An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.     

Telomere variability in the monocotyledonous plant order Asparagales

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the 'typical' Arabidopsis-type telomere (TTTAGGG)(n). This stimulated us to determine what has replaced these sequences. Using slot-blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis-type telomeric sequence has been partly or fully replaced by the human-type telomeric sequence (TTAGGG)(n). Species in Allium lack the human-type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis, Bombyx (TTAGG)(n) and Tetrahymena (TTGGGG)(n). FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human-type motif. These motifs probably form the 'true telomere' but the error rate of motif synthesis is higher compared with 'typical' plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis-like telomeres to a low-fidelity synthesis of human-like telomeres.     

Asparagales Telomerases which Synthesize the Human Type of Telomeres

The order of monocotyledonous plants Asparagales is attractive for studies of telomere evolution as it includes three phylogenetically distinct groups with telomeres composed of TTTAGGG (Arabidopsis-type), TTAGGG (human-type) and unknown alternative sequences, respectively. To analyze the molecular causes of these switches in telomere sequence (synthesis), genes coding for the catalytic telomerase subunit (TERT) of representative species in the first two groups have been cloned. Multiple alignments of the sequences, together with other TERT sequences in databases, suggested candidate amino acid substitutions grouped in the Asparagales TERT synthesizing the human-type repeat that could have contributed to the changed telomere sequence. Among these, mutations in the C motif are of special interest due to its functional importance in TERT. Furthermore, two different modes of initial elongation of the substrate primer were observed in Asparagales telomerases producing human-like repeats, which could be attributed to interactions between the telomerase RNA subunit (TR) and the substrate. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Characterization of nucleoprotein complexes in plants with human-type telomere motifs.

A conserved feature of telomeres is the 3'-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG)(n) to a type typical of human (TTAGGG)(n). Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres (Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.     

The controversial telomeres of lily plants

The molecular structure of the exceptional telomeres of six plant species belonging to the order Asparagales and two species of the order Liliales was analyzed using Southern blot and fluorescence in situ hybridization. Three different situations were found, namely: i) In the two Liliales species, Tulipa australis (Liliaceae) and Merendera montana (Colchicaceae), the chromosome ends display hybridization signals with oligonucleotides resembling telomere repeats of both plants (TTTAGGG)n and vertebrates (TTAGGG)n. ii) Asparagales species such as Phormium tenax (Hemerocallidaceae), Muscari comosum (Hyacinthaceae), Narcissus jonquilla (Amaryllidaceae) and Allium sativum (Alliaceae) lack both the plant telomere repeats and the vertebrate telomere repeats. iii) Two other Asparagales species, Aloe vera (Asphodelaceae) and an Iris hybrid (Iridaceae), display positive hybridization with the vertebrate telomere repeats but not with the plant telomere repeats. Southern blot hybridization revealed concurring results. On this basis, the composition of the telomere structure in this plant group is discussed.     

Minisatellite telomeres occur in the family Alliaceae but are lost in Allium

Although telomere sequences are considered to be highly conserved, there are switch-points in plant telomere evolution that are congruent with species' phylogenies. When Asparagales diverged, the Arabidopsis-type telomeric minisatellite repeat (TTTAGGG)(n) was first replaced by a human-type (TTAGGG)(n) repeat, and both were lost in Allium cepa (Alliaceae). We aimed to discover (1) when this loss occurred during divergence of Alliaceae and, (2) if (TTAGGG)(n) repeats were replaced by other known telomeric minisatellites. Slot-blot hybridization, fluorescent in situ hybridization, BAL31 digestion, asymmetric PCR, and cloning were used to identify and localize candidate telomeric sequences in species of Nothoscordum, Miersia, Ipheion, Tulbaghia, Gethyum, Gilliesia, Leucocoryne, Tristagma, and representatives of the three major Allium clades. Alliaceae genera other than Allium have human (TTAGGG)-type telomeric repeats that form telomeres. In Allium, only Tetrahymena-type (TTGGGG) repeats were ubiquitous in the genome, but they were not localized to telomeres. Likewise, the consensus telomeric repeats in Arabidopsis, human, Bombyx (TTAGG), Chlamydomonas (TTTTAGGG), and Oxytricha (TTTTGGGG) are absent in Allium telomeres. Alliaceae with human-type telomeres share telomere structures with related Asparagales species. We demonstrate that in the Allium ancestor human-type telomeric repeats were lost from telomeres and were not replaced by any investigated alternative minisatellite repeats. However, human and other types of minisatellite telomeric repeats are interspersed in some Allium genomes and their genomic signatures coincide with Allium clades.     

Two combinatorial patterns of telomere histone marks in plants with canonical and non‐canonical telomere repeats

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non‐canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate‐type telomere repeat TTAGGG or Allium genus‐specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non‐canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR‐dCas9‐eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C‐3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis‐like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco‐like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere‐associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.     

Telomere length in Agave tequilana Weber plants during the in vitro to ex vitro transition

Acclimatization ex vitro is a key stage of the micropropagation process, in which the vitro plants leave the sterile, high humidity environment in which they originated and form new leaves and roots, during which they suffer different types of stress. Changes in the telomere length (shortening and lengthening) have been associated with age, the development of tissue, loss of cell replication and the ability of regeneration in different plant species. However, the genetic and biological factors that are involved in the process of shortening of telomeres across the ageing of plant species are still unknown. In this study, we used terminal restriction fragments (TRF) to examine the changes of telomere length during the in vitro to ex vitro transition in vitro plants of Agave tequilana, and their relationships with age in plants grown in commercial plantations. The results showed that in vitro grown plants present the longest telomeres and that a shortening occurs during the first 6 months of ex vitro acclimatization, (compared to the plantlets that were kept in vitro). A lengthening of the telomeres was observed in the acclimatized 1-year-old plants and that this was maintained in 2 and 3-year-old plants. We also observed TRF variations in the different tissues (leaves, stems and roots) of acclimatized plants. In field plants, we did not observe any important changes in the length of the telomeres. We suggest that agaves have a mechanism that maintains telomere length at the non-critical stages during development.

     

Comparative genomic analyses in Asparagus.

Garden asparagus (Asparagus officinalis L.) belongs to the monocot family Asparagaceae in the order Asparagales. Onion (Allium cepa L.) and Asparagus officinalis are 2 of the most economically important plants of the core Asparagales, a well supported monophyletic group within the Asparagales. Coding regions in onion have lower GC contents than the grasses. We compared the GC content of 3374 unique expressed sequence tags (ESTs) from A. officinalis with Lycoris longituba and onion (both members of the core Asparagales), Acorus americanus (sister to all other monocots), the grasses, and Arabidopsis. Although ESTs in A. officinalis and Acorus had a higher average GC content than Arabidopsis, Lycoris, and onion, all were clearly lower than the grasses. The Asparagaceae have the smallest nuclear genomes among all plants in the core Asparagales, which typically have huge genomes. Within the Asparagaceae, European Asparagus species have approximately twice the nuclear DNA of that of southern African Asparagus species. We cloned and sequenced 20 genomic amplicons from European A. officinalis and the southern African species Asparagus plumosus and observed no clear evidence for a recent genome doubling in A. officinalis relative to A. plumosus. These results indicate that members of the genus Asparagus with smaller genomes may be useful genomic models for plants in the core Asparagales.     

Telomere Length Dynamics in Pearl Millet (Pennisetum glaucum [L.] R. Br.) as Observed at Different Developmental Stages

Abstract: In plants, the developmental dynamics of telomere length have only been studied in a few species to date. Contrasting results have been reported. To search for the pattern(s) operating in plants, a study of the telomere length was made in pearl millet. Telomere length in cells representing different developmental stages: a) embryo, b) leaves of 1-week-old seedlings, 1-month-old plants and boot leaf and c) germ cells (pollen) were compared. The presence of the consensus plant telomere repeat sequence (5'-TTTAGGG-3') in pearl millet telomeres was first ascertained; the sensitivity of the sequences hybridizing with (TTTAGGG)₄ oligonucleotide to time course Bal 31 exonuclease digestions were studied on dot blots and gel blots. The exonuclease digestion kinetics revealed the presence of the consensus telomere repeat sequence in pearl millet telomeres. The average telomere length (ATL) was measured from autoradiograms of Hae III digested DNA, hybridized with labelled (5'-TTTAGGG-3')₄ oligonucleotide using "UVI band" software. No significant difference in the average telomere length was observed between the embryo, leaves of 1-week-old seedlings, boot leaf and pollen. The ATL in leaves of 1-month-old plants was slightly higher. The results of the present investigation and analysis of the reports in the other plants suggest that there is an occasional increase in telomere length in some telomeres but no significant decrease due to loss during DNA replication.     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.     

Structure and polymorphism of human telomere-associated DNA

We have analyzed the DNA sequences associated with four different human telomeres. Two are members of distinct repeated sequence families which are located mainly but not exclusively at telomeres. Two are unique in the genome, one deriving from the long arm telomere of chromosome 7 and the other from the pseudoautosomal telomere. One telomere-associated repeated sequence has a polymorphic distribution among the chromosome ends, being present at a different combination of ends in different individuals. These data thus identify a new source of human genetic variation and indicate that the canonical features of the organization of telomere-associated DNA are widely conserved in evolution.     

单子叶植物高级分类阶元系统演化: matK、rbcL和18S rDNA序列的证据

基于两个叶绿体基因（matK和rbcL）和一个核糖体基因（18S rDNA）的序列分析,对代表了基部被子植物和单子叶植物主要谱系分支的86科126属151种被子植物（单子叶植物58科86属101种）进行了系统演化关系分析。研究结果表明由胡椒目Piperales、樟目Laurales、木兰目Magnoliales和林仙目Canellales构成的真木兰类复合群是单子叶植物的姐妹群。单子叶植物的单系性在3个序列联合分析中得到98％的强烈自展支持。联合分析鉴定出9个单子叶植物主要谱系（广义泽泻目Alismatales、薯蓣目Dioscorcales、露兜树目Pandanales、天门冬目Asparagalcs、百合目Liliales、棕榈目Arecales、禾本目Poales、姜目Zingiberales、鸭跖草目Commelinales）和6个其他被子植物主要谱系（睡莲目Nymphaeales、真双子叶植物、木兰目、樟目、胡椒目、林仙目）。在单子叶植物内,菖蒲目Acorales（菖蒲属Acorus）是单子叶植物最早分化的一个谱系,广义泽泻目（包括天南星科Araceae和岩菖蒲科Toficldiaccae）紧随其后分化出来,二者依次和其余单子叶植物类群构成姐妹群关系。无叶莲科Petrosaviaceac紧随广义的泽泻目之后分化出来,无叶莲科和剩余的单子叶植物类群形成姐妹群关系,并得到了较高的支持率。继无叶莲科之后分化的类群形成两个大的分支：一支是由露兜树目和薯蓣目构成,二者形成姐妹群关系：另一支是由天门冬目、百合目和鸭跖草类复合群组成,三者之间的关系在单个序列分析和联合分析中不稳定,需要进一步扩大取样范围来确定。在鸭跖草类复合群分支内,鸭跖草目和姜目的姐妹群关系在3个序列联合分析和2个序列联合分析的严格一致树中均得到强烈的自展支持,获得的支持率均是100％。但是,对于棕榈目和禾本目在鸭跖草类中的系统位置以及它们和鸭跖草目-姜目之间的关系,有待进一步解决。值得注意的是,无叶莲科与其他单子叶植物类群（除菖蒲目和泽泻目外）的系统关系在本文中获得较高的自展支持率,薯蓣目和天门冬目的单系性在序列联合分析中都得到了较好的自展支持,而这些在以往的研究中通常支持率较低。鉴于菖蒲科和无叶莲科独特的系统演化位置,本文支持将其分别独立成菖蒲目和无叶莲目Petrosavialcs的分类学界定。     

Aloe L. – a second plant family without (TTTAGGG)n telomeres

The physical ends of chromosomes are protected and stabilised by telomeres. The sequence of telomeric DNA normally consists of a simple repeating unit that is conserved in many organisms. Most plants examined have been shown to possess Arabidopsis-type telomeres consisting of many repeat copies of the sequence 5′-TTTAGGG-3′. Using fluorescent in situ hybridisation, slot blotting and the asymmetric polymerase chain reaction we demonstrate an absence of Arabidopsis-type telomeres in the genus Aloe (family Asphodelaceae). The only other plant genera so far reported without such telomeres are Allium, Nothoscordum, and Tulbaghia (family Alliaceae). As these genera and Aloe are petaloid monocots in the Asparagales, it is suggested that an absence of Arabidopsis-type telomeres may be characteristic of this related group of plants. Received: 6 September 1999; in revised form: 9 December 1999 / Accepted: 13 December 1999     

Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

Drosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85  Drosophila melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we instead suggest that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.     

Multiple developmental pathways leading to a single morph: monosulcate pollen (examples from the Asparagales)

BACKGROUND AND AIMS: Early developmental events in microsporogenesis are known to play a role in pollen morphology: variation in cytokinesis type, cell wall formation, tetrad shape and aperture polarity are responsible for pollen aperture patterning. Despite the existence of other morphologies, monosulcate pollen is one of the most common aperture types in monocots, and is also considered as the ancestral condition in this group. It is known to occur from either a successive or a simultaneous cytokinesis. In the present study, the developmental sequence of microsporogenesis is investigated in several species of Asparagales that produce such monosulcate pollen, representing most families of this important monocot clade. METHODS: The developmental pathway of microsporogenesis was investigated using light transmission and epifluorescence microscopy for all species studied. Confocal microscopy was used to confirm centripetal cell plate formation. KEY RESULTS: Microsporogenesis is diverse in Asparagales, and most variation is generally found between families. It is confirmed that the whole higher Asparagales clade has a very conserved microsporogenesis, with a successive cytokinesis and centrifugal cell plate formation. Centripetal cell wall formation is described in Tecophilaeaceae and Iridaceae, a feature that had so far only been reported for eudicots. CONCLUSIONS: Monosulcate pollen can be obtained from several developmental pathways, leading thus to homoplasy in the monosulcate character state. Monosulcate pollen should not therefore be considered as the ancestral state unless it is produced through the ancestral developmental pathway. The question about the ancestral developmental pathway leading to monosulcy remains open.     

Telomeric repeats of Tetrahymena malaccensis mitochondrial DNA: a multimodal distribution that fluctuates erratically during growth.

The linear mitochondrial DNA (mtDNA) of Tetrahymena malaccensis has tandem 52-base-pair repeats at its telomeres. The mtDNA has a multimodal distribution of telomeres. Different groups in the distribution have different numbers of telomeric repeats. The standard deviation of the size of each end group is independent of the mean size of the end group. The two sides of the mtDNA have different multimodal distributions of repeats. Cloned cell lines have multimodal distributions of mtDNA telomeres distinct from that of the original cell line. The number of telomere end groups and the average size of the end groups change in an erratic fashion as the cells are passaged and do not reach a stable equilibrium distribution in 185 generations. We propose that the mean size of a telomere end group and the size distribution of an end group are independently regulated. The system controlling the average size of end groups may be defective in T. malaccensis, since a closely related species (T. thermophila) does not have a multimodal distribution of mtDNA telomeres. T. hyperangularis, which has different telomeric repeats on each side of its mtDNA, has a multimodal distribution of mtDNA telomeres on only one side, suggesting that the mechanism controlling the average number of repeats in an end group can be sequence specific. These mitochondrial telomeres provide a new example of the more general phenomenon of expansion and contraction of arrays of repeated sequences seen, for example, with simple-sequence "satellite" DNAs; however, the mitochondrial telomeres change on a very short time scale.     

Phylogenetic Relationships of Amaryllidaceae Based on matK Sequence Data

matK gene, which is located in the chloroplast genome and evolves more quickly than the rbcL gene. A total of 31 species representing 31 of the 59 genera in the family were examined in this study. We also used 21 species from another ten families of Asparagales, four species from three families of Liliales and Acorus as outgroups. We obtained partial sequences of matK with lengths of 1,109–1,148 bp, corresponding to positions 230 to 1,343 of the Oryza sativa matK gene. The pairwise percentage sequence divergence ranged from 0 to 19.1% for all the species examined except Acorus, and 0 to 4.6% within Amaryllidaceae. Two methods of phylogenetic analysis, the Maximum Parsimony and Neighbor-Joining methods, were used. The trees obtained from these two analyses were fundamentally consistent. In both trees, the Amaryllidaceae sensu Dahlgren et al. formed a well-supported monophyletic clade with 100% bootstrap support. Amaryllidaceae were included in the Asparagales; however, its phylogenetic position within the Asparagales was not clearly resolved. Judging from the NJ tree, Agapanthus might be a sister group of the Amaryllidaceae, although bootstrap support for this was low. Character-state mapping was used to infer a center of origin and the biogeographic history of Amaryllidaceae. The result supports the hypothesis that the family evolved in Africa and subsequently spread to other continents, further suggesting that South America is the center of secondary diversification. Received 6 January 1999/ Accepted in revised form 8 April 1999