Extensive intraspecific chloroplast DNA (cpDNA) variation in the alpine Draba aizoides L. (Brassicaceae): haplotype relationships and population structure

Chloroplast DNA (cpDNA) sequence variation is currently the most widely used tool for the inference of phylogenetic relationships among plants at all taxonomic levels. Generally, noncoding regions tend to evolve faster than coding sequences and have recently been applied to the study of phylogenetic relationships among closely related taxa. An implicit assumption of many of these studies is that intraspecific cpDNA variation is either absent or low and therefore will not interfere with the reconstruction of interspecific relationships. A survey of cpDNA sequence variation in the common alpine plant species Draba aizoides L. was undertaken to assess levels of intraspecific cpDNA sequence variation. These levels were compared to levels of interspecific sequence divergence between D. aizoides and related alpine Draba species. Intraspecific cpDNA sequence divergence was extensive in D. aizoides, and intraspecific differences were often larger than interspecific differences. cpDNA haplotype relationships were explored using a maximum parsimony approach and minimum-spanning networks. Results from both methods were largely congruent but comparisons provided interesting insights into the presumed evolutionary history of cpDNA haplotypes. A combined effect of cpDNA introgression and complex lineage sorting was inferred to explain the pattern of cpDNA variation found in D. aizoides. Our results suggest that intraspecific cpDNA variation can be extensive and that intraspecific variation needs to be taken into account when inferring phylogenetic relationships among closely related taxa.     

Phylogeography of the Arctic-Alpine Saxifraga oppositifolia (Saxifragaceae) and some related taxa based on cpDNA and ITS sequence variation

Saxifraga oppositifolia (Saxifragaceae) is an important model system for the evolution of Arctic-Alpine plant species. Sequences of the psbA-trnH intergenic spacer of chloroplast DNA and of the internal transcribed spacer region, ITS1-5.8S-ITS2, of the nuclear ribosomal DNA were used to investigate the intraspecific evolution and phylogeography of this species. Samples from nearly the species' entire circumpolar distribution were included in the analysis as well as samples from the closely related taxa S. smalliana from Alaska and S. blepharophylla and S. biflora from the Alps (S. aizoides served as outgroup). These latter taxa showed a low number of parsimony informative characters, in both cpDNA and ITS sequences, which separated them from S. oppositifolia. Two main cpDNA haplotypes were detected within S. oppositifolia, one with a Eurasian distribution and one with an East Asian-North American distribution. This confirmed the existence of two cpDNA lineages with different geographical distributions in this species, which had previously been reported based on a RFLP analysis. The ITS phylogeny was not useful with respect to the intraspecific evolution and phylogeography of S. oppositifolia, because it showed a largely unresolved topology with low statistical support. The cpDNA sequence analysis, however, also suggested a putative long-distance dispersal event. All investigated taxa had cpDNA haplotypes that were congruent with their geographical origin rather than their phylogeny. This could point to putative lineage sorting in S. oppositifolia and related taxa.     

A systematic study of North American Angelica species (Apiaceae) based on nrDNA ITS and cpDNA sequences and fruit morphology

Angelica is a taxonomically complex genus widespread throughout the North Temperate Zone. Previous phylogenetic studies of the genus have focused primarily on its East Asian species. The relationships among its North American members, the monophyly of these species, and the value of fruit morphology in circumscribing its taxa have yet to be examined. This study represents the most comprehensive sampling of Angelica to date (100 species) and includes all 26 species in North America. Relationships are inferred using Bayesian inference, maximum likelihood, and maximum parsimony analyses of ITS sequences and, for multiple accessions of each North American species, cpDNA ndhF-rpl32, rpl32-trnL, and psbM-psbD sequences. The fruit morphological characters examined were those considered phylogenetically important in East Asian Angelica. The results revealed that the North American species fell into three major clades: North American Angelica clade, Archangelica clade, and the Eurasian Angelica clade. Angelica dawsonii has affinities with Lomatium brandegeei. Fourteen species within the North American Angelica clade were strongly supported as monophyletic. Two paraphyletic species resulted in new combinations in A. lineariloba and A. venenosa. Conflict between the ITS-derived and cpDNA-derived phylogenies and the lack of resolution in portions of the trees may be due to chloroplast capture and rapid species radiation. Fruit morphology supported some interspecific relationships based on molecular data, and relationships revealed by ITS and cpDNA data were roughly in accordance with fruit classification type and geographic distribution region, respectively. A diagnostic key based on fruit morphology is provided for the identification of the North American Angelica taxa.     

Genetic variation of oaks (<Emphasis Type="Italic">Quercus</Emphasis> spp.) in Switzerland. 3. Lack of impact of postglacial recolonization history on nuclear gene loci

Quercus petraea, Quercus pubescens and Quercus robur are closely related and interfertile white oaks native to Switzerland. The three species are known to share identical cpDNA haplotypes, which are indicative of the postglacial recolonization history of populations. Only two haplotypes are common in Switzerland. We compared variation of cpDNA and of isozymes in 28 oak populations from Switzerland in order to assess the impact of the postglacial population history on current genetic structures of nuclear controlled isozyme gene loci. Species delineation was based on Principal Component Analysis of leaf morphological traits. The species status of populations was reflected at isozyme gene loci, but differentiation between populations with different cpDNA haplotypes and hence different recolonization history was very low at enzyme gene loci for all species. Thus, glacial and postglacial population history was not reflected at nuclear gene loci on the temporal and spatial scale covered by the present study. Extensive gene flow through pollen among populations is likely to have blurred a previously existing genetic differentiation at biparentally inherited gene loci that possibly evolved in the different glacial refugia of the above mentioned cpDNA haplotypes.     

Patterns of intraspecific diversification in the Piriqueta caroliniana complex in southeastern North America and the Bahamas

Abstract.— We use chloroplast DNA (cpDNA) variation and nested clade phylogeographic analyses to infer the historical processes that have contributed to the high level of morphological and ecological diversification present in a group of herbaceous perennials (the Piriqueta caroliniana complex) in North America and the Bahamas. The presence of morphologically distinct and intercompatible varieties (morphotypes) that can be distinguished based on suites of taxonomic characters (e.g., leaf shape, pubescence type, stature) and contrasting habitat affinities (from marshes to dry pinelands and sand scrub) makes this group particularly appropriate for studies of intraspecific diversification. To examine the distribution of haplotypes among populations, we sampled 467 individuals from 55 locations in Florida, Georgia, and the northern Bahamas (Grand Bahama and Abaco) and screened each individual for cpDNA variation using restriction fragment length polymorphism (RFLP) and heteroduplex analyses. We develop a one-step haplotype phylogeny for this group and use the geographic distributions of haplotypes and clades to test specific phylogeographic hypotheses using the methods developed by Templeton and his colleagues (Templeton 1998). In general, the distribution of haplotypes was strongly influenced by limited dispersal distances, with the more recently derived haplotypes having much lower levels of dispersion and lower frequencies in populations than the ancestral haplotypes. The patterns of clade and haplotype dispersion and displacement and the distribution of morphotypes imply at least three cases of long-distance dispersal and one case of historical fragmentation. The historical patterns inferred for populations of Piriqueta are consistent with known biogeographical events, historical vegetation change, and the concordant patterns of multiple Pleistocene refugia that have been observed for a number of other taxa in southeastern North America.     

Microsatellite analysis of relationships among North American plums (Prunus sect. Prunocerasus, Rosaceae)

Fifteen microsatellite primer pairs developed in sweet cherry and peach were used to explore genetic relationships among North American plums (Prunus section Prunocerasus). In all, 186 putative alleles were detected with a mean value of 12.4 per locus. The Jaccard coefficient of similarity was calculated between all pairs of accessions and their genetic similarities represented by a UPGMA dendrogram. Despite the apparent closeness among native American plums as evidenced by their ability to hybridize freely and their very similar ITS and trnL-trnF nucleotide sequences, all pairs of accessions among the North American plums shared fewer than half of their alleles. Some of the relationships suggested by the UPGMA dendrogram are congruent with current taxonomic hypotheses, but others are difficult to interpret. Further resolution of relationships among American plums will require molecular markers more variable than ITS yet less variable than microsatellites.     

Phylogenetic relationships, morphological incongruence, and geographic speciation in the fontinalaceae (Bryophyta)

Nuclear ribosomal DNA (internal transcribed spacer region) and chloroplast DNA (trnL-trnF region) were sequenced from 40 samples representing all three genera (Brachelyma, Dichelyma, and Fontinalis) and 18 species of the aquatic moss family, Fontinalaceae. Phylogenetic reconstructions recovered from separate and combined analyses were used to test the hypotheses that Fontinalis and Dichelyma are monophyletic (Brachelyma is monotypic), that groups of species within Fontinalis based on leaf morphology (keeled, concave, plane) form monophyletic groups, and that species delineation based on morphological characters within Fontinalis are congruent with nr- and cpDNA gene trees. Using Brachelyma subulata to root the tree, both Dichelyma and Fontinalis are monophyletic and patristically divergent (each united by >15 synapomorphic mutations). Groups of species within Fontinalis defined by leaf morphology are polyphyletic and it is clear that leaf morphology is labile in the genus. As defined morphologically, species of Fontinalis are nonmonophyletic for both nr- and cpDNA sequences and populations of some morphological taxa are separated in widely divergent clades. Molecular evidence suggests that at least some morphospecies are artificial, defined by convergent leaf forms. The weight of the evidence indicates that F. antipyretica is positively paraphyletic, with European populations more closely related to (i.e., share a more recent common ancestor with) European endemic species than to North American populations that are morphologically conspecific. North American populations are more closely related to North American endemic species.     

Mitochondrial DNA variation and reticulate evolution of the genus Abies

The sequences of three regions of mitochondrial DNA (mtDNA) of a total length of 5226 bp were used to study the phylogeography of the genus Abies. The mtDNA haplotype network, comprising 36 studied Abies taxa, consisted of two branches; the first represented all American species plus two Asian, and the second included the remaining Eurasian species. Within these clusters, the haplotypes formed nine major groups, generally corresponding to the clades of the previously obtained phylogeny based on chloroplast DNA (cpDNA), but the relationships of these groups were significantly different; species assignment to the particular mtDNA haplotype group was more in line with its geographical distribution. In addition, the mtDNA haplotype network contains cycles indicating the recombination. It is assumed that the incongruence of cpDNA and mtDNA phylogenies is caused by the introgression capture of alien mtDNA during species hybridization and thus contains information about past migrations. The cases of incongruence of mitochondrial and chloroplast DNA suggesting a migration of Abies between Asia and North America are discussed.     

The hierarchical spatial distribution of chloroplast DNA polymorphism across the introduced range of Silene vulgaris

Silene vulgaris was introduced into North America sometime prior to 1800. In order to document the population structure that has developed since that time, collections were made from 56 local populations distributed among 9 geographical regions in eastern North America. Individual plants were characterized for chloroplast DNA (cpDNA) haplotype by restriction fragment size analysis of four noncoding regions of cpDNA amplified by polymerase chain reaction. A total of 19 cpDNA haplotypes were detected using this method. The overall gene diversity of 0.85 is quite similar to the diversity detected in these same regions of cpDNA in a previously published sample of S. vulgaris taken from across much of Europe. The spatial distribution of the North American cpDNA diversity was quantified by hierarchical F-statistics that partitioned the genetic variance into variation among local populations within regions, and variation among regions. The average FST among populations within regions was 0.66 and the FST among regions was 0.09. The among-region variation was due to both differences among regions in the frequency of two most common haplotypes, and to the presence of a number of region-specific haplotypes. In order to test for isolation by distance at the regional level, FST values were calculated for all possible pairs of regions, and regressed against the geographical distance between those regions. There was no evidence for isolation by distance. It is suggested that the local population structure is generated by recent extinction/colonization dynamics, and that the among-region structure reflects demographic events associated with range expansion following introduction to North America.     

Association between chloroplast DNA and mitochondrial DNA haplotypes in Prunus spinosa L. (Rosaceae) populations across Europe

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum-length spanning tree.     

ORIGINS OF GENOTYPIC VARIATION IN NORTH AMERICAN DANDELIONS INFERRED FROM RIBOSOMAL DNA AND CHLOROPLAST DNA RESTRICTION ENZYME ANALYSIS

Restriction enzyme analysis of ribosomal DNA (rDNA) and chloroplast DNA (cpDNA) is used to assess the relative contribution of hybridization and mutation as sources of genotypic variation in weedy asexual dandelions, with focus on the dandelion flora of North America. Of 318 North American dandelions surveyed, 145 rDNA-cpDNA clones are detected. The combined rDNA-cpDNA genotypes show that most of the polymorphic rDNA and cpDNA restriction sites or lengths in these plants are also present in weedy asexual dandelions collected from natural populations in Europe and in asexual and diploid taxa (microspecies) chosen to represent diverse Eurasian members of the genus. However, of 222 combined rDNA-cpDNA genotypes found in 427 asexual plants surveyed, only 9 genotypes are found in both North American and Eurasian dandelions. Two rDNA and three cpDNA characters are unique to individual plants in North America and are consistent with mutational origins of genotypic variation in asexual lineages. But the array of genotypic diversity, characterized by different combinations of the rDNA and cpDNA characters, show that multiple hybridization events are a more important source of genotypic variation than mutation in the asexual polyploids. The rDNA and cpDNA data also indicate polyphyletic origin of several asexual Taraxacum taxa.     

A species-level phylogenetic study of the Verbena complex (Verbenaceae) indicates two independent intergeneric chloroplast transfers

Two major impediments to infer plant phylogenies at inter- or intra- species level include the lack of appropriate molecular markers and the gene tree/species tree discordance. Both of these problems require more extensive investigations. One of the foci of this study is examining the phylogenetic utility of a combined chloroplast DNA dataset (>5.0kb) of seven non-coding regions, in comparison with that of a large fragment (ca. 3.0kb) of a low-copy nuclear gene (waxy), in a recent, rapidly diversifying group, the Verbena complex. The complex includes three very closely related genera, Verbena (base chromosome number x=7), Glandularia (x=5), and Junellia (x=10), comprising some 150 species distributed predominantly in South and North America. Our results confirm the inadequacy of non-coding cpDNA in resolving relationships among closely related species due to lack of variation, and the great potential of low-copy nuclear gene as source of variation. However, this study suggests that when both cpDNA and nuclear DNA are employed in low-level phylogenetic studies, cpDNA might be very useful to infer organelle evolutionary history (e.g., chloroplast transfer) and more comprehensively understand the evolutionary history of organisms. The phylogenetic framework of the Verbena complex resulted from this study suggests that Junellia is paraphyletic and most ancestral among the three genera; both Glandularia and Verbena are monophyletic and have been derived from within Junellia. Implications of this phylogenetic framework to understand chromosome number evolution and biogeography are discussed. Most interestingly, the comparison of the cpDNA and nuclear DNA phylogenies indicates two independent intergeneric chloroplast transfers, both from Verbena to Glandularia. One is from a diploid North American Verbena species to a polyploid North American Glandularia species. The other is more ancient, from the South American Verbena group to the common ancestor of a major Glandularia lineage, which has radiated subsequently in both South and North America. The commonly assumed introgressive hybridization may not explain the chloroplast transfers reported here. The underlying mechanism remains uncertain.     

Addressing the "hardest puzzle in American pomology:" Phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions

Prunus subg. Prunus sect. Prunocerasus (Rosaceae) is a North American taxon with 17 commonly recognized taxa. To test the hypothesis of monophyly for the section we sequenced the trnG and rpL16 introns and the trnH-psbA and trnS-trnG intergenic spacers for at least two representatives of each of the five subgenera in Prunus. Additionally we sampled heavily among Prunus subg. Prunus sections Prunus and Armeniaca and Prunus subg. Amygdalus because these groups are putatively most closely related to Prunocerasus. Once monophyly of sect. Prunocerasus was shown we added the sequences of trnL and rpS16 introns and the trnL-trnF spacer in an attempt to increase resolution within the section. The species of sect. Prunocerasus showed an initial split with P. subcordata, the only species from western North America, sister to the rest of the group. The remaining species fell into three primary clades. Within each of the three primary clades there was little phylogenetic resolution. Lastly, we present evidence that P. texana, previously classified in subg. Amygdalus, may be a plum or at least contain a Prunocerasus chloroplast. This is the first phylogenetic hypothesis presented for sect. Prunocerasus, and the clades recovered contrast sharply with previously defined groups based on morphological characters.     

Reticulate evolution in Thuja inferred from multiple gene sequences: implications for the study of biogeographical disjunction between eastern Asia and North America

The eastern Asia-North America disjunction is one of the most interesting biogeographical patterns, but its formation is still in much debate. Here nucleotide sequences of five cpDNA regions, nrDNA ITS and two low-copy nuclear genes (LEAFY, 4CL) were employed to reconstruct the phylogeny and to explore the historical biogeography of Thuja, a typical eastern Asia-North America disjunct genus. High topological discordance was observed between chloroplast and nuclear gene trees, even between different nuclear gene trees, suggesting that Thuja could have a reticulate evolutionary history due to multiple interspecific hybridization events. The eastern Asian species Thuja koraiensis might have obtained its chloroplast genome from the eastern North American species T. occidentalis by chloroplast capture, while the western North American species T. plicata is very likely to have inherited a recombinant cpDNA. Based on the phylogenetic analysis of multiple genes, DIVA-reconstruction of the distribution history, molecular clock estimation and fossil data, we inferred that Thuja could have originated from the high-latitude areas of North America in the Paleocene or earlier with subsequent expansion into eastern Asia through the Bering Land Bridge. The two eastern Asia species T. standishii and T. sutchuenensis have a sister relationship, and their split could have occurred in the Oligocene or early Miocene. In the present study, the selection of molecular markers in biogeographic studies was also discussed. Since most previous studies on the eastern Asia and North America disjunction are based on uniparentally inherited cpDNA and (or) directly sequenced nrDNA ITS data, the historical reticulate evolution in the studied groups might have been underestimated. Therefore, we suggest that multiple genes from different genomes, especially low-copy nuclear genes, be used in this research area in the future.     

Chloroplast DNA Intraspecific Phylogeography of Prairie Cordgrass (Spartina pectinata Bosc ex Link)

Chloroplast DNA (cpDNA) sequences are appropriate for studying intraspecific phylogeography. Comparing cpDNA phylogeny with different cytotypes provides insights into the origin and establishment of polyploid species. We have previously defined the geographic distribution of cytotypes in prairie cordgrass throughout the different regions of the United States. In this study, chloroplast haplotype variation is explored in 54 populations of prairie cordgrass, using nine noncoding chloroplast regions. These chloroplast analyses were combined with the cytotype surveys of prairie cordgrass to infer the phylogeography and to elucidate the origin of the different cytotypes. In this study, we identified three haplotypes, Prairie Cordgrass Group 1 (PCG1), Prairie Cordgrass Group 2 (PCG2), and Prairie Cordgrass Group 3 (PCG3). In general, related haplotypes were strongly associated with geographic distribution. Useful insertions–deletions (indels) were also found among prairie cordgrass populations. The PCG1 haplotypes collected inthe East North Central to the New England regions of the US, were polyploid (tetraploid, hexaploid, and octoploids), while the PCG2 haplotypes, found in southern South Dakota, Iowa, and Missouri, were primarily octoploids, but also included a small number of tetraploids. The PCG3 haplotypes were octoploids and were collected in North Dakota, South Dakota, and Minnesota.     

CHLOROPLAST-DNA PHYLOGENETICS AND BIOGEOGRAPHY IN A RETICULATING GROUP: STUDY IN POA (POACEAE)

Cladistic analysis of Poa chloroplast DNA (cpDNA) restriction sites tested previously hypothesized relationships within the genus. Forty-six taxa representing 19 sections or groups and three subgenera of Poa and two out-group genera, Puccinellia and Bellardiochloa, are analyzed. Five major and several minor cpDNA groups are identified. The cpDNA cladogram is generally congruent with the subgeneric taxonomy of Poa. Exceptions are reclassified or are discussed in terms of character incompatibilities and possible reticulation events. The cpDNA tree detected relationships among sections that were unresolved using traditional character sets and provides a basis for polarization of morphological character states. An assessment of biogeographic events based on the cpDNA tree suggests: 1) Poa originated in Eurasia; 2) at least six groups of species independently colonized North America; and 3) two of the latter groups colonized South America, and one closely related group colonized New Zealand and Australia. The cpDNA tree provided a conservative estimate of the number of amphi-neotropical disjunctions when compared to the known number of species disjunctions.     

Interspecific phylogenetic analysis enhances intraspecific phylogeographical inference: a case study in Pinus lambertiana

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with comprehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context.     

Association between chloroplast and mitochondrial lineages in oaks

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.      

A chloroplast DNA phylogeny of eastern Phlox (Polemoniaceae): implications of congruence and incongruence with the ITS phylogeny

The comparison of independent phylogenies is a valuable approach to the study of evolutionary pattern and process. Available data on eastern North American Phlox, including our recent ITS phylogeny, suggest that relationships are complicated in the group and that hybridization may have been a contributing factor. We used restriction site data from the chloroplast genome to develop a second phylogeny for eastern Phlox. Sampling was the same as that for the ITS study and consisted of 79 samples (including all 22 eastern Phlox species and most eastern subspecies, as well as multiple populations of many taxa). The resulting cpDNA phylogeny agrees with the ITS phylogeny in many respects, strengthening earlier conclusions. Nevertheless, incongruence between the trees is noteworthy: many samples, particularly of members of the P. pilosa and P. glaberrima complexes, are placed in different clades. A variety of tests were carried out to assess congruence in terms of topological patterns, character congruence, and homogeneity of data sets. Significant conflict between the phylogenies is discussed in light of the hypothesis that hybridization has affected relationships in this genus.