High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science.

Applying molecular methods to fungi establishing lichenized associations with green algae or cyanobacteria has repeatedly revealed the existence of numerous phylogenetic taxa overlooked by classical taxonomic approaches. Here, we report taxonomical conclusions based on multiple species delimitation and validation analyses performed on an eight-locus dataset that includes world-wide representatives of the dolichorhizoid and scabrosoid clades in section Polydactylon of the genus Peltigera. Following the recommendations resulting from a consensus species delimitation approach and additional species validation analysis (BPP) performed in this study, we present a total of 25 species in the dolichorhizoid clade and nine in the scabrosoid clade, including respectively 18 and six species that are new to science and formally described. Additionally, one combination and three varieties (including two new to science) are proposed in the dolichorhizoid clade. The following 24 new species are described: P. appalachiensis, P. asiatica, P. borealis, P. borinquensis, P. chabanenkoae, P. clathrata, P. elixii, P. esslingeri, P. flabellae, P. gallowayi, P. hawaiiensis, P. holtanhartwigii, P. itatiaiae, P. hokkaidoensis, P. kukwae, P. massonii, P. mikado, P. nigriventris, P. orientalis, P. rangiferina, P. sipmanii, P. stanleyensis, P. vitikainenii and P. willdenowii; the following new varieties are introduced: P. kukwae var. phyllidiata and P. truculenta var. austroscabrosa; and the following new combination is introduced: P. hymenina var. dissecta. Each species from the dolichorhizoid and scabrosoid clades is morphologically and chemically described, illustrated, and characterised with ITS sequences. Identification keys are provided for the main biogeographic regions where species from the two clades occur. Morphological and chemical characters that are commonly used for species identification in the genus Peltigera cannot be applied to unambiguously recognise most molecularly circumscribed species, due to high variation of thalli formed by individuals within a fungal species, including the presence of distinct morphs in some cases, or low interspecific variation in others. The four commonly recognised morphospecies: P. dolichorhiza, P. neopolydactyla, P. pulverulenta and P. scabrosa in the dolichorhizoid and scabrosoid clades represent species complexes spread across multiple and often phylogenetically distantly related lineages. Geographic origin of specimens is often helpful for species recognition; however, ITS sequences are frequently required for a reliable identification. Citation: Magain N, Miadlikowska J, Goffinet B, et al. 2023. High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science. Persoonia 51: 1-88. doi: 10.3767/persoonia.2023.51.01.

Macroevolution of Specificity in Cyanolichens of the Genus Peltigera Section Polydactylon (Lecanoromycetes, Ascomycota).

Patterns of specificity among symbiotic partners are key to a comprehensive understanding of the evolution of symbiotic systems. Specificity of mutualistic partners, within a widespread monophyletic group for which all species are sampled has rarely been explored. Here, we assess the level of specificity between the cosmopolitan lichen-forming fungus (mycobiont) from the genus Peltigera, section Polydactylon, and its cyanobacterial partner Nostoc (cyanobiont). The mycobiont and cyanobiont phylogenies are inferred from five nuclear loci and the rbcLX region, respectively. These sequences were obtained from 206 lichen thalli, representing ca. 40 closely related Peltigera species sampled worldwide, doubling the number of known species in this group. We found a broad spectrum of specificity for both partners ranging from strict specialists to generalists. Overall, mycobionts are more specialized than cyanobionts by associating mostly with one or a few Nostoc phylogroups, whereas most cyanobionts associate frequently with several Peltigera species. Specialist mycobionts are older than generalists, supporting the hypothesis that specialization of mycobionts to one or few cyanobionts, is favored through time in geographic areas where species have been established for long periods of time. The relatively recent colonization of a new geographic area (Central and South America) by members of section Polydactylon is associated with a switch to a generalist pattern of association and an increased diversification rate by the fungal partner, suggesting that switches to generalism are rare events that are advantageous in new environments. We detected higher genetic diversity in generalist mycobionts. We also found that Peltigera species specialized on a single Nostoc phylogroup have narrower geographical distributions compared with generalist species.

Genomic scanning using AFLP to detect loci under selection in the moss Funaria hygrometrica along a climate gradient in the Sierra Nevada Mountains, Spain.

The common cord moss Funaria hygrometrica has a worldwide distribution and thrives in a wide variety of environments. Here, we studied the genetic diversity in F. hygrometrica along an abiotic gradient in the Mediterranean high mountain of Sierra Nevada (Spain) using a genome scan method. Eighty-four samples from 17 locations from 24 to 2700 m were fingerprinted based on their amplified fragment length polymorphism (AFLP) banding pattern. Using PCA and Bayesian inference we found that the genetic diversity was structured in three or four clusters, respectively. Using a genome scan method we identified 13 outlier loci, which showed a signature of positive selection. Partial Mantel tests were performed between the Euclidean distance matrices of geographic and climatic variables, versus the pair-wise genetic distance of the AFLP dataset and AFLP-positive outliers dataset. AFLP-positive outlier data were significantly correlated with the gradient of the climatic variables, suggesting adaptive variation among populations of F. hygrometrica along the Sierra Nevada Mountains. We highlight the additional analyses necessary to identify the nature of these loci, and their biological role in the adaptation process.

Extant diversity of bryophytes emerged from successive post-Mesozoic diversification bursts.

Unraveling the macroevolutionary history of bryophytes, which arose soon after the origin of land plants but exhibit substantially lower species richness than the more recently derived angiosperms, has been challenged by the scarce fossil record. Here we demonstrate that overall estimates of net species diversification are approximately half those reported in ferns and ∼30% those described for angiosperms. Nevertheless, statistical rate analyses on time-calibrated large-scale phylogenies reveal that mosses and liverworts underwent bursts of diversification since the mid-Mesozoic. The diversification rates further increase in specific lineages towards the Cenozoic to reach, in the most recently derived lineages, values that are comparable to those reported in angiosperms. This suggests that low diversification rates do not fully account for current patterns of bryophyte species richness, and we hypothesize that, as in gymnosperms, the low extant bryophyte species richness also results from massive extinctions.

Mapping uncertainty and phylogenetic uncertainty in ancestral character state reconstruction: an example in the moss genus Brachytheciastrum.

The evolution of species traits along a phylogeny can be examined through an increasing number of possible, but not necessarily complementary, approaches. In this paper, we assess whether deriving ancestral states of discrete morphological characters from a model whose parameters are (i) optimized by ML on a most likely tree; (II) optimized by ML onto each of a Bayesian sample of trees; and (III) sampled by a MCMC visiting the space of a Bayesian sample of trees affects the reconstruction of ancestral states in the moss genus Brachytheciastrum. In the first two methods, the choice of a single- or two-rate model and of a genetic distance (wherein branch lengths are used to determine the probabilities of change) or speciational (wherein changes are only driven by speciation events) model based upon a likelihood-ratio test strongly depended on the sampled trees. Despite these differences in model selection, reconstructions of ancestral character states were strongly correlated to each others across nodes, often at r > 0.9, for all the characters. The Bayesian approach of ancestral character state reconstruction offers, however, a series of advantages over the single-tree approach or the ML model optimization on a Bayesian sample of trees because it does not involve restricting model parameters prior to reconstructing ancestral states, but rather allows a range of model parameters and ancestral character states to be sampled according to their posterior probabilities. From the distribution of the latter, conclusions on trait evolution can be made in a more satisfactorily way than when a substantial part of the uncertainty of the results is obscured by the focus on a single set of model parameters and associated ancestral states. The reconstructions of ancestral character states in Brachytheciastrum reveal rampant parallel morphological evolution. Most species previously described based on phenetic grounds are thus resolved of polyphyletic origin. Species polyphylly has been increasingly reported among mosses, raising severe reservations regarding current species definition.

Phylogenetic evidence of a rapid radiation of pleurocarpous mosses (Bryophyta).

Pleurocarpous mosses, characterized by lateral female gametangia and highly branched, interwoven stems, comprise three orders and some 5000 species, or almost half of all moss diversity. Recent phylogenetic analyses resolve the Ptychomniales as sister to the Hypnales plus Hookeriales. Species richness is highly asymmetric with approximately 100 Ptychomniales, 750 Hookeriales, and 4400 Hypnales. Chloroplast DNA (cpDNA) sequences were obtained to compare partitioning of molecular diversity among the orders with estimates of species richness, and to test the hypothesis that either the Hookeriales or Hypnales underwent a period (or periods) of exceptionally rapid diversification. Levels of biodiversity were quantified using explicitly historical "phylogenetic diversity" and non-historical estimates of standing sequence diversity. Diversification rates were visualized using lineage-through-time (LTT) plots, and statistical tests of alternative diversification models were performed using the methods of Paradis (1997). The effects of incomplete sampling on the shape of LTT plots and performance of statistical tests were investigated using simulated phylogenies with incomplete sampling. Despite a much larger number of accepted species, the Hypnales contain lower levels of (cpDNA) biodiversity than their sister group, the Hookeriales, based on all molecular measures. Simulations confirm previous results that incomplete sampling yields diversification patterns that appear to reflect a decreasing rate through time, even when the true phylogenies were simulated with constant rates. Comparisons between simulated results and empirical data indicate that a constant rate of diversification cannot be rejected for the Hookeriales. The Hypnales, however, appear to have undergone a period of exceptionally rapid diversification for the earliest 20% of their history.

Testing morphological concepts of orders of pleurocarpous mosses (Bryophyta) using phylogenetic reconstructions based on TRNL-TRNF and RPS4 sequences.

The ordinal classification of pleurocarpous mosses rests on characters such as branching mode and architecture of the peristome teeth that line the mouth of the capsule. The Leucodontales comprise mainly epiphytic taxa, characterized by sympodial branching and reduced peristomes, whereas the Hypnales are primarily terricolous and monopodially branching. The third order, the Hookeriales, is defined by a unique architecture of the endostome. We sampled 78 exemplar taxa representing most families of these orders and sequenced two chloroplast loci, the trnL-trnF region and the rps4 gene, to test the monophyly and relationships of these orders of pleurocarpous mosses. Estimates of levels of saturation suggest that the trnL-trnF spacer and the third codon position of the rps4 gene have reached saturation, in at least the transitions. Analyses of the combined data set were performed under three optimality criteria with different sets of assumptions, such as excluding hypervariable positions, downweighting the most likely transformations, and indirect weighting of rps4 codon positions by including amino acid translations. Multiple parallelism in nonsynonymous mutations led to little or no improvement in various indices upon inclusion of amino acid sequences. Trees obtained under likelihood were significantly better under likelihood than the trees derived from the same matrix under parsimony. Our phylogenetic analyses suggest that (1) the pleurocarpous mosses, with the exception of the Cyrtopodaceae, form a monophyletic group which is here given formal recognition as the Hypnidae; (2) the Leucodontales are at least paraphyletic; and (3) the Hypnales form, with most members of the Leucodontalean grade, a monophyletic group sister to a Hookerialean lineage. The Hypopterygiaceae, Hookeriales, and a clade composed of Neorutenbergia, Pseudocryphaea, and Trachyloma likely represent a basal clade or grade within the Hypnidae. These results suggest that mode of branching and reduced peristomes are homoplastic at the ordinal level in pleurocarpous mosses.

Quantitative trait loci: a meta-analysis.

This article presents a method to combine QTL results from different independent analyses. This method provides a modified Akaike criterion that can be used to decide how many QTL are actually represented by the QTL detected in different experiments. This criterion is computed to choose between models with one, two, three, etc., QTL. Simulations are carried out to investigate the quality of the model obtained with this method in various situations. It appears that the method allows the length of the confidence interval of QTL location to be consistently reduced when there are only very few "actual" QTL locations. An application of the method is given using data from the maize database available online at http://www. agron.missouri.edu/.

More about quantitative trait locus mapping with diallel designs.

We present a general regression-based method for mapping quantitative trait loci (QTL) by combining different populations derived from diallel designs. The model expresses, at any map position, the phenotypic value of each individual as a function of the specific-mean of the population to which the individual belongs, the additive and dominance effects of the alleles carried by the parents of that population and the probabilities of QTL genotypes conditional on those of neighbouring markers. Standard linear model procedures (ordinary or iteratively reweighted least-squares) are used for estimation and test of the parameters.

Disease resistance gene analogs as candidates for QTLs involved in pepper-pathogen interactions.

Whereas resistance genes (R-genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance (quantitative trait loci, QTLs) are still unknown. We hypothesized that genes at QTLs could share homologies with cloned R-genes. We used a PCR-based approach to isolate R-gene analogs (RGAs) with consensus primers corresponding with conserved domains of cloned R-genes: (i) the nucleotide binding site (NBS) and hydrophobic domain, and (ii) the kinase domain. PCR-amplified fragments were sequenced and mapped on a pepper intraspecific map. NBS-containing sequences of pepper, most similar to the N gene of tobacco, were classified into seven families and all mapped in a unique region covering 64 cM on the Noir chromosome. Kinase domain containing sequences and cloned R-gene homologs (Pto, Fen, Cf-2) were mapped on four different linkage groups. A QTL involved in partial resistance to cucumber mosaic virus (CMV) with an additive effect was closely linked or allelic to one NBS-type family. QTLs with epistatic effects were also detected at several RGA loci. The colocalizations between NBS-containing sequences and resistance QTLs suggest that the mechanisms of qualitative and quantitative resistance may be similar in some cases.

Circumscription and phylogeny of the Orthotrichales (Bryopsida) inferred from RBCL sequence analyses.

The affinities as well as the circumscription of the Orthotrichaceae (Bryopsida), one of the most diverse families of mosses, have been the focus of a controversy for much of the last century. We obtained rbcL sequences for 37 arthrodontous mosses, including 27 taxa of the Orthotrichales. The sequences were analyzed using maximum parsimony and neighbor joining in order to (1) test the monophyly of the Orthotrichales and the Orthotrichaceae; (2) determine their phylogenetic relationships; and (3) test the current subfamilial classification within the Orthotrichaceae. Both analyses suggest that the Orthotrichales are polyphyletic. The Erpodiaceae and the Rhachitheciaceae as well as Amphidium and Drummondia, two genera of the Orthotrichaceae, are shown to be of haplolepideous affinity. The Splachnales, the Bryales sensu lato, and the Orthotrichales form a monophyletic clade sister to the Haplolepideae. Both neighbor joining and maximum parsimony also suggest that the Orthotrichaceae are composed of two major lineages dominated either by acrocarpous or cladocarpous taxa. The monophyly of the family is, however, only well supported by Tamura's distances. The genera Macrocoma, Macromitrium, Orthotrichum, Ulota, and Zygodon all appear to be artificial assemblages. This study illustrates the contribution of rbcL sequence data to bryophyte systematics and, particularly, in determining the affinities of taxa lacking a peristome, whose characters are central to the classification of mosses.

A method to measure genetic distance between allogamous populations of alfalfa (Medicago sativa) using RAPD molecular markers.

Alfalfa (Medicago sativa L.) is a forage legume of world-wide importance whose both allogamous and autotetraploid nature maximizes the genetic diversity within natural and cultivated populations. This genetic diversity makes difficult the discrimination between two related populations. We analyzed this genetic diversity by screening DNA from individual plants of eight cultivated and natural populations of M. sativa and M. falcata using the RAPD method. A high level of genetic variation was found within and between populations. Using five primers, 64 intense bands were scored as present or absent across all populations. Most of the loci were revealed to be highly polymorphic whereas very few population-specific polymorphisms were identified. From these observations, we adopted a method based on the Roger's genetic distance between populations using the observed frequency of bands to discriminate populations pairwise. Except for one case, the between-population distances were all significantly different from zero. We have also determined the minimal number of bands and individuals required to test for the significance of between-population distances.

Characterization of mycobionts of photomorph pairs in the peltigerineae (lichenized ascomycetes) based on internal transcribed spacer sequences of the nuclear ribosomal DNA.

The "one fungus-two photomorphs" hypothesis suggests that certain lichenized fungi can establish a symbiotic relationship with either a eukaryotic or a prokaryotic photobiont. Such pairs of photomorphs are well know from cephalodiate Peltigerineae. Using an ascomycete-specific primer we amplified the internal transcribed spacer region of the nrDNA repeat of the mycobiont from total "lichen DNA" extracts of Peltigera malacea, photomorphs of P. aphthosa, P. britannica, and P. leucophlebia, Nephroma expallidum, and photomorphs of N. arcticum. Comparisons of 5.8S sequences suggest that the sequences obtained belong to the mycobiont and thus, that the ascomycete-specific primer is adequate for amplifying fungal DNA from total lichen-DNA extracts. The strict identity of nucleotide sequences of the internal transcribed spacer region of the nrDNA repeat between joined-photomorphs supports the one fungus-two photomorphs hypothesis. Photomorph may thus primarily reflect phenotypic plasticity of photomorphic fungi in response to changing environmental conditions. The cyanomorph recently reported for P. leucophlebia is shown to be based on a misidentified specimen of P. aphthosa. Comparisons of the ITS sequences further supports recognizing P. aphthosa, P. britannica, and P. leucophlebia at the species rather than the infraspecific level.

Comparing power of different methods for QTL detection.

We compared the powers of two methods for detection of quantitative trait loci (QTL) using genetic markers, in the simple case of an interval between two codominant markers and a backcross population. The first method is the interval mapping approach, based on the use of likelihood ratio tests performed in many positions within the interval considered and the second is the classical analysis of variance (ANOVA) testing only on the positions of the two markers. For both approaches we took into account the correlation between tests performed at different markers or positions in the interval. Appropriate thresholds and powers of tests were then calculated using analytical formulations. Simulations were also done to check the validity of the approximations used to calculate the power of the interval mapping test. Results show that the interval mapping test is slightly more powerful (about 5%) than ANOVA for small intervals (less than 20 cM) and that, for quite large effects of the QTL, the advantage of interval mapping increases as the distance between markers increases. It is more than 30% for intervals of about 70 cM.

Maximum-likelihood models for mapping genetic markers showing segregation distortion. 1. Backcross populations.

A maximum-likelihood approach is used in order to estimate recombination fractions between markers showing segregation distortion in backcross populations. It is assumed that the distortions are induced by viability differences between gametes or zygotes due to one or more selected genes. We show that Bailey's (1949) estimate stays consistent and efficient under more general assumptions than those defined by its author. This estimate should therefore be used instead of the classical maximum-likelihood estimate. The question of detection of linkage is also discussed. We show that the order of markers on linkage groups may be affected by segregation distortion.

Maximum-likelihood models for mapping genetic markers showing segregation distortion. 2. F2 populations.

In F2 populations, gametic and zygotic selection may affect the analysis of linkage in different ways. Therefore, specific likelihood equations have to be developed for each case, including dominant and codominant markers. The asymptotic bias of the "classical" estimates are derived for each case, in order to compare them with the standard errors of the suggested estimates. We discuss the utility and the efficiency of a previous model developed for dominant markers. We show that dominant markers provide very poor information in the case of segregation distortion and, therefore, should be used with circumspection. On the other hand, the estimation of recombination fractions between codominant markers is less affected by selection than is that for dominant markers. We also discuss the analysis of linkage between dominant and codominant markers.

Constructing confidence intervals for QTL location.

We describe a method for constructing the confidence interval of the QTL location parameter. This method is developed in the local asymptotic framework, leading to a linear model at each position of the putative QTL. The idea is to construct a likelihood ratio test, using statistics whose asymptotic distribution does not depend on the nuisance parameters and in particular on the effect of the QTL. We show theoretical properties of the confidence interval built with this test, and compare it with the classical confidence interval using simulations. We show in particular, that our confidence interval has the correct probability of containing the true map location of the QTL, for almost all QTLs, whereas the classical confidence interval can be very biased for QTLs having small effect.

Approximate thresholds of interval mapping tests for QTL detection.

A general method is proposed for calculating approximate thresholds of interval mapping tests for quantitative trait loci (QTL) detection. Simulation results show that this method, when applied to backcross and F2 populations, gives good approximations and is useful for any situation. Programs which calculate these thresholds for backcross, recombinant inbreds and F2 for any given level and any chromosome with any given distribution of codominant markers were written in Fortran 77 and are available under request. The approach presented here could be used to obtain, after suitable calculations, thresholds for most segregating populations used in QTL mapping experiments.

Detecting a major gene in an F2 population.

The aim of this paper is to study the behavior of the likelihood ratio test for the detection of a major gene in an F2 population. The model is a mixture of three normal distributions where the proportions are known. The information matrix is not positive definite, and therefore classical results cannot be used. An example concerning beans is given.