relationships among avian orders and indicate that the utility of intron data for other phylogenetic questions should be examined.     

Evolution of the AMP-forming acetyl-CoA synthetase gene in the Drosophilidae family

Analysis of the AMP-forming ACS gene was performed in 12 species of the Drosophilidae family. Systematically four introns, aligned at the same positions, were detected, but none of them showed a position similar to those known for species outside the Drosophilidae family. The average length of introns varied from 63 to 75 bp but in two species Drosophila takahashii and D. kikkawai the length of the second intron was 343 and 210 bp, respectively. In coding regions, about 80% of the third codon positions were substituted while first and second positions showed, respectively, 14% and 6% substitutions. Interestingly, the divergence observed at the protein level between species was very low. The phylogenetic tree based on the DNA sequences of the exons was mainly in agreement with taxonomic classification and previous molecular phylogenies except for D. ananassae, which appeared more closely related to D. subobscura and D. funebris than to the species of the melanogaster group.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

中国石蒜属种间亲缘关系ITS序列分析

本文利用核糖体DNA内转录间隔区(ITS)序列对石蒜属13个种(含变种)的亲缘关系进行分析。结果表明,各样品的ITS1长度为259~260 bp,ITS2为230 bp,分别有多个特异性信息位点。以ITS序列为依据对石蒜属植物亲缘关系进行分析,表明石蒜属13个种可分为三大类,其中类Ⅰ包括中国石蒜、地笑、安徽石蒜和长筒石蒜,核型为M+T型;类Ⅱ包括矮小石蒜、换锦花、玫瑰石蒜和红蓝石蒜,核型为ST型;类Ⅲ包括稻草石蒜、乳白石蒜、短蕊石蒜和两种人工杂交种,核型为ST+M+T。系统进化树与核型分析结果相似,第Ⅲ类可能为自然杂交种。     

药用真菌金耳的rDNA ITS序列分析与鉴别

研究了两个居群的金耳Tremella aurantialba以及近似品的rDNAITS区碱基全序列的特征及其差异,首次报道了金耳的ITS和5.8SrDNA完整序列,序列总长度为467~468,长度变异较少。聚类分析表明两个居群金耳亲缘关系非常密切,金耳药材和近似品金黄银耳形成一个稳定的独立分支,近似品黄金银耳形成另一个分支。ITS序列的差异为金耳的鉴别提供了可靠的分子标记,为金耳菌类药材基原入药建立了遗传基础。     

Assessing the potential of regions of the nuclear and mitochondrial genome to develop a "molecular tool box" for the detection and characterization of Phytophthora species

Four different intergenic regions of mitochondrial DNA (mt-IGS), a fragment of the intergenic spacer (IGS) region of the rDNA (rDNA-IGS), and a fragment of the ras-related protein (Ypt1) gene were amplified and sequenced from a panel of 31 Phytophthora species representing the most significant forest pathogens and the breadth of diversity in the genus. Over 80 kbp of novel sequences were generated and alignments showed very variable (introns and non-coding regions) as well as conserved coding regions. The mitochondrial DNA regions had an AT/GC ratio ranging from 67.2 to 89.0% and were appropriate for diagnostic development and phylogeographic analysis. The IGS fragment was less variable but still appropriate to discriminate amongst some important forest pathogens. The introns of the Ypt1 gene were sufficiently polymorphic for the development of molecular markers for almost all Phytophthora species, with more conserved flanking coding regions appropriate for the design of Phytophthora genus-specific primers. In general, phylogenetic analysis of the sequence alignments grouped species in clades that matched those based on the ITS regions of the rDNA. In many cases the resolution was improved over ITS but in other cases sequences were too variable to align accurately and yielded phylograms inconsistent with other data. Key studies on the intraspecific variation and primer specificity remain. However the research has already yielded an enormous dataset for the identification, detection and study of the molecular evolution of Phytophthora species.     

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.     

Using nuclear genes to reconstruct angiosperm phylogeny at the species level: A casestudy with Brassicaceae species

Angiosperm phylogeny has been investigated extensively using organellar sequences; recent efforts using nuclear genes have also been successful in reconstructing angiosperm phylogenies at family or deeper levels. However, it is not clear whether nuclear genes are also effective in understanding relationships between species in a genus. Here we present a case study of phylogeny at generic and specific levels with nuclear genes, using Brassicaceae taxa as examples. Brassicaceae includes various crops and the model plant Arabidopsis thaliana. A recent study showed that nuclear genes can provide well-resolved relationships between tribes and larger lineages in Brassicaceae, but few species were included in any given genus. We present a phylogeny with multiple species in each of five genera within Brassicaceae for a total of 65 taxa, using three protein-coding nuclear genes, MLH1, SMC2, and MCM5, with up to approximately 10 200 base pairs (in both exons and introns). Maximum likelihood and Bayesian analyses of the separate gene regions and combined data reveal high resolution at various phylogenetic depths. The relationships between genera here were largely congruent with previous results, with further resolution at the species level. Also, we report for the first time the affinity of Cardamine rockii with tribe Camelineae instead of other Cardamine members. In addition, we report sequence divergence at three levels: across angiosperms, among Brassicaceae species, and between Arabidopsis ecotypes. Our results provide a robust species-level phylogeny for a number of Brassicaceae members and support an optimistic perspective on the phylogenetic utility of conserved nuclear data for relatively recent clades.     

Molecular phylogeny of selected Old World Astragalus (Fabaceae): incongruence among chloroplast trnL-F, ndhF and nuclear ribosomal DNA ITS sequences

This study reports maximum parsimony and Bayesian phylogenetic analyses of selected Old World Astragalus using two chloroplast fragments including trnL-F and ndhF and the nuclear ribosomal internal transcribed spacer (nrDNA ITS). A total of 52 taxa including 34 euploid Old World and New World Astragalus , one aneuploid species from the Neo-Astragalus clade as a representative and 14 other Astragalean taxa, plus Cheseneya astragalina and two species of Caragana as outgroups were analyzed for both trnL-F and nrDNA ITS regions. ndhF was analyzed in 30 taxa and the same number for the combination of these three datasets were examined. In general, the trnL-F dataset and the ndhF and nrDNA ITS datasets generated more or less the same clades within Astragalus . However, in the trnL-F and ndhF phylogenies, Astragalus species are not gathered in a single clade, the so-called Astragalus s.s., as indicated by the nrDNA ITS tree. Visual inspection of these three phylogenies revealed that they were inconsistent regarding the position and relationships of Astragalus hemsleyi , A. ophiocarpus , A. annularis–A. epiglottis / Astragalus pelecinus, A. echinatus and A. arizonicus . Incongruence length difference test suggested that the trnL-F , ndhF and nrDNA ITS datasets were incongruent. In spite of this, phylogenetic analyses of the combined datasets as one unit or as three partitions generated trees that were topologically similar as a mix of the cpDNA and the nrDNA ITS trees. However, the combined dataset provided more resolved and statistically supported clades. The recently described A. memoriosus appeared closely related to A. stocksii (both from sect. Caraganella ) based on both trnL-F and nrDNA ITS sequences.     

根据ITS序列证据重建防己科蝙蝠葛族的系统发育

研究了国产防己科蝙蝠葛族tirb．Menispermeae9属20种和外类群青牛胆族trib．Tinosporeae 2属3种植物完整的ITS(包括5.8S rDNA)序列。trib．Menispermeae的ITS长527～601 bp,排序后长667bp。当gap处理为missing时具281个有信息位点。PAUP软件分析结果表明：①trib．Menispermeae是一个单系类群,该分支得到hootstrap l00％的支持;②确定了存疑种Pachygone valida的系统学位置,该种是Coc—culus属的成员;③Sinomenium和Menispermum两属有很近的系统学关系,组成族内稳定的一支,它们的ITS序列同源性极高,ITS1比族内其它属长41～73bp;④Stephania和Cyclea也是系统发育关系很近的两个类群。前者具两个主要分支,其IIS1、ITS2的G+C含量差异较大,在种类组成上,该两大支与传统上Stephania属内处理的2个亚属——千金藤亚属subgen．Stephania和山乌龟亚属subgen．Tuberiphania基本一致;Cyclea属内种间的ITS序列差异小,同源性极高。     

Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey

We investigated the phylogenetic relationships in Tulipa in Turkey using DNA sequences from the plastid trnL‐trnF region and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. We generated trnL‐trnF and nuclear ITS sequences for 11 Tulipa spp. from Turkey and compared the utility of trnL‐trnF and ITS sequences for phylogenetic analysis. Neighbor‐joining, Bayesian and maximum parsimony methods were implemented using the same matrices. Our study of Tulipa based on molecular data revealed congruent results with previous studies. Despite the relatively lower resolution of trnL‐trnF than that of ITS, both sequence matrices generated similar results. Three clades were clearly distinguished, corresponding to subgenera Tulipa, Eriostemones and Orithyia. It is not fully resolved whether Clusianae should be recognized as a separate section of subgenus Tulipa or a distinct subgenus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 270–279.     

Molecular evolutionary processes and conflicting gene trees: The hominoid case

Molecular evolutionary processes modify DNA over time, creating both newly derived substitutions shared by related descendant lineages (phylogenetic signal) and “false” similarities which confound phylogenetic reconstruction (homoplasy). However, some types of DNA regions, for example those containing tandem duplicate repeats, are preferentially subject to homoplasy-inducing processes such as sporadically occurring concerted evolution and DNA insertion/deletion. This added level of homoplasic “noise” can make DNA regions with repeats less reliable in phylogenetic reconstruction than those without repeats. Most molecular datasets which distinguish among African hominoids support a human-chimpanzee clade; the most notable exception is from the involucrin gene. However, phylogenetic resolution supporting a chimpanzee-gorilla clade is based entirely on involucrin DNA repeat regions. This is problematic because (1) involucrin repeats are difficult to align, and published alignments are contradictory; (2) involucrin repeats are subject to DNA insertion/deletion; (3) gorillas are polymorphic in that some do not have repeats reported to be synapomorphies linking chimpanzees and gorillas. Gene tree/species tree conflicts can occur due to the sorting of ancestrally polymorphic alleles during speciation. Because hominoid females transfer between groups, mitochondrial and nuclear gene flow occur to the same extent, and the probability of conflict between mitochondrial and nuclear gene trees is theoretically low. When hominoid intraspecific mitochondrial variability is taken into account [based on cytochrome oxidase subunit II (COII) gene sequences], humans and chimpanzees are most closely related, showing the same relative degree of separation from gorillas as when single individuals representing species are analyzed. Conflicting molecular phylogenies can be explained in terms of molecular evolutionary processes and sorting of ancient polymorphisms. This perspective can enhance our understanding of hominoid molecular phylogenies. © 1994 Wiley-Liss, Inc.     

Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.      

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

Structure,molecular evolution,and phylogenetic utility of the 5(') region of the external transcribed spacer of 18S-26S rDNA in Lessingia (Compositae,Astereae)

The 18S-26S nuclear rDNA external transcribed spacer (ETS) has recently gained attention as a region that is valuable in phylogenetic analyses of angiosperms primarily because it can supplement nucleotide variation from the widely used and generally shorter internal transcribed spacers (ITS-1 and ITS-2) and thereby improve phylogenetic resolution and clade support in rDNA trees. Subrepeated ETS sequences (often occurring in the 5(') region) can, however, create a challenge for systematists interested in using ETS sequence data for phylogeny reconstruction. We sequenced the 5(')ETS for members of Lessingia (Compositae, Astereae) and close relatives (26 taxa total) to characterize the subrepeat variation across a group of closely related plant lineages and to gain improved understanding of the structure, molecular evolution, and phylogenetic utility of the region. The 5(')ETS region of Lessingia and relatives varied in length from approximately 245 to 1009 bp due to the presence of a variable number of subrepeats (one to eight). We assessed homology of the subrepeats using phylogenetic analysis and concluded that only two of the subrepeats and a portion of a third ( approximately 282 bp in total) were orthologous across Lessingia and could be aligned with confidence and included in further analyses. When the partial 5(')ETS data were combined with 3(')ETS and ITS data in phylogenetic analyses, no additional resolution of relationships among taxa was obtained beyond that found from analysis of 3(')ETS + ITS sequences. Inferred patterns of concerted evolution indicate that homogenization is occurring at a faster rate in the 3(')ETS and ITS regions than in the 5(')ETS region. Additionally, homogenization appears to be acting within but not among subrepeats of the same rDNA array. We conclude that challenges in assessing subrepeat orthology across taxa greatly limit the utility of the 5(')ETS region for phylogenetic analyses among species of Lessingia.