Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

从核rDNA序列探讨隙蛛亚科Coelotinae的分类地位

隙蛛亚科Coelotinae主要分布于东亚地区,其中我国的已有种类占到全世界种数的一半以上,因此对于我国隙蛛类蜘蛛的研究已经成为世界暗蛛科研究的重点之一。隙蛛亚科属于无筛器类群,于1893年,由Cambridge以隙蛛属为模式属而建立,归属于无筛器的漏斗蛛科。之后,虽然经历了数次修订     

Molecular phylogenetic relationships inAveneae (Poaceae) species and other grasses as inferred from ITS1 and ITS2 rDNA sequences

A phylogenetic analysis was conducted on sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA in 23 species ofAveneae (Poaceae subfam.Pooideaae). These sequences ofHelictotrichon spp.,Arrhenatherum elatius, Avena spp.,Trisetum spp.,Koeleria spp.,Holcus lanatus, Alopecurus vaginatus together with published ITS sequences of furtherAveneae, Poeae, Triticeae, andBromeae were analysed by the neighbor-joining distance method to assess the molecular phylogenetic relationship in perennial and annualAveneae. The results suggest unexpectedly close affinities of the agronomically important genusAvena to comparatively small-flowered taxa ofAveneae. GenusArrhenatherum and small-flowered subgenera ofHelictotrichon are close extant relatives. The large genusHelictotrichon is para- if not polyphyletic, only its subgenera are monophyletic.Trisetum is clearly separated fromHelictotrichon and forms together withKoeleria and perhaps others a monophyletic lineage which is characterised by a conspicuous 9-bp deletion in ITS1. The impact of the ITS data on the delineation of some genera and subtribes ofAveneae and on the recognition of their biogeographical and ecological patterns is outlined.     

Molecular systematics of Helicoma, Helicomyces and Helicosporium and their teleomorphs inferred from rDNA sequences

Three genera of asexual, helical-spored fungi, Helicoma, Helicomyces and Helicosporium traditionally have been differentiated by the morphology of their conidia and conidiophores. In this paper we assessed their phylogenetic relationships from ribosomal sequences from ITS, 5.8S and partial LSU regions using maximum parsimony, maximum likelihood and Bayesian analysis. Forty-five isolates from the three genera were closely related and were within the teleomorphic genus Tubeufia sensu Barr (Tubeufiaceae, Ascomycota). Most of the species could be placed in one of the seven clades that each received 78% or greater bootstrap support. However none of the anamorphic genera were monophyletic and all but one of the clades contained species from more than one genus. The 15 isolates of Helicoma were scattered through the phylogeny and appeared in five of the clades. None of the four sections within the genus were monophyletic, although species from Helicoma sect. helicoma were concentrated in Clade A. The Helicosporium species also appeared in five clades. The four Helicomyces species were distributed among three clades. Most of the clades supported by sequence data lacked unifying morphological characters. Traditional characters such as the thickness of the conidial filament and whether conidiophores were conspicuous or reduced proved to be poor predictors of phylogenetic relationships. However some combinations of characters including conidium colour and the presence of lateral, tooth-like conidiogenous cells did appear to be predictive of genetic relationships.     

Phylogenetic relationships among members of the subfamily Sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its type subsection was significantly included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aziopsis and Phedimus, based on which they can be recognized as isolated genera.     

Phylogenetic relationships among members of the subfamily sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its typical subsection was reliably included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aizopsis and Phedimus, based on which they can be recognized as isolated genera.     

Basal relationships of Coleoptera inferred from 18S rDNA sequences

The basal relationships of the hyperdiverse insect order Coleoptera (beetles) have proven difficult to resolve. Examination of beetle suborder relationships using 18S ribosomal DNA reveals a previously unproposed relationship among the four major lineages: [(Archostemata(Myxophaga(Adephaga, Polyphaga)))]. Adding representatives of most other insect orders results in a non-monophyletic Coleoptera. However, constraining Coleoptera and its suborders to be monophyletic, in analyses of beetle and outgroup sequences, also results in the above beetle relationships, with the root placed between Archostemata and the remaining suborders.     

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed, differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided.     

Molecular phylogenetic relationship of toads distributed in the Far East and Europe inferred from the nucleotide sequences of mitochondrial DNA genes

The toads of the Bufo bufo species group are widely distributed in the Eurasian continent and Japanese Archipelago. In this study, we analyzed the mtDNA gene sequences of this species group and estimated the divergence time to clarify the evolutionary relationships and biogeography of toads distributed in the Far East and Europe. The phylogenetic tree indicated that this group produced Bufo bufo in Europe, whereas it produced B. japonicus in the Far East. B. japonicus was divided into three major clades corresponding to a group consisting of B. j. gargarizans in China, B. j. bankorensis in Taiwan, and B. j. miyakonis on Miyako Isl. and eastern and western groups of Japanese B. j. japonicus subspecies group. The eastern and western groups were divided into several subclades which tended to reflect the region-specific geographic distribution of all localities except B. j. japonicus from Hakodate. The estimated branching times of these clades suggest that geological events may have influenced the divergence of the toads distributed in the Far East and Europe.     

Molecular phylogeny of ocelloid-bearing dinoflagellates (Warnowiaceae) as inferred from SSU and LSU rDNA sequences

Background  

Dinoflagellates represent a major lineage of unicellular eukaryotes with unparalleled diversity and complexity in morphological features. The monophyly of dinoflagellates has been convincingly demonstrated, but the interrelationships among dinoflagellate lineages still remain largely unresolved. Warnowiid dinoflagellates are among the most remarkable eukaryotes known because of their possession of highly elaborate ultrastructural systems: pistons, nematocysts, and ocelloids. Complex organelles like these are evolutionary innovations found only in a few athecate dinoflagellates. Moreover, the taxonomy of warnowiids is extremely confusing and inferences about the evolutionary history of this lineage are mired by the absence of molecular phylogenetic data from any member of the group. In this study, we provide the first molecular phylogenetic data for warnowiids and couple them with a review of warnowiid morphological features in order to formulate a hypothetical framework for understanding character evolution within the group. These data also enabled us to evaluate the evolutionary relationship(s) between warnowiids and the other group of dinoflagellates with complex organelles: polykrikoids.     

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed, differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided.     

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.     

Multiple origins of anural development in ascidians inferred from rDNA sequences

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians. Correspondence to: W.R. Jeffery     

Molecular phylogeny of the Caryophyllaceae (Caryophyllales) inferred from chloroplast matK and nuclear rDNA ITS sequences

Caryophyllaceae is a principally holarctic family including around 2200 species often classified into the three subfamilies Alsinoideae, Caryophylloideae, and Paronychioideae. Complex and possibly homoplasious morphological characters within the family make taxa difficult to delimit and diagnose. To explore part of the morphological evolution within the family, we investigated the phylogeny of the Caryophyllaceae by means of analyzing plastid and nuclear sequence data with parsimony and Bayesian methods. We describe a mode of tracing a stable phylogenetic signal in ITS sequences, and a significant common signal is shared with the plastid data. Parsimony and Bayesian analyses yield some differences in tree resolution. None of the subfamilies appear monophyletic, but the monophyly of the Caryophylloideae is not contradicted. Alsinoideae are paraphyletic, with Arenaria subg. Eremogone and Minuartia subg. Spergella more closely related to the Caryophylloideae. There is strong support for the inclusion of Spergula-Spergularia in an Alsinoideae-Caryophylloideae clade. Putative synapomorphies for these groupings are twice as many stamens as number of sepals and a caryophyllad-type of embryogeny. Paronychioideae form a basal grade, where tribe Corrigioleae are sister to the rest of the family. Free styles and capsules with simple teeth are possibly plesiomorphic for the family.     

Phylogenetic relationships ofSphaerophoraceae (Ascomycetes) inferred from SSU rDNA sequences

SSU rDNA was sequenced from the lichenized fungiBunodophoron scrobiculatum andLeifidium tenerum (Sphaerophoraceae), andStereocaulon ramulosum andPilophorus acicularis (Stereocaulaceae) and analysed by maximum parsimony with 44 homologous ascomycete sequences in a cladistic study. A small insertion (c. 60 nt.) was found in the sequence ofLeifidium tenerum. Sphaerophoraceae constitutes a strongly supported monophyletic group which groups together withLecanora dispersa and theStereocaulaceae. Together withPorpidia crustulata, this larger group is a sistergroup to thePeltigerineae. This analysis thus supports theLecanorales as monophyletic, includingSphaerophoraceae and thePeltigerineae, but does not provide strong support for this monophyly. The analysis also suggests that the prototunicate ascus in theSphaerophoraceae is a reversion to the plesiomorphic state. Based on morphological, anatomical and chemical reasons,Sphaerophoraceae is proposed to belong to one of the groups presently included in the paraphyletic suborderCladoniineae within theLecanorales.     

Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic analyses of ITS rDNA and actin gene sequences

Ampelomyces quisqualis complex is well known as the most common and widespread hyperparasite of the family Erysiphaceae, the cause of powdery mildew diseases. As commercial biopesticide products it is widely used to control the disease in field and plastic houses. Although genetic diversity within Ampelomyces isolates has been previously recognized, a single name A. quisqualis is still applied to all pycnidial intracellular hyperparasites of powdery mildew fungi. In this study, the phylogenetic relationships among Ampelomyces isolates originating from various powdery mildew fungi in Korea were inferred from Bayesian and maximum parsimony analyses of the sequences of ITS rDNA region and actin gene. In the phylogenetic trees, the Ampelomyces isolates could be divided into four distinct groups with high sequence divergences in both regions. The largest group, Clade 1, mostly accommodated Ampelomyces isolates originating from the mycohost Podosphaera spp. (sect. Sphaerotheca). Clade 2 comprised isolates from several genera of powdery mildews, Golovinomyces, Erysiphe (sect. Erysiphe), Arthrocladiella, and Phyllactinia, and was further divided into two subclades. An isolate obtained from Podosphaera (sect. Sphaerotheca) pannosa was clustered into Clade 3, with those from powdery mildews infecting rosaceous hosts. The mycohosts of Ampelomyces isolates in Clade 4 mostly consisted of species of Erysiphe (sect. Erysiphe, sect. Microsphaera, and sect. Uncinula). The present phylogenetic study demonstrates that Ampelomyces hyperparasite is indeed an assemblage of several distinct lineages rather than a sole species. Although the correlation between Ampelomyces isolates and their mycohosts is not obviously clear, the isolates show not only some degree of host specialization but also adaptation to their mycohosts during the evolution of the hyperparasite.     

Morphology and biogeography of Apiaceae subfamily Saniculoideae as inferred by phylogenetic analysis of molecular data

The phylogenetic placements of several African endemic genera at the base of Apiaceae subfamilies Saniculoideae and Apioideae have revolutionized ideas of relationships that affect hypotheses of character evolution and biogeography. Using an explicit phylogeny of subfamily Saniculoideae, we reconstructed the evolutionary history of phenotypic characters traditionally important in classification, identified those characters most useful in supporting relationships, and inferred historical biogeography. The 23 characters examined include those of life history, vegetative morphology, inflorescences, and fruit morphology and anatomy. These characters were optimized over trees derived from maximum parsimony analysis of chloroplast DNA trnQ-trnK sequences from 94 accessions of Apiaceae. The results revealed that many of these characters have undergone considerable modification and that traditional assumptions regarding character-state polarity are often incorrect. Infrasubfamilial relationships inferred by molecular data are supported by one to five morphological characters. However, none of these morphological characters support the monophyly of subfamilies Saniculoideae or Apioideae, the clade of Petagnaea, Eryngium and Sanicula, or the sister-group relationship between Eryngium and Sanicula . Southern African origins of Saniculoideae and of its tribes Steganotaenieae and Saniculeae are supported based on dispersal-vicariance analysis.     

Genetic diversity within the Albugo candida complex (Peronosporales, Oomycota) inferred from phylogenetic analysis of ITS rDNA and COX2 mtDNA sequences

Albugo candida is a destructive fungus infecting brassicaceous hosts. The genetic diversity within the A. candida complex from various host plants was investigated by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and the cytochrome c oxidase subunit II (COX2) region of mtDNA. The aligned nucleotide sequences of A. candida shared significantly high distances, up to 20.4 and 8.9%, in two genes. The phylogenetic trees, obtained using the Bayesian method and maximum parsimony analysis, showed two separate groups that corresponded to the host genera. Group I included A. candida isolates infecting Arabis, Autrieta, Berteroa, Biscutella, Brassica, Cardaminopsis, Diplotaxis, Eruca, Erysimum, Heliophila, Iberis, Lunaria, Raphanus, Sinapis, Sisymbrium, and Thlaspi. Group II contained all isolates from Capsella, Descurainia, Diptychocarpus, Draba, and Lepidium. The genetic similarities between the two genes among isolates within Group I were 99.0-100% and 99.6-100%, while those within Group II were 90.4-100% and 91.1-100%, respectively, showing considerably lower values than for Group I. The A. candida isolates from Capsella bursa-pastoris in Korea are clearly separated by sequence analysis for the two genes compared to those from Wales, England, and the USA. Based on the molecular data from the two genes, we suggest the high degree of genetic diversity exhibited within A. candida complexes warrants their division into several distinct species.     

Molecular phylogeny of Lactarius volemus and its allies inferred from the nucleotide sequences of nuclear large subunit rDNA

The phylogenetic relationships of Lactarius volemus and its relatives were investigated using the nucleotide sequences of the nuclear-encoded large subunit ribosomal DNA (LSU rDNA). Thirty-six sequences from L. volemus, L. corrugis, and L. hygrophoroides, including three sequences obtained from the GenBank database, were used in this study. Samples studied were divided into four major subclades (A–D) in both neighbor-joining (NJ) and maximum-parsimony (MP) trees. Lactarius volemus and L. corrugis formed one large clade in both NJ and MP trees (bootstrap value, 100%), which was divided into three subclades (A–C). Subclade A included three clusters of L. volemus strains, i.e., velvet, red, and Chinese types. Subclade B included the common and red types of L. corrugis. Subclade C included the common and yellow types of L. volemus. Subclade D included only one type of L. hygrophoroides. An analysis of the fatty acid composition supported the divisions found in the molecular analysis. Analyses of nucleotide sequence, fatty acid composition, morphological characteristics, and the taste of the fruiting bodies all led us to conclude that the common, velvet, red, and Chinese types of L. volemus, and the common and red types of L. corrugis, may each belong to different species, subspecies, or varieties. Further studies with more material from a wide range of regions are required to conduct taxonomic revision of these types. The LSU rDNA region may be a useful tool to investigate phylogenetic relationships within the section Dulces of the genus Lactarius.     

Phylogeny of subfamily Epidendroideae (Orchidaceae) inferred from ndhF chloroplast gene sequences

This project undertakes the first molecular-based phylogenetic study of subfamily Epidendroideae (Orchidaceae). Approximately 1200 nucleotides (from the 3' half of the chloroplast gene ndhF for 34 orchid taxa and a lilioid monocot, Clivia miniata (Amaryllidaceae), were subjected to phylogenetic analysis using parsimony and maximum likelihood methods. Oryza sativa (Poaceae), a nonlilioid monocot, was designated as outgroup. Trees from both parsimony and maximum likelihood methods suggest that subfamily Epidendroideae is monophyletic, with Listera (Neottieae) as sister. Although subtribal relationships are typically well resolved and have strong branch support, intertribal relationships are generally poorly resolved. Perhaps this general lack of resolution among tribes reflects a rapid species radiation that coincided with anatomical, physiological, and anatomical adaptations that initiated large-scale epiphytism in the ancestral Epidendroideae. Six taxa in this study exhibit deletions that are not evenly divisible by three and result in extensive sequence frameshifts. For example, one deletion is 227 bp in length and is flanked by the short direct repeat sequence; TCAATAGGAATTTCTTTT. Multiple deletions and frameshifts suggest that ndhF may be a pseudogene, in at least some orchid taxa.