trnL-trnF Intergenic Spacer Research Articles

Toward a Phylogeny of Mimosa (Leguminosae: Mimosoidae): A Preliminary Analysis of Southern South American Species Based on Chloroplast DNA Sequence1

The pantropical and subpantropical genus Mimosa L. comprises more than 500 species, of which nearly 480 are reported for the American Continent. Mimosa is subdivided into five sections, four of which are represented in southern South America: Batocaulon DC., Habbasia DC., Calothamnos Barneby, and Mimosa. Previous taxonomic studies of the species from the austral region have found classification conflicts among (a) sections Batocaulon and Habbasia, (b) sections Calothamnos and Mimosa, and (c) series and subseries within section Mimosa. This paper reports a preliminary phylogenetic analysis of chloroplast nucleotide sequences of the trnL intron and the trnL-trnF intergenic spacer from 34 species of Mimosa and related genera. Key morphological characters were mapped onto the phylogenetic hypothesis and discussed. Sequence analysis indicates that the genus Mimosa is monophyletic; it is derived from Piptadenia viridiflora (Kunth) Benth. The four sections proposed by Barneby are not natural groups. The cladogram retrieved indicates that the representatives of Mimosa sect. Batocaulon are not clustered, the xerophylous representatives of this section are basal, and the remaining species are related to those species of section Habbasia, suggesting that section Batocaulon ser. Stipellares is more recently derived. The species of Mimosa sect. Calothamnos that were analyzed are nested in section Mimosa. The results seem to support retention of this section within section Mimosa as was noted previously by Bentham. The chloroplast sequence data suggest that the representatives from sections Calothamnos and Mimosa share a common ancestor with those from section Habbasia and section Batocaulon ser. Stipellares.

Phylogeny, Species Richness, and Ecological Specialization in Cyperaceae Tribe Cariceae

Cyperaceae tribe Cariceae is characterized by both species richness and habitat diversity, making it an ideal system to study ecological specialization and niche differentiation. We present a phylogenetic hypothesis for the tribe based on nuclear and chloroplast DNA sequence comparisons (ETS-1f, ITS, trnL intron, trnL-trnF intergenic spacer) for 140 representative species from five continents, and use this hypothesis to suggest patterns of both niche conservatism and niche differentiation, particularly within the large subgenus Carex. We identify a new major clade, comprising forest species of East Asian Carex section Siderostictae (subgenus Carex) as sister to the rest of tribe Cariceae. Within Carex subgenus Carex, species tolerant of water-saturated habitats occur in only a few, apparently derived groups, with varying species richness. Clades of predominantly wetland species tend to have broad geographic distribution, often with sister species on different continents, suggesting recent dispersal. In contrast, species within several clades are predominantly forest specialists with distinct Asian and North American lineages. Niche segregation along environmental gradients, such as soil moisture or acidity, is quite common among closely related wetland species, but more difficult to demonstrate within upland forest groups. More complete sampling of species within both wetland and forest groups, combined with comparable sampling of environmental preferences and testing against null models, will be needed for more rigorous exploration of the observed patterns.

Chloroplast DNA analysis in Tunisian fig cultivars ( Ficus carica L.): Sequence variations of the trnL-trnF intergenic spacer

We investigated the nucleotide variation of a non-coding, chloroplast DNA (cpDNA) region to infer relationships among Tunisian fig cultivars. In this study, we examine the level of genetic diversity and its distribution using sequences of the trnL and trnF genes intergenic spacer. The non-coding region displays 28 substitution sites. Insertions and deletions involving 6 sites were found. By using the Kimura-2 method, nucleotide sequences have been aligned using the MEGA program to calculate pairwise divergence of trnL-trnF spacer sequences between cultivars. The size of this non-coding region varied from 430 to 464 bases. The relatively high A + T values (63.7–64.4%) of trnL-trnF intergenic spacer in Ficus carica may explain the high proportion of the identified transversions ( t i/ t v = 0.9). These results suggest the occurrence of nucleotide diversity with a large variation level of chloroplast non-coding region. The analysed data illustrate a considerable level of variability in the genetic pool of the local germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical correspondence. Fourteen haplotypes were detected among 20 individuals examined, yielding a haplotype diversity of 0.983 and a high level of nucleotide diversity (0.0100). The observed variation pattern of plastid DNA provides evidence that the fig germplasm has been undergoing rapid expansion. Neutrality tests rejected the neutrality assumption in the total sample. The cytoplasm variability indicates a narrow genetic base in the cultivated common fig. Despite the high level value of the apparent diversity, we may conclude that fig chloroplast genome provides a new conceptual and practical opportunity to evaluate genetic diversity and to identify local cultivars, making it a valuable source to include into potential breeding programs.

Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.

A Phylogenetic Study in American Podocominae (Asteraceae: Astereae) Based on Morphological and Molecular Data

Abstract A molecular and morphological phylogenetic analysis of South American Podocominae is herein performed. Taxa of both South American and Australasian Podocominae as well as members of other subtribes of Astereae were included in the analyses. Sequence data from the nuclear internal transcribed spacer (ITS), and the plastid trnL–trnF intergenic spacer regions were used in the molecular analysis to assess relationships among the South American genera of Podocominae. The trnL–trnF intergenic spacer provided only six informative characters. In contrast, the ITS region was more variable. Our results showed that: 1) neither the South American genera of Podocominae nor the whole subtribe Podocominae are monophyletic; 2) South American and Australasian genera, initially placed in the subtribe Podocominae on the basis of morphological characters, show no evidence of close relationship in our phylogenetic analyses based on molecular data; 3) Our results do not confirm Podocoma in its current concept as monop...

A New Species of <I>Microgramma</I> (Polypodiaceae) from Brazil and Recircumscription of the Genus Based on Phylogenetic Evidence

We describe a new species of Microgramma (M. microsoroides) from the Atlantic Forest of Brazil, present a phylogeny of Microgramma, and provide a new generic circumscription. Microgramma comprises ca. 30 species in the Neotropics and perhaps two species in Africa and on islands in the Indian Ocean. Several species are contentious with regard to generic placement, and have been treated within Pleopeltis, Polypodium, and Solanopteris. From all known species of Microgramma, the new species differs by the sori forming irregular rows between adjacent primary veins. This finding is quite surprising, inasmuch as all other species in the genus have sori in two very strict rows, one row on each side of the midrib. The monophyly of Microgramma, as newly redefined, is strongly supported in chloroplast DNA-based phylogenetic analyses, using the genes rbcL and rps4, the rps4-trnS intergenic spacer, and the trnL-trnF intergenic spacer on 13 samples from 12 species. The new species is nested within Microgramma as sister to Microgramma megalophylla. Recognition of the relationships of M. microsoroides calls into question certain soral characters traditionally used to define genera of Polypodiaceae, as well as genera of ferns in other families. Sorus placement is notoriously variable in some genera and in many different families of Polypodiales.

Phylogenetic Relationships of Zeugites (Poaceae: Centothecoideae) Inferred from Plastid and Nuclear DNA Sequences and Morphology

The genus Zeugites P. Browne encompasses ten species of neotropical grasses. To infer the phylogenetic relationships of the species, parsimony analyses of morphological data and DNA sequences from a nuclear region (ITS) and two non-coding plastid regions (the trnL intron and the trnL-trnF intergenic spacer) were undertaken for each data set separately and in combination. The combined analysis is presented as our preferred phylogenetic hypothesis because of its better resolution and higher support for the clades. Zeugites is monophyletic only if Calderonella sylvatica is included in it. Our analyses recover two major clades within Zeugites: Clade I, formed by Zeugites pittieri, Zeugites latifolia, Zeugites sylvatica (Calderonella sylvatica), Zeugites sagittata, and Zeugites capillaris; and Clade II, including Zeugites hackelii, Zeugites smilacifolia, Zeugites munroana, Zeugites pringlei, Zeugites mexicana, and Zeugites americana. The monospecific genus Pohlidium is strongly supported as sister of Zeugites (including Calderonella) but it is maintained as a different genus due to noticeable differences in spikelet and inflorescence morphology.

Phylogenetic Relationships of Zeugites (Poaceae: Centothecoideae) Inferred from Plastid and Nuclear DNA Sequences and Morphology

The genus Zeugites P. Browne encompasses ten species of neotropical grasses. To infer the phylogenetic relationships of the species, parsimony analyses of morphological data and DNA sequences from a nuclear region (ITS) and two non-coding plastid regions (the trnL intron and the trnL-trnF intergenic spacer) were undertaken for each data set separately and in combination. The combined analysis is presented as our preferred phylogenetic hypothesis because of its better resolution and higher support for the clades. Zeugites is monophyletic only if Calderonella sylvatica is included in it. Our analyses recover two major clades within Zeugites: Clade I, formed by Zeugites pittieri, Zeugites latifolia, Zeugites sylvatica (Calderonella sylvatica), Zeugites sagittata, and Zeugites capillaris; and Clade II, including Zeugites hackelii, Zeugites smilacifolia, Zeugites munroana, Zeugites pringlei, Zeugites mexicana, and Zeugites americana. The monospecific genus Pohlidium is strongly supported as sister of Zeugites (including Calderonella) but it is maintained as a different genus due to noticeable differences in spikelet and inflorescence morphology.

Monophyly and Phylogenetic Relationships in <I>Lymania</I> (Bromeliaceae: Bromelioideae) Based on Morphology and Chloroplast DNA Sequences

A cladistic analysis of Lymania was conducted using morphology and sequences from three chloroplast DNA regions: the matK coding region and the psbA-trnH and trnL-trnF intergenic spacers. The monophyly of the genus and the phylogenetic relationships among related genera were examined. Of the nine Lymania species, eight are endemic to southern Bahia, Brazil. Lymania is the first genus in Bromeliaceae subfamily Bromelioideae to be subjected to a combined morphological and molecular analysis. The genera of Bromelioideae have been particularly difficult to classify and there has been disagreement about their interrelationships and monophyly. Morphological data show better resolution than molecular data alone. The partition homogeneity test supported a combined analysis of the two data sets, yielding a single most parsimonious tree. In the combined analysis, monophyly of Lymania is moderately supported, and the genus is closely related to species of Aechmea subg. Lamprococcus and subg. subg. Ortigiesia. The morphological distinctiveness coupled with low molecular divergence indicates relatively recent and rapid speciation within Lymania. The combined analysis of morphological and molecular data as done in this study provides a framework for future research on other Bromelioideae genera that could foster better taxonomic rearrangements.

Phylogenetic Relationships in Tribe Cariceae (Cyperaceae) Based on Nested Analyses of Four Molecular Data Sets

Phylogenetic reconstruction for Carex and relatives in tribe Cariceae is complicated by species richness and nearly cosmopolitan distribution. In this investigation, our main objective was to estimate evolutionary relationships in tribe Cariceae using DNA sequence data from two spacer regions in nuclear ribosomal genes (ITS and ETS-1f) combined with noncoding chloroplast DNA (trnL intron, trnL–trnF intergenic spacer, and trnE–trnD intergenic spacers). Parsimony analyses of separate and combined data and Bayesian analysis of the combined data matrix revealed strong support for monophyly of tribe Cariceae and for monophyly of two major lineages, one comprising principally Carex subgen. Carex and Vigneastra, and the other representing subgen. Vignea. A third clade with representatives from Kobresia and Uncinia, along with Cymophyllus fraserianus, Carex curvula, and several unispicate Carex received weak-to-moderate support. A small clade comprising Schoenoxiphium and two unispicate carices was placed as sister to the clades comprising multispicate Carex species in the parsimony analysis, but sister to the clade of Kobresia, Uncinia, and unispicate Carex in the Bayesian analysis. Two large widespread groups within subgen. Carex, sect. Hymenochlaenae and sect. Physocarpae s.l. (‘‘bladder sedges"), were highly polyphyletic, while ten clades that grouped species from two or more sections were each strongly supported as monophyletic. Within subgen. Vignea, three sections were strongly supported as monophyletic while sects. Phaestoglochin and Vulpinae were polyphyletic. Adding the variable ETS-1f region improved resolution and bootstrap support values over previous studies, but many of the characters supporting major branches came from the trnL region.

Variasi Genetik pada Marga Dicksonia Koleksi Kebun Raya Bali – Deteksi dengan PCR - SSCP

Dicksonia is one of tree ferns species that can be found in Indonesian highlands. Due to their wide distribution this species was suggested to have genetic variation among their population. Samples from Bali, West Java (Cibodas) and East Nusa Tenggara were compared using the PCR-SSCP methods to detect genetic variation of trnL-trnF intergenic spacer from the cpDNA plastid genome. Sample from Bali and West Java (Cibodas) did not morphologically and genetically variated, but they were variated to samples from East Nusa Tenggara.

Heterogeneity of Three Molecular Data Partition Phylogenies of Mints Related to M. ×piperita (Mentha; Lamiaceae)

Phylogenetic reconstructions with molecular tools are now widely used, thanks to advances in PCR and sequencing technologies. The choice of the molecular target still remains a problem because too few comparative data are available. This is particularly true for hybrid taxa, where differential introgression of genome parts leads to incongruity between data sets. We have studied the potential of three data partitions to reconstruct the phylogeny of mints related to M. x piperita. These included nuclear DNA (ITS), chloroplast DNA (non-coding regions trnL intron, intergenic spacers trnL-trnF, and psbA-trnH), and AFLP and ISSR, markers. The taxonomic sampling was composed of hybrids, diploid and polyploid genomes. Since the genealogy of cultivated mint hybrids is known, they represent a model group to compare the usefulness of various molecular markers for phylogeny inference. Incongruities between ITS, chloroplast DNA, and AFLP-ISSR phylogenetic trees were recorded, although DNA fingerprinting data were congruent with morphological classification. Evidence of chloroplast capture events was obtained for M. x piperita. Direct sequencing of ITS led to biased results because of the existence of pseudogenes. Sequencing of cloned ITS further failed to provide evidence of the existence of the two parental copy types for M. x piperita, a sterile hybrid that has had no opportunity for concerted evolution of ITS copies. AFLP-ISSR data clustered M. x piperita with the parent that had the largest genome. This study sheds light on differential of introgression of different genome regions in mint hybrids.

Phylogenetics of Papaver and Related Genera Based on DNA Sequences from ITS Nuclear Ribosomal DNA and Plastid trnL Intron and trnL–F Intergenic Spacers

Representatives from Papaver, Roemeria, Stylomecon and Meconopsis were studied to elucidate phylogenetic relationships between Papaver and these closely allied genera. Two molecular data sets were used individually and combined and included sequences from the internally transcribed spacer region (ITS) of 18S-26S nuclear ribosomal DNA and the trnL intron and the trnL-trnF intergenic spacer region of plastid DNA. Parsimony analysis demonstrated that the genus is not monophyletic unless the closely related Roemeria, Stylomecon and Meconopsis cambrica are included in a revised circumscription of Papaver. Three distinct clades are resolved in a combined ITS and trnL-F analysis. Clade 1 consists of Papaver sect. Meconella and Asian Meconopsis. Clade 2 contains a group here identified as Papaver s.s., comprising sections Carinatae, Meconidium, Oxytona, Papaver, Pilosa, Pseudopilosa and Rhoeadium. Clade 3 consists of Papaver sect. Argemonidium and Roemeria refracta. A number of diagnostic indels support these groupings. Within clade 2, sects. Papaver and Rhoeadium are either not monophyletic or lack evidence supporting their monophyly. The results of this molecular analysis indicate that a number of morphological characters such as valvate capsule dehiscence, dark or light filaments and sessile stigmatic discs have arisen in parallel. The phylogenetic trees are incongruent with the existing taxonomy of Papaver, and a revised classification is suggested.

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences.

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.

Species identification using sequences of the trnL intron and the trnL-trnF IGS of chloroplast genome among popular plants in Taiwan

Forensic botanical comparison can be hampered by the lack of appropriate DNA databases. While DNA sequence databases for many mitochondrial loci have been established for the identification of animal species, less is known regarding the genomes of plants. We report on the use of the trnL intron and the trnL-trnF intergenic spacer (IGS) in the chloroplast genome and establish a DNA sequence database for plant species identification. The DNA sequences at these two loci from commonly encountered plants, including monocots and dicots, were aligned to establish a DNA database of local plants. The database comprises 373 individual sequences representing 80 families, 206 genera and 269 species. These plant species can be grouped to species level using both sequence and length polymorphisms at these loci. To validate the database for future forensic purposes, we sequenced 20 blind samples and searched the local database and the databases of GenBank and EMBL. Fifteen of these 20 samples used in blind trial testing matched their respective species from our local DNA database but only 6 matched species registered in the GenBank and EMBL databases. The sequences of two species used in the blind trial did not match any sequence registered in any of these databases. Cluster analysis was performed to demonstrate the family and genus distribution of samples. Neighbor-joining trees of the two DNA regions from 70 samples of the local database and 10 of the species used in the blind trials were constructed and clustered to both family and genus. The bootstrap values of the trnL intron were higher than most of those of the trnL-trnF IGS. The sequence database described in this study can be used to identify plant species using DNA sequences of the trnL intron and trnL-trnF IGS of chloroplast genome and illustrates its value in plant species identification.

Phylogenetics of Quiinaceae (Malpighiales): evidence from trnL-trnF sequence data and morphology

We present the first parsimony analyses of the Neotropical family Quiinaceae using nucleotide sequence data from the non-coding trnL intron and trnL-trnF intergenic spacer of the plastid genome, analysed separately as well as in combination with morphology. Both molecules and combined data recover Quiinaceae as a well-supported monophyletic group. Quiinaceae form a polytomy together with their potential sister groups, the monophyletic Ochnaceae s.str. and the monotypic Medusagynaceae from the Seychelles in the Indian Ocean. Froesia is resolved as sister to the rest of the family. Other members of the family, Lacunaria, Quiina, and Touroulia, are all recovered as monophyletic despite the inclusion of strikingly distinctive representatives (L. oppositifolia and Q. pteridophylla). Relationships among the last three genera, however, are yet uncertain. Optimising characters of breeding system onto the molecular phylogeny reveals that bisexual flowers (Froesia) are the ancestral state in Quiinaceae, whereas androdioecy (Quiina, Touroulia) and dioecy (Lacunaria) are derived breeding systems.

Molecular evidence for the natural hybrid origin of Phalaenopsis × intermedia Lindl.

SummaryIn order to confirm the hybrid origin of Phalaenopsis intermedia Lindl., internal transcribed spacers (ITSs) of ribosomal DNA (rDNA) and three fragments of chloroplast DNA (cpDNA) were studied. Nineteen clones of ITS sequences from three accessions of P. intermedia Lindl. were aligned with nine species of sections of Phalaenopsis and Stauroglottis that were candidate parents. A phylogenetic tree, derived from the ITS data, was constructed by the Neighbor-joining (NJ) method. Two major groups were shown for 19 clones of ITS sequences of P. intermedia based on the phylogenetic tree. The average genetic distance between the aforementioned two groups and the candidate parents was calculated based on the Kimura 2-parameter method. One group had the lowest genetic distance from the ITS repeat sequences of P. aphrodite Rchb.f., and another group had the lowest distance from that of P. equestris (Schauer) Rchb.f. The results indicated that both P. aphrodite and P. equestris are parents of P. intermedia based on the ITS data. In addition, analysis of three fragments of cpDNA, namely the trnL intron, the trnL-trnF intergenic spacer (IGS), and the atpB-rbcL IGS, showed that P. intermedia had the lowest genetic distance from P. aphrodite. Both the ITS and cpDNA data, as well as reference to the effects of maternal inheritance of cpDNA, suggest that P. aphrodite was the maternal parent and P. equestris was the paternal parent of P. intermedia. Therefore, molecular evidence supports P. intermedia being a natural hybrid derived from P. aphrodite and P. equestris.

Phlogeny and Biogeography of the Prayer Plant Family

Marantaceae are the second largest family in the order Zingiberales, with approximately 31 genera and 535 species. Earlier studies based on morphological and molecular characters could not confidently determine the relationships among major lineages of the family, nor could they identify the basal branch of the family tree. Phylogenetic analyses of DNA sequence data from all three genomic compartments (chloroplast: matK, ndhF, rbcL, rps16 intron, and trnL-trnF intergenic spacer; mitochondrion: cox1; nucleus: ITS region and the 5'-end of 26S) for a restricted set of taxa were conducted under parsimony criteria to define the root node and to assess geographical distribution patterns. Our results support the recognition of five major lineages, most of which are restricted to a single geographical region (tropical America, tropical Africa, or tropical Asia). The phylogenies and character reconstructions (Fitch parsimony optimization, Bremer ancestral areas, and DIVA) support an African origin for the family, followed by a minimum of two dispersal events to the New World tropics and four or more dispersal events to the Asian tropics. Less likely are two alternative hypotheses: (1) vicariance of a western Gondwanan group (the Americas and Africa) followed by several dispersals to Asia and Africa, or (2) an American origin followed by several dispersals to Africa and Asia. The low specific diversity in Africa may be due to higher extinction rates as a result of shrinking lowland tropical forests during the Tertiary.

Evolution of the Euphrasia transmorrisonensis complex (Orobanchaceae) in alpine areas of Taiwan

AbstractAims To unravel isolation and differentiation of the genetic structure of the Euphrasia transmorrisonensis complex, a showy herb, among alpine regions of mountain peaks in subtropical Taiwan and to infer its evolutionary history.Location Alpine ecosystems of high‐montane regions of Taiwan.Methods Phylogenetic analyses of the trnL intron and the trnL–trnF intergenic spacer of chloroplast (cp) DNA, and the intertranscribed spacer (ITS) of nuclear ribosomal (nr) DNA between 18S and 26S were carried out on 18 populations of the E. transmorrisonensis complex in Taiwan.Results In total, 10 haplotypes for cpDNA and 14 haplotypes for nrDNA were detected. Three population groups located in the northern, north‐eastern, and south‐central regions of the Central Mountain Range (CMR) were revealed according to the frequencies of haplotypes and haplotype lineages of nrDNA. Balancing selection might have played a role in the evolution of Euphrasia in Taiwan.Main conclusions By integrating the spatial‐genetic patterns of cpDNA and nrDNA, two possible evolutionary histories of Euphrasia in Taiwan were inferred. The favourable hypotheses for interpreting the data suggest at least three origins of the E. transmorrisonensis complex in Taiwan, corresponding to each nuclear lineage in the northern (II), northern/north‐eastern (I), and central/southern regions (III) with subsequent hybridization between lineages I and II and lineages II and III. These lineage boundaries are strengthened by the finding that haplotypes of C derived from cpDNA were found in the geographical region of lineage II of nrDNA, while haplotypes of A derived from cpDNA were found in the region of lineage III of nrDNA. Thus, the origin of chloroplasts exclusive to lineages II and III supports their long‐term isolation from one another.

The Chloroplast trnT–trnF Region in the Seed Plant Lineage Gnetales

The trnT-trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT-trnL and trnL-trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25-35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL-trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL-trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA(Thr)(UGU), tRNA(Leu)(UAA), and tRNA(Phe) (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints.