Interspecific Sequence Divergence Research Articles

Phylogenetic relationships among Thunnus species inferred from rDNA ITS1 sequence

Intra and interspecific nucleotide sequence variation of rDNA first internal transcribed spacer (ITS1) was analysed using all eight species of the genus Thunnus plus two out‐group species within the same family, skipjack tuna Katsuwonus pelamis and striped bonito Sarda orientalis. Intraspecific nucleotide sequence variation in ITS1, including intra‐genomic variation, was low, ranging from 0·003 to 0·014 [Kimura's two parameter distance (K2P)], whereas variation between species within the genus Thunnus ranged from 0·009 to 0·05. The Atlantic and Pacific northern bluefin tunas Thunnus thynnus thynnus and Thunnus thynnus orientalis, recently proposed to be distinct species, were found to share nearly identical ITS1 sequences (mean K2P = 0·006) well within the range of intraspecific variation. The northern bluefin tuna appeared to be a sister group to albacore Thunnus alalunga, with all other Thunnus species in a distinct clade. The ITS1 phylogeny was consistent with mtDNA phylogeny in clustering the three tropical Thunnus species (T. albacares, T. atlanticus and T. tonggol). Southern bluefin Thunnus maccoyii and bigeye Thunnus obesus tunas showed a closer affinity to this tropical tuna group than to the northern bluefin tuna and albacore. The molecular data supported mitochondrial introgression between species and contradicted morphological subdivision of the genus into two subgenera Neothunnus and Thunnus.

Rapid nested PCR-based detection of Ramularia collo-cygni direct from barley

Ramularia collo-cygni is a barley pathogen of increasing importance in Northern and Central Europe, New Zealand and South America. Accurate visual and microscopic identification of the pathogen from diseased tissue is difficult. A nested PCR-based diagnostic test has been developed as part of an initiative to map the distribution of the pathogen in Scotland. The entire nuclear ribosomal internal transcribed spacer and 5.8S rRNA gene regions from 14 isolates of diverse global origin exhibited complete homology following sequence characterization. Two pairs of species-specific primers, based on inter-specific sequence divergence with closely related species, were designed and empirically evaluated for diagnostic nested PCR. Nested primers Rcc3 and Rcc4 consistently amplified a single product of 256 bp from DNA of 24 R. collo-cygni isolates of diverse global provenance, but not from other Ramularia species, or other fungi commonly encountered in cereal pathosystems, as well as Hordeum or Secale DNA preparations. Using this approach, R. collo-cygni was successfully identified from naturally infected barley leaf, awn and grain samples of diverse geographical provenance, in particular from symptoms that lacked the presence of characteristic conidiophores. It is envisaged that this assay will become established as an important tool in continuing studies into the ecology, aetiology and epidemiology of this poorly understood yet economically damaging plant pathogen.

DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera: Lymantriidae)

Reliable and rapid identification of exotic pest species is critical to biosecurity. However, identification of morphologically indistinct specimens, such as immature life stages, that are frequently intercepted at borders is often impossible. Several DNA-based methods have been used for species identification; however, a more universal and anticipatory identification system is needed. Consequently, we tested the ability of DNA "barcodes" to identify species of tussock moths (Lymantriidae), a family containing several important pest species. We sequenced a 617 base pair fragment of the mitochondrial gene cytochrome c oxidase 1 for 20 lymantriid species. We used these, together with other Noctuoidea species sequences from GenBank and the Barcoding of Life Database to create a "profile" or reference sequence data set. We then tested the ability of this profile to provide correct species identifications for 93 additional lymantriid specimens from a data set of mock unknowns. Of the unknowns, 100% were correctly identified by the cytochrome c oxidase 1 profile. Mean interspecific sequence (Kimura 2-parameter) divergence was an order of magnitude greater (14%) than mean intraspecific divergence (<1%). Four species showed deeper genetic divergences among populations. We conclude that DNA barcodes provide a highly accurate means of identifying lymantriid species and show considerable promise as a universal approach to DNA-based identification of pest insects.

Cryptic species and population structuring of the Atlantic and Pacific seabob shrimp species, Xiphopenaeus kroyeri and Xiphopenaeus riveti

Seabob shrimps of the genus Xiphopenaeus are important fishery resources along the Atlantic and Pacific coasts of Central and South America. The genus was considered to comprise two species: the Atlantic Xiphopenaeus kroyeri (Heller, Sitzungsber Math Naturwiss cl kaiserliche Akad Wiss Wien 45:389–426, 1862), and the Pacific Xiphopenaeus riveti (Bouvier, Bull Mus Hist Nat Paris 13:113–116, 1907). In a recent review, Xiphopenaeus was regarded as a monotypic genus, on the basis that no clear morphological differences could be found between Pacific and Atlantic specimens (Perez Farfante and Kensley, Mem Mus Nat Hist Nat Paris 175:1–79, 1997). In the present work, nuclear (allozymes), and mitochondrial (Cytochrome Oxidase I) genes were used to demonstrate the validity of X. riveti and reveal the presence of two cryptic species of Xiphopenaeus within X. kroyeri in the Atlantic Ocean. The high levels of molecular divergence among these species contrast with their high morphological resemblance. Interspecific sequence divergences (Kimura 2-parameter distance) varied from 0.106 to 0.151, whereas intraspecific distances ranged from 0 to 0.008 in Xiphopenaeus sp. 1, from 0 to 0.003 in Xiphopenaeus sp. 2, and from 0.002 to 0.005 in X. riveti. In addition, five diagnostic allozyme loci were found between sympatric samples of Xiphopenaeus sp. 1 and 2 along the Brazilian coast. The results suggest that Xiphopenaeus sp. 2 from the Atlantic is more closely related to the Pacific X. riveti than to the Atlantic Xiphopenaeus sp. 1. Furthermore, a high level of genetic structuring (Xiphopenaeus sp. 1: FST=0.026; P<0.05; Xiphopenaeus sp. 2: FST=0.055; P<0.01) was found in the Brazilian Xiphopenaeus populations, indicating the presence of different genetic stocks in both Atlantic species. These findings have important commercial implications as they show that the fisheries of the two Atlantic species must be managed separately, and that each one is comprised of different populations.

Identification of wild species of sunflower by a specific plastid DNA sequence

SUMMARY Four sunflower species, Helianthus annuus , H. argophyllus , H. debilis and H. tuberosus , were characterized at the molecular level using the plastid trnH-psbA intergenic spacer. The trnH-psbA sequence was selected with the aim of developing a “DNA barcode” system (Kress et al. , 2005) as a tool for species and specimen identification. The plastid region was PCR amplified with specific primers and sequenced with an ABI Prism 3730 Automated DNA sequencer. Intraspecific and interspecific sequence variation was evaluated to assess the resolution of the technique. Sequencing of both forward and reverse strands allowed for a high base calling accuracy and overcame the problem of polymerase slippage within microsatellite regions. After sequence editing, a very low (or absent) intraspecific variability was detected, whereas interspecific variability due to SNPs, indels and SSR length was sufficient for an unambiguous identification of each species. RESUMEN Fue hecha la caracterización de cuatro especies de girasol, Helianthus annuus , H. argophyllus , H. debilis y H. tuberosus , a nivel molecular, por la técnica ’plastid trnH-psbA intergenic spacer’. La secuencia trnH-psbA fue elegida con el objetivo de crear el sistema de “código de barras DNA” (Kress et al. , 2005), que serviría como herramienta para la identificación de las especies vegetales y muestras. La región plastidial está fortificada con la técnica PCR junto con la utilización de los “primers” específicos y secuencionado en el secuenciador automático de DNA “ABI Prism 3730”. La calificación de variabilidad de las secuencias de intra- e interespecies fue realizada con el fin de calificar la precisión de la técnica utilizada. El secuencionamiento de las partes de DNA en las dos direcciones, fue satisfactoriamente exacto, y fue superado el problema de distorsión de la imagen dentro de las regiones microsatélites. Tras la ordenación de las secuencias, la determinada variabilidad de interespecies era baja o no existía. La variabilidad de interespecies SNP, de los fragmentos indel y de longitud de los fragmentos SSR, era suficiente para la identificación irrebatible de cada especie por separado. RÉSUMÉ Les quatre espèces de tournesol, Helianthus annuus , H. argophyllus , H. debilis et H. tuberosus ont été caractérisées au niveau moléculaire par l’espaceur intergénique plastide trnH-psbA. La séquence trnH-psbA a été choisie dans le but de développer un système de „code-barres ADN“ (Kress et al. , 2005) comme outil d’identification d’espèces et d’échantillons. La région plastide a été amplifiée par la méthode PCR avec des amorces spécifiques et séquencée avec un séquenceur ADN automatique “ABI prisme 3730”. La variation de séquence intra et interspécifique a été évaluée pour déterminer la précision de la technique utilisée. Le séquençage des parties d’ADN dans les deux directions a été suffisamment exact et le problème de dérapage polymérase à l’intérieur des régions microsatellites a été maîtrisé. Après organisation de la séquence, une variabilité interspécifique faible ou inexistante a été détectée tandis que la variabilité interspécifique due aux SNP, indels et longueur de SSR suffisait à identifier sans ambiguïté chaque espèce.

Differentiation ofDendrobiumSpecies used as ”Huangcao Shihu” by rDNA ITS Sequence Analysis

The genus Dendrobium Sw. is composed of 74 species and two varieties in China, and 32 species carry the name "Huangcao Shihu" on the herbal medicine market, making the identification of the origin of "Huangcao Shihu" difficult for consumers. Here, the ITS regions were sequenced and evaluated to differentiate the 18 Dendrobium species used as "Huangcao Shihu". Diversity in DNA sequences among various species was found with the inter-specific sequence divergence ranging from 3.2% to 37.9% in ITS1 and 5.0% to 26.6% in ITS2. Moreover, the variations within species were very low, ranging in sequence divergence from 0 to 3.0% in ITS1 and 0 to 4.0% in ITS2. Therefore, these species could be easily distinguished at the DNA level. Furthermore, based on the divergent ITS regions, five pairs of species-specific primers were designed and used for the rapid PCR identification of five Dendrobium species listed in the Chinese Pharmacopoeia.

Development of primers to characterize the mitochondrial control region of Galápagos land and marine iguanas (Conolophus and Amblyrhynchus)

AbstractPrimers were developed for the amplification and sequencing of the mitochondrial control region of Galápagos land (Conolophus) and marine (Amblyrhynchus) iguanas. Sequences were obtained for four land iguana samples from two islands and for 28 marine iguana samples from three islands. A series of 70–80 bp tandem repeats adjacent to the control region are described and preliminary quantification of intra‐ and interspecific sequence divergence is included.

Speciation history of the North American funnel web spiders, Agelenopsis (Araneae: Agelenidae): Phylogenetic inferences at the population–species interface

Intra- and interspecific relationships of 12 out of 13 described species as well as a potential new species in the spider genus Agelenopsis (Araneae: Agelenidae) were analyzed using sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and 16S ribosomal RNA. Approximately half of the species examined formed well-supported monophyletic groups, whereas the rest of the species were part of well-supported monophyletic species groups. Rather than viewing cases where species were not identified as being monophyletic as poor taxonomy, these cases more likely represent recent speciation and offer insights into the process of speciation. The clade with the lowest levels of interspecific sequence divergence was found in eastern North America, whereas western species displayed much higher levels of interspecific divergence. These patterns appear to extend below the species level as well, with southwestern species exhibiting the highest levels of intraspecific sequence divergence and geographic structuring. The relationship between Agelenopsis and Barronopsis, a genus once considered a sub-genus of Agelenopsis, was also examined. The two genera are reciprocally monophyletic but more generic level sampling is needed to confirm an apparent sister relationship between the two.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Classification of BRCA1 missense variants of unknown clinical significance

Background:BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast–ovarian cancer families. Several missense variants have been identified throughout the...

Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation

Introduction: Interpretation of results from mutation screening of tumour suppressor genes known to harbour high risk susceptibility mutations, such as APC, BRCA1, BRCA2, MLH1, MSH2, TP53, and PTEN, is becoming...

Sex-specific SCAR markers in the dioecious plant Rumex nivalis (Polygonaceae) and implications for the evolution of sex chromosomes.

We developed SCAR primers based on isolated and sequenced male-specific fragments as identified in an AFLP analysis of the dioecious plant Rumex nivalis. PCR amplification using these primers on females and males resulted in fragments exclusively present in males. Co-amplification of the nuclear rDNA internal transcribed spacer 2 together with the male-specific fragment was applied as an internal control for successful PCR reactions to avoid false-negative sex scoring. With a length of about 164 bp, the AFLP fragment was of a similar size as the tandemly arranged, repetitive sequences of 180 bp located on the Y chromosomes of Rumex acetosa. The genetic distances between the Y-chromosomal sequences of R. nivalis and R. acetosa, both members of the section Acetosa, were substantial. We found intra-individual divergence among cloned sequences of the male-specific fragment in R. nivalis. The patterns of interspecific and intra-individual sequence variation found are in accordance with proposed modes of the evolution of sex chromosomes. Y chromosomes possibly arose only once in the genus Rumex and consist mainly of heterochromatic DNA. Due to the almost complete absence of selection on them, Y chromosomes are likely to accumulate large numbers of mutations.

Phylogenetic relationships of nearctic Reticulitermes species (Isoptera: Rhinotermitidae) with particular reference to Reticulitermes arenincola Goellner

DNA sequence comparisons of the mitochondrial COII, 16S, and 12S rRNA genes were used to infer phylogenetic relationships among the six known US Reticulitermes species ( Reticulitermes flavipes, Reticulitermes arenincola, Reticulitermes tibialis, Reticulitermes hageni, Reticulitermes virginicus, and Reticulitermes hesperus) and the closely related European species Reticulitermes santonensis. The interspecific pairwise sequence divergence, based on uncorrected “ p” distance, varied up to 10% across the COII, 4.9% across the 16S, and 3% across the 12S fragments. Phylogenetic trees were constructed using maximum parsimony, likelihood, and distance methods. The combined results suggest several phylogenetic relationships including: (i) R. flavipes, R. arenincola, and European R. santonensis are possibly conspecific; (ii) R. virginicus and R. hageni are closely related species; and (iii) R. tibialis and R. hesperus are closely related species. Interestingly, while there is apparent synonymity between R. flavipes and R. arenincola by DNA sequence, there are clear morphological differences in the soldier caste. This finding suggests a combination of molecular and morphological approaches are necessary for accurate species identification. These data lend resolution to the complex problem of Reticulitermes systematics, and will assist future efforts directed toward characterizing species distribution and ecology.

CERAMIUM INKYUII SP. NOV. (CERAMIACEAE, RHODOPHYTA) FROM KOREA: A NEW SPECIES BASED ON MORPHOLOGICAL AND MOLECULAR EVIDENCE1

Ceramium inkyuii sp. nov. is newly described based on samples collected from the east coast of Korea and compared with similar species such as C. paniculatum and C. tenerrimum. The new species is characterized by pseudo‐dichotomously branched thalli with a twist in the upper part, a single row of spines on the abaxial side, strongly inrolled apices, and the presence of gland cells. In contrast, C. paniculatum has alternate branches and lacks gland cells, and C. tenerrimum is spineless and also lacks gland cells. Ceramium inkyuii was observed to be an annual species producing tetrasporangia in the spring to summer and cystocarps in the fall. Plastid‐encoded rbcL and nuclear small subunit (SSU) rDNA sequences were determined in four samples of C. inkyuii from different locations and six samples of four putative relatives. All four C. inkyuii replicates from three different locations had identical sequences of each gene, and the interspecific sequence divergences were enough to warrant its natural entity. The phylogenies of the rbcL and SSU rDNA sequences also indicate the monophyly of C. inkyuii. The spinous C. inkyuii was more closely related to the spineless C. tenerrimum than to the spinous C. paniculatum.

First report of the European flat oyster Ostrea edulis, identified genetically, from Oyster Harbour, Albany, south-western Western Australia

Ostrea angasi is the only large native ostreine species in southern Australian waters (i.e. New South Wales, Victoria, Tasmania, South Australia and Western Australia (WA)). It has also been recorded as a fossil from Late Pliocene calcarenites of the Roe Plain along the south-eastern coast of WA. Wild populations were harvested for food before and after European settlement. A sample of flat oysters from Oyster Harbour, Albany, WA, was typed for 16S and cytochrome oxidase 1 (CO1) mitochondrial (mt) DNA markers previously shown to be diagnostic for species of Ostreidae. Ostrea edulis was identified in the sample at an approximate 30% occurrence with O. angasi. Interspecific partial 16S and CO1 mtDNA sequence divergences, estimated using Kimura's two-parameter model, were 0.83% and 1.45%, respectively. The occurrence of O. edulis among native O. angasi populations has not hitherto been suspected and, thus, there has not been a legitimate morphological separation of the two. These results emphasise the value of molecular markers in: (1) discriminating morphologically plastic and closely related species; and, thus (2) the monitoring of species introduced into morphologically similar stocks. We caution against such introductions because of the possibility of the importation of oyster diseases (e.g. bonamiasis) and of this and other adverse impacts upon native species.

A simple restriction fragment length polymorphism (RFLP) assay to discriminate common Porphyra (Bangiophyceae, Rhodophyta) taxa from the Northwest Atlantic

The identification of Porphyra species hashistorically been difficult because of the lack of distinguishing morphologicaland ecological characters. We developed a restriction fragment lengthpolymorphism (RFLP) assay, based on inter-specific sequence variation inthe ribulose bisphosphate carboxylase oxygenase largesubunit (rbcL) gene andrbcL-rbcS intergenic spacer, toprovide a simple and effective tool for screening and sorting large collectionsof Porphyra from the Northwest Atlantic. A singlerestriction digest (Hae III) discriminates betweenmultiplePorphyra species including one cryptic taxon; anadditionalenzyme (Hind III) was necessary to distinguish between theclosely related P. leucosticta and an introducedspecies P. yezoensis.

Molecular Genetic Evidence for a New Species of Bream of the Genus Acanthopagrus Peters (Perciformes: Sparidae)

Fishes of the genus Acanthopagrus are found throughout the coastal waters of Asia and Australia with several species being of commercial significance. In this study, genetic comparisons are made between widely disjunct populations of Acanthopagrus australis (Gunther) from Australian and Taiwanese waters and among samples of A. butcheri (Munro), A. berda, (Forskal), A schlegeli (Day) and A. latus (Houttuyn) using mitochondrial DNA sequences obtained from the control region. The mean interspecific pairwise sequence divergence for all species is 17%, while the divergence between A. australis from Australia and that of Taiwan is slightly larger at 18%. These values are considerably higher than those found for intraspecific control region comparisons in some fish species. Phylogenetic analyses indicate that A. australis from Australia is more closely related to the Australian species A. butcheri than to A. australis from Taiwan. These findings suggest that the northern and southern hemisphere forms of A. australis are not monophyletic, with the former possibly representing a new undescribed species of Acanthopagrus.

Phylogeography of the Caribbean Rock Iguana (Cyclura): Implications for Conservation and Insights on the Biogeographic History of the West Indies

The Caribbean rock iguana, Cyclura, has had an unstable intrageneric taxonomy and an unclear phylogenetic position within the family Iguanidae. We use mtDNA sequence data to address these issues and explore the phylogeographic history of the genus. ND4 to leucine tRNA sequence data were collected from multiple individuals of each of the eight species of Cyclura (including 15 of 16 subspecies) and from four localities of Iguana iguana (representative of this species' broad geographic range). This data set was combined with sequence data from Sites et al. (1996, Mol. Biol. Evol. 13, 1087–1105) and analyzed under maximum-parsimony and maximum-likelihood optimization criteria. The ND4 region provided good resolution for the majority of nodes, as indicated by high bootstrap support. In agreement with several recent molecular studies, Cyclura is recovered as monophyletic and is not closely related to any other genus, whereas Iguana is strongly supported as the sister taxon to Sauromalus. This result is statistically more likely than other published hypotheses of Iguanid relationships. Cyclura shows a southeast to northwest speciation sequence in the Caribbean, with the most ancient lineage on the Puerto Rican Bank. The amount of interspecific sequence divergence within Cyclura (maximum 11.4%) is very high in comparison to data from other iguanid taxa at this locus, suggesting that this group either has been in the Caribbean for a very long time or has gone through a very rapid rate of evolution at this locus. Using dates from other published studies, we calculate a molecular clock that suggests that Cyclura colonized the Caribbean between 15 and 35 mya. Several questions regarding subspecific taxonomy are raised in the analysis and await further investigation using a more rapidly evolving marker such as nuclear microsatellites.

Molecular evidence for allopolyploid speciation and a single origin of the narrow endemic Draba ladina (Brassicaceae)

Draba ladina (Brassicaceae) is a small alpine flower endemic to the Swiss Alps. It occurs exclusively at elevations between 2600 and 3000 m and is restricted to less than a dozen mountains in the Lower Engadin. Morphological characters and polyploidy suggest a hybrid origin. Potential diploid progenitor species are distributed widely and often occur sympatrically. To study the evolutionary history of D. ladina we assessed intra- and interspecific sequence variation at noncoding chloroplast DNA loci and nuclear rDNA ITS sequences in D. ladina and its presumed progenitor species D. aizoides, D. dubia, and D. tomentosa. A single ITS (Internal Transcribed Spacer) genotype was found in each of D. aizoides and D. dubia and two in D. tomentosa. Additivity of ITS sequences of D. aizoides and D. tomentosa was found in D. ladina, supporting the hypothesis of an allotetraploid origin. Intraspecific cpDNA variation was found in all diploid species, but not in D. ladina. The single chloroplast DNA haplotype found in the latter was closest to one cpDNA haplotype found in D. tomentosa, suggesting that D. tomentosa was the maternal parent. These results suggest that D. ladina is a relatively young, presumably postglacial, taxon with a single allopolyploid origin.

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.