MOLECULAR EVOLUTIONARY DIVERGENCE AMONG NORTH AMERICAN CAVE CRICKETS. II. DNA-DNA HYBRIDIZATION

Single-copy DNA divergence among 23 populations of cave crickets belonging to two genera (Euhadenoecus and Hadenoecus) has been determined by DNA-DNA hybridization employing the TEACL method. These same populations have been studied for allozyme variation (Caccone and Sbordoni, 1987). In addition, a European relative (Dolichopoda laetitiae) has been included as an outgroup for rooting the phylogeny. One of the most remarkable findings is the large degree of DNA divergence among these species and populations. A ΔT_m of up to 5°C has been found between populations of the same species; even further divergence is indicated by a lowered normalized percentage of reassociation. A phylogeny was constructed and tested for synchrony of rates, i.e., a molecular clock. Statistically, we could not reject the clock hypothesis. Attempts to calibrate the clock led to the conclusion that these insects are among the fastest evolving (with respect to single-copy DNA) groups yet studied—at least as fast as Drosophila and sea urchins—where a ΔT_m of 1°C indicates 0.5 to 1.5 MY since the last common ancestor. In general, the phylogeny derived from the DNA data agrees with that derived from isozymes. Nei's D and ΔT_m are correlated; in this group a D of 0.1 corresponds to a ΔT_m of about 1.5°C. This indicates that, relative to total single-copy DNA, the protein-coding regions of the genome are slowly evolving.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Taxonomic and molecular studies on Drosophila sinobscura and D. hubeiensis,two sibling species of the D. obscura group*

Allozyme electrophoresis and three different DNA sequences (ATOC180 satellite DNA, 5SrDNA repeats, and parts of the Adh gene) were used to compare the two closely related East Asian sibling species Drosophila sinobscura and D. hubeiensis producing fertile hybrids in the laboratory. The data were also applied to establish their phylogenetic relationships to the other species of the D. obscura group. Genetic divergence in 5SrDNA repeats and specifically in the Adh gene separate the two species clearly from each other and justify their species status. Both species are related to the European species of the D. obscura group but the presence of members of the ATOC 180 satellite DNA family, specific and common to the species triad D. ambigua, D. tristis and D. obscura, in the genomic DNA of D. sinobscura and D. hubeiensis put the two sibling species in their close neighbourhood.     

Molecular phylogeny of the <Emphasis Type="Italic">Drosophila obscura</Emphasis> species group,with emphasis on the Old World species

Background  

Species of the Drosophila obscura species group (e.g., D. pseudoobscura, D. subobscura) have served as favorable models in evolutionary studies since the 1930's. Despite numbers of studies conducted with varied types of data, the basal phylogeny in this group is still controversial, presumably owing to not only the hypothetical 'rapid radiation' history of this group, but also limited taxon sampling from the Old World (esp. the Oriental and Afrotropical regions). Here we reconstruct the phylogeny of this group by using sequence data from 6 loci of 21 species (including 16 Old World ones) covering all the 6 subgroups of this group, estimate the divergence times among lineages, and statistically test the 'rapid radiation' hypothesis.     

Molecular Evolution of P Transposable Elements in the Genus Drosophila. II. The obscura Species Group

A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.     

Chloroplast DNA restriction site variation in theVernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly ofVernonia

Chloroplast DNA restriction site variation was examined for 35 taxa in theVernonieae and four outgroup tribes, using 17 restriction enzymes mapped for ca. 900 restriction sites per species; 139 mutations were found to be phylogenetically informative. Phylogenetic trees were constructed using Wagner and weighted parsimony, and evaluated by bootstrap and decay analyses. Relationships of Old and New World taxa indicate complex geographical relationships; there was no clear geographic separation by hemisphere. The relationships between Old and New World Vernonias found here support prior morphological analyses. The sister group to all New and most Old World taxa was composed of a small group of Old World species including yellow-flowered, trinervate-leaved species previously postulated to be basal in the tribe. The majority of both New and Old World taxa are derived from a lineage beginning with the monotypic genusStokesia, an endemic of the southeastern United States. The genusVernonia was also found to be paraphyletic within both the New and Old World. Available data do not support either the separation ofVernonia or the tribeVernonieae into geographically distinct lineages. The pattern of relationships within theVernonieae for taxa from North America, Asia, Africa, Central and South America is most similar to that of several other groups of both plants and animals with a boreotropical origin, rather than an origin in Gondwanaland. Such a pattern of distribution suggests more ancient vicariant events than are routinely postulated for theAsteraceae.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Nuclear and mitochondrial ribosomal RNA variability in the obscura group of Drosophila

Parts of 28S (nuclear) and 12S (mitochondrial) ribosomal RNA of Palearctic, Nearctic and African species of the obscura group have been sequenced by the direct method of sequencing. Rates of nucleotide substitutions in both molecules were compared. The nucleotide divergence is higher in the mitochondrial rRNA. Average distances of species taken in pairwise were compared to results obtained with the melanogaster subgroup: the divergence of nuclear rRNA appears lower, that of the mtDNA higher whereas genetic distances (allozymes) and sncDNA distances are similar. Noticeable variability of evolutionary rates can be observed even in low taxonomical levels. Phylogenetic trees for the obscura group are in general agreement with those obtained with other characters.     

Ribosomal DNA evidence for the diversification of Tropaeolum sect. Chilensia (Tropaeolaceae)

Maximum parsimony and Bayesian likelihood analysis of ribosomal DNA internal transcribed spacer (ITS) sequences from 55 samples representing 25 species, subspecies, and/or hybrids of Tropaeolum sect. Chilensia yielded a tree with a large proportion of well-supported nodes at the interspecific and intersubspecific level. However, the tree shows samples from two species, T.azureum and T. brachyceras, arising in two different positions. Samples representing subspecies of T. hookerianum and T. leptophyllum suggest that these species are polyphyletic. The data corroborate evidence for hybridization between T. brachyceras and T. tricolor. Consideration of interfertility data, past and present Chilean ecology, and empirical evidence for the behavior of phenotypic and genotypic characters in known hybrids suggest a high likelihood that reticulate evolution has played a role in the diversification of T. sect. Chilensia. This reticulate evolution may explain the discordance between the ITS tree and the conventional taxonomy. High divergence in ITS sequences between T. sect. Chilensia and other members of Tropaeolum prohibits reliable outgroup-rooting, but midpoint rooting places the root at a partition comprising taxa whose distribution conforms to a relictual eastern-western South American disjunction described for other taxa. Within the Chilean taxa, the analysis suggests that biogeographic diversification has been from the mesophytic southern habitats northward to central mediterranean and northern arid zones.     

Correlation of Tm and sequence of DNA duplexes with delta H computed by an improved empirical potential method

T_m values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH _calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between T_m and ΔH _calc (correlation coefficient, 0.99), with T_m values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH _calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate T_m and (relative) ΔH _calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔH_calc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that T_m of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.     

MITOCHONDRIAL DNA PHYLOGENIES FOR THE DROSOPHILA OBSCURA GROUP

Species belonging to the obscura group of the genus Drosophila have long held a central position in evolutionary studies, especially in experimental population genetics. Despite the considerable amount of accumulated knowledge, many of the phylogenetic relationships of the species in the group remain unclear. Here we present DNA sequence data for the mitochondrial gene cytochrome oxidase I (COI) for 13 species native to both the Old and New Worlds. We combine these data with seven other mitochondrial gene sequences from previous studies, for a total of over 3 kb per species. Strongly supported conclusions include: (1) the two North American subgroups, pseudoobscura and affinis, are each monophyletic; and (2) among Eurasian species two unambiguous clades are identified, one containing D. tristis, D. ambigua, and D. obscura and the other containing D. guanche, D. subobscura, and D. madeirensis. Constructing firm hypotheses connecting these four major clades is problematic with all datasets. Major ambiguities are the number of invasions giving rise to the North American obscura species and the relationships among the Eurasian species. The inadequacy of the mtDNA data to resolve these ambiguities does not reside in lack of changes; the transversions-only parsimony tree has 283 informative characters. Rather, the problems are likely intrinsic to the history of the group: while radiating in temperate Eurasia, North America was colonized once or twice, followed by one or two radiations in the New World.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Colonization of North America by Drosophila subobscura: Ecological analysis of three communities of drosophilids in California

We have carried out bimonthly collections of Drosophila species during a 16 month period in three localities of California (Eureka, Davis, and Gilroy). D. subobscura, which has colonized North America only a few years ago, is well established and has an annual abundance cycle similar to that observed in the Old World. The differences between the three localities account for 26.4% of the species diversity. Davis and Gilroy exhibit similarities attributable to their similar environmental conditions. Seasonality accounts for 46% and 50.4 % of the species diversity in Davis and Gilroy respectively, but only for 39.4% in Eureka. Differences between years explain only a small percentage of the total species diversity. A correspondence analysis of the association between species and months of collection shows ecological similarities between D. subobscura and the Nearctic members of the obscura species group.     

Ancient and Recent Horizontal Invasions of Drosophilids by P Elements

P elements of two different subfamilies designated as M- and O-type are thought to have invaded host species in the Drosophila obscura group via horizontal transmission from external sources. Sequence comparisons with P elements isolated from other species suggested that the horizontal invasion by the O-type must have been a rather recent event, whereas the M-type invasion should have occurred in the more distant past. To trace the phylogenetic history of O-type elements, additional taxa were screened for the presence of O- and M-type elements using type-specific PCR primers. The phylogeny deduced from the sequence data of a 927-bp section (14 taxa) indicate that O-type elements have undergone longer periods of regular vertical transmission in the lineages of the saltans and willistoni groups of Drosophila. However, starting from a species of the D. willistoni group they were transmitted horizontally into other lineages. First the lineage of the D. affinis subgroup was infected, and finally, in a more recent wave of horizontal spread, species of three different genera were invaded by O-type elements from the D. affinis lineage: Scaptomyza, Lordiphosa, and the sibling species D. bifasciata/D. imaii of the Drosophila obscura subgroup. The O-type elements isolated from these taxa are almost identical (sequence divergence <1%). In contrast, no such striking similarities are observed among M-type elements. Nevertheless, the sequence phylogeny of M-type elements is also not in accordance with the phylogeny of their host species, suggesting earlier horizontal transfer events. The results imply that P elements cross species barriers more frequently than previously thought but require a particular genomic environment and thus seem to be confined to a rather narrow spectrum of host species. Consequently, different P element types acquired by successive horizontal transmission events often coexist within the same genome. Received: 15 May 2000 / Accepted: 19 July 2000     

Plastome sequences of two New World bamboos—Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)—extend phylogenomic understanding of Bambusoideae

• Premise of the study: Two New World species of Bambusoideae, Arundinaria gigantea and Crytpochloa strictiflora, were investigated in a phylogenomic context. Complete plastome sequences have been previously determined and analyzed for nine bambusoid species that exclusively represent Old World lineages. The addition of New World species provides more complete information on relationships within Bambusoideae. • Methods: Plastomes from A. gigantea and C. strictiflora were sequenced using Sanger methods. Phylogenomic and divergence estimate analyses were conducted on both species with 23 other Poaceae. • Key Results: Phylogenomic and divergence analyses suggested that A. gigantea diverged from within Arundinarieae between 1.94–3.92 mya and that C. strictiflora diverged as the sister to tropical woody species between 24.83 and 40.22 mya. These results are correlated with modern relative diversities in the two lineages. • Conclusions: The two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa. The overall evidence for A. gigantea is consistent with recent dispersal, and that for C. strictiflora is consistent with vicariance.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Determination of DNA base compositions from melting profiles in dilute buffers

Equations were determined for the dependency of the melting temperature (T_m) of DNA upon the logarithm of the sodium ion concentration, for four DNA samples of widely different base compositions (θ_GC). The slopes of these T_m versus log M equations wore found to decrease with increasing θ_{G C}of the samples. An empirical equation relating T_m, log M (Na⁺) and θ_{G C} was derived, which also accounts for differences in T_m versus log M slopes. Data from the literature for some synthetic polynucleotides and for the crab(Cancer pagarus) satellite poly AT are discussed in relation to the above finding. The changes in T_m versus log M slopes with θ_{G C} are interpreted in terms of changes in the thermodynamic parameters ΔS and ΔH with base composition.     

Mitochondrial DNA evolution in theobscura species subgroup ofDrosophila

Summary Mitochondrial DNA (mtDNA) restriction site maps for nine species of theDrosophila obscura subgroup and forDrosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for theobscura subgroup species, one Nearctic, represented byDrosophila pseudoobscura, and four Palearctic, two grouping the related triads of speciesDrosophila subobscura, Drosophila madeirensis, Drosophila guanche, andDrosophila ambigua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each,Drosophila bifasciata, andDrosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related speciesD. subobscura, D. madeirensis, andD. guanche, the pairD. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for theobscura subgroup species was from 15.5 kb forD. pseudoobscura to 17.1 forD. tristis.     

Cryptic species in the genus Phylloscopus (Old World leaf warblers)

In almost all ecological and evolutionary research, it is important to assess the number of species under study. Cryptic species, which are morphologically similar, present a special problem because they need to be identified through studies of behavioural or genetic variation. Here we review the important factors in the recent discovery of three species and elevation of nine previously known taxa to species status within the genus Phylloscopus (Old World leaf‐warblers) and we examine the case of three morphologically similar taxa (inornatus, humei and mandellii) that until recently were considered to be members of the single species Phylloscopus inornatus. We have identified several locations at which humei and inornatus coexist at high density, and the results of playback experiments, observations of interactions, and spectrogram analysis of vocalizations at these sites all indicate that there are significant behavioural differences and a lack of recognition between humei and inornatus. Estimated relationships within the species complex based on mitochondrial control region DNA sequences show that there is little variation within the geographic ranges of either humei or inornatus compared with the deep split between the two taxa. These observations support the division of humei and inornatus into separate species. The third taxon, mandellii, which is geographically separated from both humei and inornatus, is behaviourally and genetically more similar to humei. Under the biological species concept, it is difficult to determine the species status of allopatric taxa. Alternative species concepts, such as the phylogenetic and recognition concepts, are more easily applied to allopatric taxa but lead to differing conclusions over whether mandellii and humei are separate species. This confusion arises from the fact that the two taxa are in an early stage of allopatric divergence. The review of other recently designated species reveals that song divergence is of primary importance in their designation and that song variation, playback experiments and genetic analysis lead to similar conclusions regarding species status.     

a molecular phylogeny of apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

Phylogenetic relationships among 40 New World and Old World members of Apiaceae subfamily Apioideae, representing seven of the eight tribes and eight of the ten subtribes commonly recognized in the subfamily, were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of 18-26S nuclear ribosomal DNA. Although the sequences are alignable, with only 11% of sites excluded from the analyses because of alignment ambiguity, divergence values in pairwise comparisons of unambiguous positions among all taxa were high and ranged from 0.5 to 33.2% of nucleotides in ITS 1 and from 0 to 33.2% of nucleotides in ITS 2. Average sequence divergence across both spacer regions was 18.4% of nucleotides. Phylogenies derived from ITS sequences estimated using neighbor-joining analysis of substitution rates, and maximum likelihood and parsimony methods give trees of essentially similar topology and indicate that: (1) there is little support for any existing system of classification of the subfamily that is based largely on morphological and anatomical features of the mericarp; (2) there is a major phylogenetic division within the subfamily, with one clade comprising the genus Smyrnium and those taxa belonging to Drude's tribes Dauceae, Scandiceae, and Laserpitieae and the other clade comprising all other examined taxa; and (3) the genera Arracacia, Coaxana, Coulterophytum, Enantiophylla, Myrrhidendron, Prionosciadium, and Rhodosciadium, all endemic to Mexico and Central America, comprise a clade but their relationships to other New World taxa are equivocal. A phylogeny derived from parsimony analysis of chloroplast DNA rpoC1 intron sequences is consistent with, but considerably less resolved than, relationships derived from these ITS regions. This study affirms that ITS sequences are useful for phylogenetic inference among closely related members of Apioideae but, owing to high rates of nucleotide substitution, are less useful in resolving relationships among the more ancestral nodes of the phylogeny.