Macroscale analysis of mistletoe host ranges in the Andean-Patagonian forest.

The number of host species infected by a mistletoe (host range) is critical in that it influences prevalence, virulence and overall distribution of the parasite; however, macroecological analyses of this life history feature are lacking for many regions. The Andean-Patagonian forest, found along the southern Andes from 35 °S to Tierra del Fuego at 55 °S, contains 12 mistletoe species in three families (Loranthaceae, Misodendraceae and Santalaceae). By tabulating herbarium records, the host ranges and geographical distributions of these mistletoes were explored. Our results show that these parasites occur on 43 plant species in 24 families but with varying degrees of specificity. All Misodendrum species and Desmaria mutabilis (Loranthaceae) are specialists that use Nothofagus as their primary hosts. Tristerix and Notanthera (Loranthaceae) and Antidaphne and Lepidoceras (Santalaceae) are generalists parasitizing more than six host species from several genera and families. Although many of the mistletoe species are sympatric, there is low overlap in host use. Our data show that in the southern South American bioregion, generalist mistletoes have smaller geographic ranges than specialists. This contrast with a previous hypothesis that predicted mistletoes with large geographic ranges would also have large host ranges, and conversely, less diverse regions would have more specialised mistletoes.

First Report of the Mistletoe Tristerix verticillatus on Schinus fasciculatus from the Sierra de San Luis, Argentina.

There are 11 species of mistletoes in the genus Tristerix (Loranthaceae) endemic to the Andes in South America (1,2). Tristerix verticillatus (Ruiz & Pav.) Barlow & Wiens is distributed on the east side of the Andes from Bolivia to Argentina at high elevations (>1,500 m). On the west side of the Andes, it is only found in Chile where it occurs at low to high elevations (50 to 3,000 m). Along its range, the mistletoe parasitizes more than 25 species, mainly Anarcadiaceae and Rhamnaceae. In February of 2007, it was observed to be parasitizing Schinus fasciculatus (Griseb.) I.M. Johnst. (Anacardiaceae) at two locations in the Sierra de San Luis, Argentina. One location was 6 km south of Las Chacras on Route 31 (32°35'56″S, 65°47'6″W, elevation 1,185 m) and the other was 12 km north of El Trapiche on Route 9 (33°1'21″S, 66°4'11″W, elevation 1,260 m). At these localities, the mistletoe was in full flower and parasitized only one host species, S. fasciculatus. No mortality associated with infection by this mistletoe was observed at either location. Specimens of the mistletoe were collected from both locations and deposited at the Centro Regional Universitario Bariloche Herbarium (BCRU), Río Negro, Argentina. To our knowledge, this is the first report of a Tristerix sp. outside the Andes and extends the eastern distribution of the genus by ≈270 km and also of S. fasciculatus serving as a host for T. verticillatus since previous collections were from other species of Schinus. References: (1) G. C. Amico et al. Am. J. Bot. 94:558, 2007. (2) J. Kuijt, Syst. Bot. Monogr. 19, 1988.

First Report of Arceuthobium hondurense in Oaxaca, Mexico.

Honduran dwarf mistletoe (Arceuthobium hondurense Hawksw. & Wiens) has only been found in four locations in Honduras: west of Zamorano, east of Lepaterique, Cusuco National Park, and Celaque National Park (1,2). At one time it was believed that this mistletoe could be in danger of extinction (1). However, it has also been reported in two locations in Chiapas, Mexico (3). In December 2000, ≈1 km north of Suchixtepec, Oaxaca, Mexico, near Route 175 (elevation 2,770 m), we collected a dwarf mistletoe parasitizing Pinus tecunumanii (Schw.) Eguiluz et Perry that was morphologically similar to A. hondurense (1). This population initially had been classified as A. nigrum Hawksw. & Wiens (1), but is now classified as A. hondurense based on morphology (male flower color and stigma length) (1) and analysis of nuclear rDNA internal transcribed spacer (ITS) sequences from Honduras (GenBank Accession No. AF325969) and Oaxaca (GenBank Accession No. AY055215). A comparison of these two A. hondurense ITS sequences indicated they are very closely related to each other, whereas both are distinct from A. nigrum (GenBank Accession No. L25693). Specimens of A. hondurense from Oaxaca were deposited at the Deaver Herbarium, Northern Arizona University, Flagstaff. To our knowledge, this is the first report of A. hondurense in Oaxaca, Mexico, and extends its known distribution west across the Isthmus of Tehuantepec from central Chiapas by ≈400 km. References: (1) F. G. Hawksworth and D. Wiens. Dwarf Mistletoes: Biology, Pathology, and Systematics. USDA Agric. Handb. 709, 1996. (2) R. Mathiasen et al. Phytologia 85:268, 1998. (3) R. Mathiasen et al. Plant Dis. 85:444, 2001.

First Report of Arceuthobium hondurense in Mexico.

Honduran dwarf mistletoe (Arceuthobium hondurense Hawksw. & Wiens) is a rare dwarf mistletoe previously known only from Honduras (1,2). In March 2000 we collected a dwarf mistletoe from approximately 7 km west of San Cristobal de las Casas, Chiapas, Mexico near Route 190 (elevation 2,440 m), which was morphologically similar to A. hondurense (1). This population had initially been classified as A. nigrumHawksw. & Wiens (1). However, our morphological measurements and analysis of nuclear rDNA ITS sequences of A. hondurense plants from Honduras (GenBank No. AF325969) and the plants from Chiapas (AF325970) have confirmed that the Chiapan population is A. hondurense and not A. nigrum. An additional population of A. hondurense was discovered in Chiapas approximately 11 km west of Oxchuc near Route 186 (elevation 2160 m). Both of the Chiapan populations of A. hondurense were parasitizing Pinus tecunumanii(Schw.) Eguiluz et Perry. Specimens of A. hondurense from Chiapas were deposited at the Deaver Herbarium, Northern Arizona University, Flagstaff. This is the first report of A. hondurense in Mexico and extends its known distribution from northwestern Honduras (3) by approximately 500 km. Although A. hondurense has not been observed in the pine forests of Guatemala, it is probable that it also occurs there (1). References: (1) F. G. Hawksworth and D. Wiens. 1996. Dwarf Mistletoes: Biology, Pathology, and Systematics. USDA Agric. Handb. 709. (2) R. Mathiasen et al. Phytologia 36:211, 1998. (3) R. Mathiasen et al. Plant Dis. 84:372, 2000.

Multigene phylogeny of land plants with special reference to bryophytes and the earliest land plants.

A widely held view of land plant relationships places liverworts as the first branch of the land plant tree, whereas some molecular analyses and a cladistic study of morphological characters indicate that hornworts are the earliest land plants. To help resolve this conflict, we used parsimony and likelihood methods to analyze a 6, 095-character data set composed of four genes (chloroplast rbcL and small-subunit rDNA from all three plant genomes) from all major land plant lineages. In all analyses, significant support was obtained for the monophyly of vascular plants, lycophytes, ferns (including PSILOTUM: and EQUISETUM:), seed plants, and angiosperms. Relationships among the three bryophyte lineages were unresolved in parsimony analyses in which all positions were included and weighted equally. However, in parsimony and likelihood analyses in which rbcL third-codon-position transitions were either excluded or downweighted (due to apparent saturation), hornworts were placed as sister to all other land plants, with mosses and liverworts jointly forming the second deepest lineage. Decay analyses and Kishino-Hasegawa tests of the third-position-excluded data set showed significant support for the hornwort-basal topology over several alternative topologies, including the commonly cited liverwort-basal topology. Among the four genes used, mitochondrial small-subunit rDNA showed the lowest homoplasy and alone recovered essentially the same topology as the multigene tree. This molecular phylogeny presents new opportunities to assess paleontological evidence and morphological innovations that occurred during the early evolution of terrestrial plants.

Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny.

As the oldest extant lineages of land plants, bryophytes provide a living laboratory in which to evaluate morphological adaptations associated with early land existence. In this paper we examine reproductive and structural innovations in the gametophyte and sporophyte generations of hornworts, liverworts, mosses and basal pteridophytes. Reproductive features relating to spermatogenesis and the architecture of motile male gametes are overviewed and evaluated from an evolutionary perspective. Phylogenetic analyses of a data set derived from spermatogenesis and one derived from comprehensive morphogenetic data are compared with a molecular analysis of nuclear and mitochondrial small subunit rDNA sequences. Although relatively small because of a reliance on water for sexual reproduction, gametophytes of bryophytes are the most elaborate of those produced by any land plant. Phenotypic variability in gametophytic habit ranges from leafy to thalloid forms with the greatest diversity exhibited by hepatics. Appendages, including leaves, slime papillae and hairs, predominate in liverworts and mosses, while hornwort gametophytes are strictly thalloid with no organized external structures. Internalization of reproductive and vegetative structures within mucilage-filled spaces is an adaptive strategy exhibited by hornworts. The formative stages of gametangial development are similar in the three bryophyte groups, with the exception that in mosses apical growth is intercalated into early organogenesis, a feature echoed in moss sporophyte ontogeny. A monosporangiate, unbranched sporophyte typifies bryophytes, but developmental and structural innovations suggest the three bryophyte groups diverged prior to elaboration of this generation. Sporophyte morphogenesis in hornworts involves non-synchronized sporogenesis and the continued elongation of the single sporangium, features unique among archegoniates. In hepatics, elongation of the sporophyte seta and archegoniophore is rapid and requires instantaneous wall expandability and hydrostatic support. Unicellular, spiralled elaters and capsule dehiscence through the formation of four regular valves are autapomorphies of liverworts. Sporophytic sophistications in the moss clade include conducting tissue, stomata, an assimilative layer and an elaborate peristome for extended spore dispersal. Characters such as stomata and conducting cells that are shared among sporophvtes of mosses, hornworts and pteridophytes are interpreted as parallelisms and not homologies. Our phylogenetic analysis of three different data sets is the most comprehensive to date and points to a single phylogenetic solution for the evolution of basal embryophytes. Hornworts are supported as the earliest divergent embryophyte clade with a moss/liverwort clade sister to tracheophytes. Among pteridophytes, lycophytes are monophyletic and an assemblage containing ferns, Equisetum and psilophytes is sister to seed plants. Congruence between morphological and molecular hypotheses indicates that these data sets are tracking the same phylogenetic signal and reinforces our phylogenetic conclusions. It appears that total evidence approaches are valuable in resolving ancient radiations such as those characterizing the evolution of early embryophytes. More information on land plant phylogeny can be found at: http: //www.science.siu.edu/ landplants/index.html.

The phylogeny of land plants inferred from 18S rDNA sequences: pushing the limits of rDNA signal?

Previous studies of the phylogeny of land plants based on analysis of 18S ribosomal DNA (rDNA) sequences have generally found weak support for the relationships recovered and at least some obviously spurious relationships, resulting in equivocal inferences of land plant phylogeny. We hypothesized that greater sampling of both characters and taxa would improve inferences of land plant phylogeny based on 18S rDNA sequences. We therefore conducted a phylogenetic analysis of complete (or nearly complete) 18S rDNA sequences for 93 species of land plants and 7 green algal relatives. Parsimony analyses with equal weighting of characters and characters state changes and parsimony analyses weighting (1) stem bases half as much as loop bases and (2) transitions half as much as transversions did not produce substantially different topologies. Although the general structure of the shortest trees is consistent with most hypotheses of land plant phylogeny, several relationships, particularly among major groups of land plants, appear spurious. Increased character and taxon sampling did not substantially improve the performance of 18S rDNA in phylogenetic analyses of land plants, nor did analyses designed to accommodate variation in evolutionary rates among sites. The rate and pattern of 18S rDNA evolution across land plants may limit the usefulness of this gene for phylogeny reconstruction at deep levels of plant phylogeny. We conclude that the mosaic structure of 18S rDNA, consisting of highly conserved and highly variable regions, may contain historical signal at two levels. Rapidly evolving regions are informative for relatively recent divergences (e.g., within angiosperms, seed plants, and ferns), but homoplasy at these sites makes it difficult to resolve relationships among these groups. At deeper levels, changes in the highly conserved regions of small-subunit rDNAs provide signal across all of life. Because constraints imposed by the secondary structure of the rRNA may affect the phylogenetic information content of 18S rDNA, we suggest that 18S rDNA sequences be combined with other data and that methods of analysis be employed to accommodate these differences in evolutionary patterns, particularly across deep divergences in the tree of life.

Phylogenetic relationships of land plants using mitochondrial small-subunit rDNA sequences.

Phylogenetic relationships among embryophytes (tracheophytes, mosses, liverworts, and hornworts) were examined using 21 newly generated mitochondrial small-subunit (19S) rDNA sequences. The "core" 19S rDNA contained more phylogenetically informative sites and lower homoplasy than either nuclear 18S or plastid 16S rDNA. Results of phylogenetic analyses using parsimony (MP) and likelihood (ML) were generally congruent. Using MP, two trees were obtained that resolved either liverworts or hornworts as the basal land plant clade. The optimal ML tree showed hornworts as basal. That topology was not statistically different from the two MP trees, thus both appear to be equally viable evolutionary hypotheses. High bootstrap support was obtained for the majority of higher level embryophyte clades named in a recent morphologically based classification, e.g., Tracheophyta, Euphyllophytina, Lycophytina, and Spermatophytata. Strong support was also obtained for the following monophyletic groups: hornworts, liverworts, mosses, lycopsids, leptosporangiate and eusporangiate ferns, gymnosperms and angiosperms. This molecular analysis supported a sister relationship between Equisetum and leptosporangiate ferns and a monophyletic gymnosperms sister to angiosperms. The topologies of deeper clades were affected by taxon inclusion (particularly hornworts) as demonstrated by jackknife analyses. This study represents the first use of mitochondrial 19S rDNA for phylogenetic purposes and it appears well-suited for examining intermediate to deep evolutionary relationships among embryophytes.

The nuclear ribosomal DNA intergenic spacers of wild and cultivated soybean have low variation and cryptic subrepeats.

The intergenic ribosomal DNA spacers (IGSs) from cultivated soybean (Glycine max) and wild soybean (Glycine soja) were sequenced and compared with six other legumes. These IGS sequences were 1821 bp in length in G. soja and G. max cultivars Arksoy, Ransom, and Tokyo, and 1823 bp long in the G. max cultivar Columbus. These represent the smallest published plant IGS sequences to data. Two clones from each of the above five cultivars were sequenced and only 22 sites (1.2%) were polymorphic, thereby supporting previous work that showed low genetic variation in cultivated soybean. The amount of variation observed between different clones derived from the same individual was equal to the amount seen between different cultivars. The soybean IGS sequence was aligned with six other published legume sequences and two homologous regions were identified. The first spans positions 706-1017 in the soybean IGS sequence and ends at a putative promoter site that appears conserved among all legumes. The second is located within the 5' external transcribed spacer, spans positions 1251-1823 in soybean, and includes sequences first identified as subrepeats IV-1 and IV-2 in Vicia angustifolia. Sequences homologous to these two subrepeats were identified among all legume species examined and are here designated "cryptic subrepeats" (CS-1, CS-2) given the range in similarity value (79-96% for CS-1 and 60-95% for CS-2). Comparisons of CS-1 and CS-2 sequences within individual species show that divergence (substitutional mutations, insertions, and deletions) is sufficiently high to obscure recognition of the repeat nature of these sequences by routine dot plot analytical methods. The lack of subrepeats in the 5' half of the soybean IGSs raises questions regarding the role they play in transcription termination or enhancement.

Structural analyses of plastid-derived 16S rRNAs in holoparasitic angiosperms.

Higher-order structures have been constructed for plastid-encoded small-subunit (SSU, 16S), rRNAs from representatives of seven nonphotosynthetic holoparasitic angiosperm families: Apodanthaceae, Cynomoriaceae, Cytinaceae, Balanophoraceae, Hydnoraceae, Mitrastemonaceae, and Rafflesiaceae. Whereas most pairwise comparisons among angiosperms differ by 2-3% in substitutions, the 16S rRNAs of the holoparasites show an increasingly greater number of mutations: Cynomorium (7.3%), Cytinus (8.0%), Bdallophyton (12.7%), Mitrastema (14.9%), Hydnora (19.4%), Pilostyles (30.4%) and Corynaea (35.9%). Despite this high level of sequence variation, SSU structures constructed for all species except Pilostyles possess the typical complement of 50 helices (that contain numerous compensatory mutations) thereby providing indirect evidence supporting their functionality. Pilostyles, likely with the most unusual plastid 16S rRNA yet documented, lacks four major helices and contains lengthy insertions for four others. Sequences of products generated via RT-PCR show that these structural modifications are present on a mature (transcribed) rRNA. The trend toward increasing numbers of base substitutions in the holoparasites is accompanied by a marked increase in A+U content of the rRNA. This 'A/T drift' phenomenon of rDNA is especially apparent in Corynaea whose SSU rDNA sequence is 72% A+T. A comparison of Cytinus to tobacco showed that substitution rates appear to be dependent upon the composition of neighboring bases. Transversions represented 26% of the mutations when flanking bases were G or C whereas transversions increased to 36% when the flanking bases were A to T. The underlying molecular mechanism associated with these high substitution rates is presently unknown, however, relaxation of selection pressure on ribosome function resulting in altered DNA replication and/or repair systems may be involved.

Do nonasterid holoparasitic flowering plants have plastid genomes?

Past work involving the plastid genome (plastome) of holoparasitic plants has been confined to Scrophulariaceae (or Orobanchaceae) which have truncated plastomes owing to loss of photosynthetic and other genes. Nonasterid holoparasites from Balanophoraceae (Corynaea), Hydnoraceae (Hydnora) and Cytinaceae (Cytinus) were tested for the presence of plastid genes and a plastome. Using PCR, plastid 16S rDNA was successfully amplified and sequenced from the above three holoparasites. The sequence of Cytinus showed 121 single base substitutions relative to Nicotiana (8% of the molecule) whereas higher sequence divergence was observed in Hydnora and Corynaea (287 and 513 changes, respectively). Secondary structural models for these 16S rRNAs show that most changes are compensatory, thus suggesting they are functional. Probes constructed for 16S rDNA and for four plastid-encoded ribosomal protein genes (rps2, rps4, rps7 and rpl 16) were used in Southern blots of digested genomic DNA from the three holoparasites. Positive hybridizations were obtained using each of the five probes only for Cytinus. For Smal digests, all plastid gene probes hybridized to a common fragment ca. 20 kb in length in this species. Taken together, these data provide preliminary evidence suggestive of the retention of highly diverged and truncated plastid genome in Cytinus. The greater sequence divergence for 16S rDNA and the negative hybridization results for Hydnora and Corynaea suggests two possibilities: the loss of typically conserved elements of their plastomes or the complete absence of a plastome.

Characterization of mitochondrial small-subunit ribosomal RNAs from holoparasitic plants.

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3-4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3-7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented.

High rates of nucleotide substitution in nuclear small-subunit (18S) rDNA from holoparasitic flowering plants.

Relative rate tests, using Gnetum as a reference taxon, were conducted on nuclear 18S rRNA sequences from 10 angiosperms including autotrophic nonparasites (Arabidopsis, Asarum, Glycine, Malpighia, and Zea), a chlorophyllous hemiparasite (Arceuthobium--Viscaceae), and achlorophyllous holoparasites (Balanophora--Balanophoraceae, Prosopanche--Hydnoraceae, and Rafflesia and Rhizanthes--Rafflesiaceae). Compared with Glycine, the mean number of substitutions per site (K) for five autotrophic angiosperms is 0.036 whereas for the holoparasites K = 0.126, i.e., 3.5 times higher. Comparisons of autotrophic species with short and long generation times showed no differences in K; hence, divergent rRNA sequences in the holoparasites are likely attributable to other mechanisms. These might include genetic bottlenecks, effective population size, and/or molecular drive. High substitution rates appear to be associated only with those parasitic angiosperms that have developed a highly modified haustorial system and extreme nutritional dependence upon the host. At present, high substitution rates in these parasites confound attempts to determine their phylogenetic position relative to other angiosperms.

From field to film: rapid sequencing methods for field-collected plant species.

Methods that have proven effective in the rapid extraction of plant DNA, PCR amplification and sequencing of ribosomal and chloroplast genes are presented. Techniques that can be used under field conditions to preserve DNA for subsequent extraction are reviewed. Tissues that are fresh, heat-desiccated, silica gel-desiccated and cetyltrimethylammonium bromide (CTAB) buffer preserved are compared for DNA quality and quantity. The "delayed CTAB" method yields high molecular weight DNA, thereby providing an alternative to preservation using silica gel. Optimized methodologies for PCR amplification and double-stranded sequencing of the product are detailed including gel purification of PCR products using DEAE membranes.

An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA.

The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot.

Phylogenetic relationships of the Santalales and relatives.

Determining relationships among parasitic angiosperms has often been difficult owing to frequent morphological reductions in floral and vegetative features. We report 18S (small-subunit) rRNA sequences for representative genera of three families within the Santalales (Olacaceae, Santalaceae, and Viscaceae) and six outgroup dicot families (Celastraceae, Cornaceae, Nyssaceae, Buxaceae, Apiaceae, and Araliaceae). Using Wagner parsimony analysis, one most parsimonious tree resulted that shows the Santalales to be a holophyletic taxon most closely related to Euonymus (Celastraceae). The santalalean taxa showed approximately 13% more transitional mutations than the group of seven other dicot species. This suggests a higher fixation rate for mutations in these organisms, possibly owing to a relaxation of selection pressures at the molecular level in parasitic vs nonparasitic plants. Outgroup relationships are generally in accord with current taxonomic classifications, such as the grouping of Nyssaceae and Cornaceae together (Cornales) and the grouping of Araliaceae with Apiaceae (Apiales). These data provide the first nucleotide sequences for any parasitic flowering plant and support the contention that rRNA sequence analysis can result in robust phylogenetic comparisons at the family level and above.