Sequential horizontal gene transfers from different hosts in a widespread Eurasian parasitic plant, Cynomorium coccineum.

PREMISE: Parasitic plants with large geographic ranges, and different hosts in parts of their range, may acquire horizontally transferred genes (HGTs), which might sometimes leave a footprint of gradual host and range expansion. Cynomorium coccineum, the only member of the Saxifragales family Cynomoriaceae, is a root holoparasite that occurs in water-stressed habitats from western China to the Canary Islands. It parasitizes at least 10 angiosperm families from different orders, some of them only in parts of its range. This parasite therefore offers an opportunity to trace HGTs as long as parasite-host pairs can be obtained and sequenced. METHODS: By sequencing mitochondrial, plastid, and nuclear loci from parasite-host pairs from throughout the parasite's range and with prior information from completely assembled mitochondrial and plastid genomes, we detected 10 HGTs of five mitochondrial genes. RESULTS: The 10 HGTs appear to have occurred sequentially as C. coccineum expanded from East to West. Molecular-clock models yield Cynomorium stem ages between 66 and 156 Myr, with relaxed clocks converging on 66-67 Myr. Chinese Sapindales, probably Nitraria, were the first source of transferred genes, followed by Iranian and Mediterranean Caryophyllales. The most recently acquired gene appears to come from a Tamarix host in the Iberian Peninsula. CONCLUSIONS: Data on HGTs that have accumulated over the past 15 years, along with this discovery of multiple HGTs within a single widespread species, underline the need for more whole-genome data from parasite-host pairs to investigate whether and how transferred copies coexist with, or replace, native functional genes.

Massive intracellular gene transfer during plastid genome reduction in nongreen Orobanchaceae.

Plastid genomes (plastomes) of nonphotosynthetic plants experience extensive gene losses and an acceleration of molecular evolutionary rates. Here, we inferred the mechanisms and timing of reductive genome evolution under relaxed selection in the broomrape family (Orobanchaceae). We analyzed the plastomes of several parasites with a major focus on the genus Orobanche using genome-descriptive and Bayesian phylogenetic-comparative methods. Besides this, we scanned the parasites' other cellular genomes to trace the fate of all genes that were purged from their plastomes. Our analyses indicate that the first functional gene losses occurred within 10 Myr of the transition to obligate parasitism in Orobanchaceae, and that the physical plastome reduction proceeds by small deletions that accumulate over time. Evolutionary rate shifts coincide with the genomic reduction process in broomrapes, suggesting that the shift of selectional constraints away from photosynthesis to other molecular processes alters the plastid rate equilibrium. Most of the photosynthesis-related genes or fragments of genes lost from the plastomes of broomrapes have survived in their nuclear or mitochondrial genomes as the results of multiple intracellular transfers and subsequent fragmentation. Our findings indicate that nonessential DNA is eliminated much faster in the plastomes of nonphotosynthetic parasites than in their other cellular genomes.

Combining FISH and model-based predictions to understand chromosome evolution in Typhonium (Araceae).

BACKGROUND AND AIMS: Since the advent of molecular phylogenetics, numerous attempts have been made to infer the evolutionary trajectories of chromosome numbers on DNA phylogenies. Ideally, such inferences should be evaluated against cytogenetic data. Towards this goal, we carried out phylogenetic modelling of chromosome number change and fluorescence in situ hybridization (FISH) in a medium sized genus of Araceae to elucidate if data from chromosomal markers would support maximum likelihood-inferred changes in chromosome numbers among close relatives. Typhonium, the focal genus, includes species with 2n = 65 and 2n = 8, the lowest known count in the family. METHODS: A phylogeny from nuclear and plastid sequences (96 taxa, 4252 nucleotides) and counts for all included species (15 of them first reported here) were used to model chromosome number evolution, assuming discrete events, such as polyploidization and descending or ascending dysploidy, occurring at different rates. FISH with three probes (5S rDNA, 45S rDNA and Arabidopsis-like telomeres) was performed on ten species with 2n = 8 to 2n = 24. KEY RESULTS: The best-fitting models assume numerous past chromosome number reductions. Of the species analysed with FISH, the two with the lowest chromosome numbers contained interstitial telomeric signals (Its), which together with the phylogeny and modelling indicates decreasing dysploidy as an explanation for the low numbers. A model-inferred polyploidization in another species is matched by an increase in rDNA sites. CONCLUSIONS: The combination of a densely sampled phylogeny, ancestral state modelling and FISH revealed that the species with n = 4 is highly derived, with the FISH data pointing to a Robertsonian fusion-like chromosome rearrangement in the ancestor of this species.

A new method for handling missing species in diversification analysis applicable to randomly or nonrandomly sampled phylogenies.

Chronograms from molecular dating are increasingly being used to infer rates of diversification and their change over time. A major limitation in such analyses is incomplete species sampling that moreover is usually nonrandom. While the widely used γ statistic with the Monte Carlo constant-rates test or the birth-death likelihood analysis with the δ AICrc test statistic are appropriate for comparing the fit of different diversification models in phylogenies with random species sampling, no objective automated method has been developed for fitting diversification models to nonrandomly sampled phylogenies. Here, we introduce a novel approach, CorSiM, which involves simulating missing splits under a constant rate birth-death model and allows the user to specify whether species sampling in the phylogeny being analyzed is random or nonrandom. The completed trees can be used in subsequent model-fitting analyses. This is fundamentally different from previous diversification rate estimation methods, which were based on null distributions derived from the incomplete trees. CorSiM is automated in an R package and can easily be applied to large data sets. We illustrate the approach in two Araceae clades, one with a random species sampling of 52% and one with a nonrandom sampling of 55%. In the latter clade, the CorSiM approach detects and quantifies an increase in diversification rate, whereas classic approaches prefer a constant rate model; in the former clade, results do not differ among methods (as indeed expected since the classic approaches are valid only for randomly sampled phylogenies). The CorSiM method greatly reduces the type I error in diversification analysis, but type II error remains a methodological problem.

Maximum likelihood inference implies a high, not a low, ancestral haploid chromosome number in Araceae, with a critique of the bias introduced by 'x'.

BACKGROUND AND AIMS: For 84 years, botanists have relied on calculating the highest common factor for series of haploid chromosome numbers to arrive at a so-called basic number, x. This was done without consistent (reproducible) reference to species relationships and frequencies of different numbers in a clade. Likelihood models that treat polyploidy, chromosome fusion and fission as events with particular probabilities now allow reconstruction of ancestral chromosome numbers in an explicit framework. We have used a modelling approach to reconstruct chromosome number change in the large monocot family Araceae and to test earlier hypotheses about basic numbers in the family. METHODS: Using a maximum likelihood approach and chromosome counts for 26 % of the 3300 species of Araceae and representative numbers for each of the other 13 families of Alismatales, polyploidization events and single chromosome changes were inferred on a genus-level phylogenetic tree for 113 of the 117 genera of Araceae. KEY RESULTS: The previously inferred basic numbers x = 14 and x = 7 are rejected. Instead, maximum likelihood optimization revealed an ancestral haploid chromosome number of n = 16, Bayesian inference of n = 18. Chromosome fusion (loss) is the predominant inferred event, whereas polyploidization events occurred less frequently and mainly towards the tips of the tree. CONCLUSIONS: The bias towards low basic numbers (x) introduced by the algebraic approach to inferring chromosome number changes, prevalent among botanists, may have contributed to an unrealistic picture of ancestral chromosome numbers in many plant clades. The availability of robust quantitative methods for reconstructing ancestral chromosome numbers on molecular phylogenetic trees (with or without branch length information), with confidence statistics, makes the calculation of x an obsolete approach, at least when applied to large clades.

Slowdowns in diversification rates from real phylogenies may not be real.

Studies of diversification patterns often find a slowing in lineage accumulation toward the present. This seemingly pervasive pattern of rate downturns has been taken as evidence for adaptive radiations, density-dependent regulation, and metacommunity species interactions. The significance of rate downturns is evaluated with statistical tests (the gamma statistic and Monte Carlo constant rates (MCCR) test; birth-death likelihood models and Akaike Information Criterion [AIC] scores) that rely on null distributions, which assume that the included species are a random sample of the entire clade. Sampling in real phylogenies, however, often is nonrandom because systematists try to include early-diverging species or representatives of previous intrataxon classifications. We studied the effects of biased sampling, structured sampling, and random sampling by experimentally pruning simulated trees (60 and 150 species) as well as a completely sampled empirical tree (58 species) and then applying the gamma statistic/MCCR test and birth-death likelihood models/AIC scores to assess rate changes. For trees with random species sampling, the true model (i.e., the one fitting the complete phylogenies) could be inferred in most cases. Oversampling deep nodes, however, strongly biases inferences toward downturns, with simulations of structured and biased sampling suggesting that this occurs when sampling percentages drop below 80%. The magnitude of the effect and the sensitivity of diversification rate models is such that a useful rule of thumb may be not to infer rate downturns from real trees unless they have >80% species sampling.

Relationships within the Araceae: comparison of morphological patterns with molecular phylogenies.

PREMISE OF THE STUDY: The first family-wide molecular phylogeny of the Araceae, a family of about 3800 published species in 120 genera, became available in 1995, followed by a cladistic analysis of morpho-anatomical data in 1997. The most recent and comprehensive family-wide molecular phylogeny was published in 2008 and included species from 102 genera. We reanalyzed the molecular data with a more complete genus sampling and compared the resulting phylogeny with morphological and anatomical data, with a view to contributing to a new formal classification of the Araceae. METHODS: We analyzed 113 aroid genera and 4494 aligned nucleotides that resulted from adding 11 genera to the 2008 molecular matrix. We also analyzed 81 morphological characters in the context of the molecular phylogeny, using an extended version of the 1997 morpho-anatomical data set. KEY RESULTS: The resulting maximum-likelihood phylogeny is well resolved and supported, and most of the 44 larger clades also have morphological or anatomical synapomorphies as well as ecological or geographic cohesion. Of the 44 clades, 16 are here newly circumscribed and informally named. However, some relationships remain poorly supported within the Aroideae subfamily. The most problematic placement is Calla within Aroideae, which conflicts with the distribution of morphological, anatomical, and palynological character states. CONCLUSIONS: The comparison of the molecular analysis with morphological and anatomical data presented here represents an important basis for a new formal classification for the Araceae and for the understanding of the evolution of this ancient family, a monocot group known in the fossil record from the early Cretaceous.

Reevaluation of the cox1 group I intron in Araceae and angiosperms indicates a history dominated by loss rather than horizontal transfer.

The origin and modes of transmission of introns remain matters of much debate. Previous studies of the group I intron in the angiosperm cox1 gene inferred frequent angiosperm-to-angiosperm horizontal transmission of the intron from apparent incongruence between intron phylogenies and angiosperm phylogenies, patchy distribution of the intron among angiosperms, and differences between cox1 exonic coconversion tracts (the first 22 nt downstream of where the intron inserted). We analyzed the cox1 gene in 179 angiosperms, 110 of them containing the intron (intron(+)) and 69 lacking it (intron(-)). Our taxon sampling in Araceae is especially dense to test hypotheses about vertical and horizontal intron transmission put forward by Cho and Palmer (1999. Multiple acquisitions via horizontal transfer of a group I intron in the mitochondrial coxl gene during evolution of the Araceae family. Mol Biol Evol. 16:1155-1165). Maximum likelihood trees of Araceae cox1 introns, and also of all angiosperm cox1 introns, are largely congruent with known phylogenetic relationships in these taxa. The exceptions can be explained by low signal in the intron and long-branch attraction among a few taxa with high mitochondrial substitution rates. Analysis of the 179 coconversion tracts reveals 20 types of tracts (11 of them only found in single species, all involving silent substitutions). The distribution of these tracts on the angiosperm phylogeny shows a common ancestral type, characterizing most intron(+) and some intron(-) angiosperms, and several derivative tract types arising from gradual back mutation of the coconverted nucleotides. Molecular clock dating of small intron(+) and intron(-) sister clades suggests that coconversion tracts have persisted for 70 Myr in Araceae, whose cox1 sequences evolve comparatively slowly. Sequence similarity among the 110 introns ranges from 91% to identical, whereas putative homologs from fungi are highly different, but sampling in fungi is still sparse. Together, these results suggest that the cox1 intron entered angiosperms once, has largely or entirely been transmitted vertically, and has been lost numerous times, with coconversion tract footprints providing unreliable signal of former intron presence.

Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers.