Multilocus Molecular Phylogenetic Analysis Research Articles

Curvularia americana and Curvularia tropicalis cause leaf and crown necrosis on Bermuda grass in Italy

Curvularia americana and C. tropicalis are described as causes of leaf and crown necroses on Bermuda grass (Cynodon dactylon x Cynodon transvaalensis) in Veneto, Northern Italy. These pathogens were characterized using morphological characters, and a multilocus molecular phylogenetic analysis based on the nuclear ribosomal internal transcribed spacer (ITS) region, the partial glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor-1α (tef1-α) genes. Pathogenicity tests and the fulfilment of Koch’s postulates confirmed C. americana and C. tropicalis as foliar pathogens of Bermuda grass. This is the first report of C. americana and C. tropicalis as pathogens of Bermuda grass, and the first record these two fungi in Europe.

Three in one: molecular phylogeny of the genus Helodrilus (Crassiclitellata: Lumbricidae) with a description of two new genera and two new species

Abstract The earthworm genus Helodrilus comprises about 20 species with distributions ranging from the Iberian Peninsula to the Caucasus, through Central Europe, the Balkans, Anatolia and the Levant. The species prefer moist habitats and are often found in the mud of river banks, in swamps or in caves. Although a high degree of morphological variability is observed in the taxonomic characters, the genus shares a common feature: the absence of nephridial bladders. In this study, we clarify the phylogeny and taxonomy of the genus Helodrilus using an integrated approach including a multilocus molecular phylogenetic analysis and thorough morphological examination of several species across the putative Helodrilus area of distribution. The findings of this study have led to the genus Helodrilus being split and to the description of two new genera and two new species: Coventina Szederjesi, Sarbu & Csuzdi gen. nov. and Imetescolex movilensis Szederjesi, Sarbu & Csuzdi gen. & sp. nov. The highly homoplasic nature of the absence of nephridial bladders was also revealed.

Contribution to the knowledge of the genus Ovipennis Hampson, 1900 with descriptions of two new species from Southern and Southwestern China (Lepidoptera, Erebidae, Arctiinae, Lithosiini).

The knowledge of the genus Ovipennis Hampson, 1900 is updated. Nanarsine milani (ern, 2009) is recorded from China and Vietnam for the first time and transferred to the genus Ovipennis based on multilocus molecular phylogenetic and morphological analyses: O. milani (ern, 2009), comb. n. Two new species related to Ovipennis semilutea species-group are described: O. mixta Huang, Zhao, Volynkin Han, sp. n. from Guizhou and Hunan and O. multicornuta Huang, Volynkin, Zhao Han, sp. n. from Sichuan. Adults and genitalia of the aforementioned taxa are illustrated.

Fusarium abutilonis and F. guadeloupense, two novel species in the Fusarium buharicum clade supported by multilocus molecular phylogenetic analyses

ABSTRACT This study was conducted to elucidate evolutionary relationships and species diversity within the Fusarium buharicum species complex (FBSC). We also evaluate the potential of these species to produce mycotoxins and other bioactive secondary metabolites. Maximum likelihood and maximum parsimony analyses of sequences from portions of four marker loci (ITS rDNA, TEF1, RPB1, and RPB2) and the combined 4495 bp data set support recognition of seven genealogically exclusive species within the FBSC. Two of the three newly discovered species are formally described as F. abutilonis and F. guadeloupense based on concordance of gene genealogies and morphological data. Fusarium abutilonis induces leaf, stem, and root lesions on several weedy Malvaceae (Abution theophrasti, Anoda cristata, Sida spinosa) and a fabaceous host (Senna obtusifolia) in North America and also was recovered from soil in New Caledonia. Fusarium abutilonis, together with its unnamed sister, Fusarium sp. ex common marsh mallow (Hibiscus moscheutos) from Washington state, and F. buharicum pathogenic to cotton and kenaf in Russia and Iran, respectively, were strongly supported as a clade of malvaceous pathogens. The four other species of the FBSC are not known to be phytopathogenic; however, F. guadeloupense was isolated from human blood in Texas and soil in Guadeloupe. The former isolate is unique because it represents the only known case of a fusarial infection disseminated hematogenously by a species lacking microconidia and the only documented fusariosis caused by a member of the FBSC. Whole genome sequence data and extracts of cracked maize kernel cultures were analyzed to assess the potential of FBSC isolates to produce mycotoxins, pigments, and phytohormones.

Exploring the basal topology of the heteronemertean tree of life: establishment of a new family, along with turbotaxonomy of Valenciniidae (Nemertea: Pilidiophora: Heteronemertea)

Abstract With about 480 known species, Pilidiophora (phylum Nemertea) has been regarded as consisting of Hubrechtiiformes (~20 spp.) and Heteronemertea (~460 spp.). Most (~87%) of the latter belong to Lineidae (> 400 spp.), while the systematics of non-lineid heteronemerteans remains largely untouched. To explore the phylogeny of non-hubrechtiiformid, non-lineid pilidiophorans, we performed multilocus molecular phylogenetic analyses with five genes (18S, 28S, H3, 16S and COI) from 41 newly sequenced specimens. Pilidiophora comprised Hubrechtellidae, Lineidae, Valenciniidae, the new family Eopilidiidae and a not formally named family-level heteronemertean taxon. Eopilidion misakiense gen. et sp. nov. is described from a specimen from Japan, but whether it belongs to Heteronemertea remains unclear. Apart from this new species, the following 11 new species of valenciniids are established, based on molecular sequences and external morphology (localities in parentheses): Baseodiscus giribeti sp. nov. (Panama), B. kakuii sp. nov. (Japan), B. komatsui sp. nov. (Japan), B. narusei sp. nov. (Japan), B. ohtsukai sp. nov. (Japan), B. paracelensis sp. nov. (Vietnam), B. profundus sp. nov. (Russian Far East), B. urgorrii sp. nov. (Spain), B. zebra sp. nov. (Japan), Oxypolella hiebertae sp. nov. (Vietnam) and Valencinura jambio sp. nov. (Japan).

Paraphoma Garibaldii Sp. Nov. Causing Leaf Spot Disease of Campanula Rapunculoides in Italy

Leaf and stem spots are among the most important diseases compromising ornamental plants worldwide. In this study, Paraphoma garibaldii sp. nov. is described from leaf lesions on Campanula rapunculoides in Piedmont, Northern Italy. The new species was characterised using a polyphasic approach including morphological characterisation and a multilocus molecular phylogenetic analysis based on partial nucleotide sequences of the translation elongation factor 1-α (tef1), the internal transcribed spacers (ITS) region and the β-tubulin (tub2) markers. Pathogenicity tests and the fulfilment of Koch's postulates confirm P. garibaldii as a novel foliar pathogen of Campanula rapunculoides . Presently, the fungal infection due to Paraphoma garibaldii is known from a single location in Italy, and further surveys are required to determine its distribution and relative importance.

Tricholoma alpinum, sp. nov., under five-needle pines in alpine and subalpine zones in Japan

ABSTRACT Alpine regions in Japan are characterized by the Siberian dwarf pine, Pinus pumila. Although these regions are conserved as national parks due to their unique biome, few reports of the fungal biota are available. We examined mushroom fungi under such vegetation and found a unique yellowish-capped Tricholoma species. Multilocus molecular phylogenetic analyses and morphological observation of specimens showed that the Tricholoma species is very similar to T. fumosoluteum known in North America. The Japanese yellowish-capped T. aff. fumosoluteum had larger basidiospores and basidia and shorter pileipellis hyphae compared with T. fumosoluteum. Therefore, we describe the Japanese entity as a new species, T. alpinum.

A new species of Solaropsis Beck, 1837 (Gastropoda: Stylommatophora: Solaropsidae) from the Brazilian Amazon

A specimen of Solaropsidae from the collection of the Academy of Natural Sciences of Drexel University (Philadelphia, USA) was recognised as a potential new species based on shell morphology. With support from a multi-locus molecular phylogenetic analysis, a new species is described here: Solaropsis penthesileae sp. nov. It is native to the Amazon Rainforest in Pará state, northern Brazil, and it is closely related to S. nimbus (Simone). It differs in its more discoid shell, with a wider body whorl that bears a stronger median angulation in its lower spire. The specimen was collected in 1998 and is an example of the long shelf-life invertebrates may have in museum collections before they are identified and formally described. It is also a reminder of the importance of those collections for biodiversity studies.

Neofusicoccum species causing branch cankers on avocado in Crete (Greece)

Branch cankers is one of the most important threats to avocado production. During the late summer of 2016, sampling was conducted in the main avocado growing area in Chania Province, Crete, Greece, to study the occurrence of branch cankers and identify the causal agents. Four fungal isolates were identified using a polyphasic approach including morphological characters and a multilocus molecular phylogenetic analysis based on partial sequences of the internal transcribed spacer region (ITS) translation elongation factor 1alpha gene (tef-1α), beta-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2). The isolates were identified as Neofusicoccum parvum and N. stellenboschiana. Pathogenicity tests showed both the Neofusicoccum species as pathogenic. To our knowledge, this is the first report of these pathogens causing branch cankers on avocado in Greece.

Alien worm in worm: a new genus of endoparasitic polychaete (Phyllodocidae, Annelida) from scale worms (Aphroditidae and Polynoidae, Annelida)

Up to now, 19 species in eight genera and two families (Dorvilleidae and Oenonidae) of polychaetes are known to be endoparasitic in the body of other polychaetes. In this paper, we describe Endovermis seisuiae gen. et sp. nov. as the first representative of Phyllodocidae parasitizing other polychaetes. Specimens of E. seisuiae were found in the body cavity of Aphrodita sp. (Aphroditidae) and Lepidonotus sp. (Polynoidae) collected from muddy sediment, 216–218 m deep, off central Japan, the western North Pacific. Our multi-locus molecular phylogenetic analysis based on COI, 16S, 18S, and 28S sequences along with other phyllodocids for which sequences were available in public databases suggested that Endovermis as sister to the clade formed by Paranaitis Southern, 1914 and Eteone Savigny, 1922. urn:lsid:zoobank.org:pub:B4C50ABC-C3D5-4899-AAB6-1304FC08B0CF

Type studies of Rossbeeverabispora, and a new species of Rossbeevera from south China.

The type of Rossbeeverabispora and additional collections from the type location and adjacent areas were studied. Molecular data for R.bispora derived from the new collections are provided. In addition, R.griseobrunnea, a new species of Rossbeevera, is described from Xiangtoushan National Nature Reserve, Guangdong Province of China. The new species is characterized by its globose to subglobose sequestrate basidiomata with grayish white to grayish brown pileus, pale bluish discoloration in some parts of the hymenophore when injured becoming rusty brown to dark brown after being exposed to the air, fusoid (star-shaped in cross section) basidiospores 17–20 × 9–12 μm, and subcutis elements in the pileus. Based on multi-locus (ITS+nrLSU+tef1-α+rpb2) molecular phylogenetic analyses, both species appear as sister to R.paracyanea. We present color photos, macro- and micro-description, SEM basidiospores, molecular affinities of the species and compare them with morphologically similar taxa within the genus. A key to the species known from northern and southern hemispheres is provided.

Does biological intimacy shape ecological network structure? A test using a brood pollination mutualism on continental and oceanic islands.

Biological intimacy-the degree of physical proximity or integration of partner taxa during their life cycles-is thought to promote the evolution of reciprocal specialization and modularity in the networks formed by co-occurring mutualistic species, but this hypothesis has rarely been tested. Here, we test this "biological intimacy hypothesis" by comparing the network architecture of brood pollination mutualisms, in which specialized insects are simultaneously parasites (as larvae) and pollinators (as adults) of their host plants to that of other mutualisms which vary in their biological intimacy (including ant-myrmecophyte, ant-extrafloral nectary, plant-pollinator and plant-seed disperser assemblages). We use a novel dataset sampled from leafflower trees (Phyllanthaceae: Phyllanthus s. l. [Glochidion]) and their pollinating leafflower moths (Lepidoptera: Epicephala) on three oceanic islands (French Polynesia) and compare it to equivalent published data from congeners on continental islands (Japan). We infer taxonomic diversity of leafflower moths using multilocus molecular phylogenetic analysis and examine several network structural properties: modularity (compartmentalization), reciprocality (symmetry) of specialization and algebraic connectivity. We find that most leafflower-moth networks are reciprocally specialized and modular, as hypothesized. However, we also find that two oceanic island networks differ in their modularity and reciprocal specialization from the others, as a result of a supergeneralist moth taxon which interacts with nine of 10 available hosts. Our results generally support the biological intimacy hypothesis, finding that leafflower-moth networks (usually) share a reciprocally specialized and modular structure with other intimate mutualisms such as ant-myrmecophyte symbioses, but unlike nonintimate mutualisms such as seed dispersal and nonintimate pollination. Additionally, we show that generalists-common in nonintimate mutualisms-can also evolve in intimate mutualisms, and that their effect is similar in both types of assemblages: once generalists emerge they reshape the network organization by connecting otherwise isolated modules.

Neocosmospora perseae sp. nov., causing trunk cankers on avocado in Italy

Trunk and branch cankers are among the most important diseases compromising avocado production worldwide. A novel species, Neocosmospora perseae sp. nov. is described isolated from trunk lesions on Persea americana in the main avocado producing area of Sicily, Italy. The new species is characterised using a polyphasic approach including morphological characters and a multilocus molecular phylogenetic analysis based on partial sequences of the translation elongation factor-1α, the internal transcribed spacer regions plus the large subunit of the rDNA cistron, and the RNA polymerase II second largest subunit. Pathogenicity tests and the fulfilment of Koch's postulates confirm N. perseae as a novel canker pathogen of Persea americana.

Molecular phylogeny of Neotropical rock frogs reveals a long history of vicariant diversification in the Atlantic forest

The Brazilian Atlantic coastal forest is one of the most heterogeneous morphoclimatic domains on earth and is thus an excellent region in which to examine the role that habitat heterogeneity plays in shaping diversification of lineages and species. Here we present a molecular phylogeny of the rock frogs of the genus Thoropa Cope, 1865, native to the Atlantic forest and extending to adjacent campo rupestre of Brazil. The goal of this study is to reconstruct the evolutionary history of the genus using multilocus molecular phylogenetic analyses. Our topology reveals 12 highly supported lineages among the four nominal species included in the study. Species T. saxatilis and T. megatympanum are monophyletic. Thoropa taophora is also monophyletic, but nested within T. miliaris. Populations of T. miliaris cluster in five geographically distinct lineages, with low support for relationships among them. Although all 12 lineages are geographically structured, some T. miliaris lineages have syntopic distributions with others, likely reflecting a secondary contact zone between divergent lineages. We discuss a biogeographic scenario that best explains the order of divergence and the distribution of species in Atlantic forest and adjacent areas, and outline the implications of our findings for the taxonomy of Thoropa.

Multilocus molecular identification and phylogenetic analysis of Colletotrichum tamarilloi as the causal agent of Tamarillo (Solanum betaceum) anthracnose in the Ecuadorian highlands

Anthracnose caused by Colletotrichum spp., is one of the most serious diseases affecting tamarillo (Solanum betaceum) production in the Ecuadorian highlands. The objective of this study was to characterise Colletotrichum isolates obtained from tamarillo to clarify its taxonomic and phylogenetic position. Based on phenotypic and morphologic characterisation, the isolates of this study were consistently grouped within the Colletotrichum acutatum complex. Multilocus molecular phylogenetic analysis based on combined sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences using Bayesian probabilities, indicated that 16 of 20 isolates from Imbabura, Cotopaxi, Tungurahua, Bolivar, Chimborazo, Azuay and Loja provinces, belonged to C. tamarilloi. This study represents the first reported case of anthracnose of S. betaceum caused by C. tamarilloi in the Ecuadorian highlands. Interestingly, pathogenicity tests and multilocus molecular analysis revealed a new infraspecific and more aggressive species formed by four isolates obtained from the Pichincha province. These findings probably provided interesting information related to mutant isolates and a possible description of a new pathogenic species affecting tamarillo fruits.

Guyanagarika, a new ectomycorrhizal genus of Agaricales from the Neotropics

A new genus and three new species of Agaricales are described from the Pakaraima Mountains of Guyana in the central Guiana Shield. All three of these new species fruit on the ground in association with species of the ectomycorrhizal (ECM) tree genus Dicymbe (Fabaceae subfam. Caesalpinioideae) and one species has been shown to form ectomycorrhizas. Multi-locus molecular phylogenetic analyses place Guyanagarika gen. nov. within the Catathelasma clade, a lineage in the suborder Tricholomatineae of the Agaricales. We formally recognize this ‘Catathelasma clade’ as an expanded family Catathelasmataceae that includes the genera Callistosporium, Catathelasma, Guyanagarika, Macrocybe, Pleurocollybia, and Pseudolaccaria. Within the Catathelasmataceae, Catathelasma and Guyanagarika represent independent origins of the ectomycorrhizal habit. Guyanagarika is the first documented case of an ECM Agaricales genus known only from the Neotropics.

Four new morel (Morchella) species in the elata subclade (M. sect. Distantes) from Turkey

Four Turkish Morchella species identified in published multilocus molecular phylogenetic analyses are described here as new, using detailed macro- and microscopic data: M. mediterraneensis (Mel-27), M. fekeensis (Mel-28), M. magnispora (Mel-29), and M. conifericola (Mel-32). A distribution map of morels identified to date in Turkey is also provided.

Aspergillus section Nidulantes (formerly Emericella): Polyphasic taxonomy, chemistry and biology.

Aspergillus section Nidulantes includes species with striking morphological characters, such as biseriate conidiophores with brown-pigmented stipes, and if present, the production of ascomata embedded in masses of Hülle cells with often reddish brown ascospores. The majority of species in this section have a sexual state, which were named Emericella in the dual name nomenclature system. In the present study, strains belonging to subgenus Nidulantes were subjected to multilocus molecular phylogenetic analyses using internal transcribed spacer region (ITS), partial β-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) sequences. Nine sections are accepted in subgenus Nidulantes including the new section Cavernicolus. A polyphasic approach using morphological characters, extrolites, physiological characters and phylogeny was applied to investigate the taxonomy of section Nidulantes. Based on this approach, section Nidulantes is subdivided in seven clades and 65 species, and 10 species are described here as new. Morphological characters including colour, shape, size, and ornamentation of ascospores, shape and size of conidia and vesicles, growth temperatures are important for identifying species. Many species of section Nidulantes produce the carcinogenic mycotoxin sterigmatocystin. The most important mycotoxins in Aspergillus section Nidulantes are aflatoxins, sterigmatocystin, emestrin, fumitremorgins, asteltoxins, and paxillin while other extrolites are useful drugs or drug lead candidates such as echinocandins, mulundocandins, calbistrins, varitriols, variecolins and terrain. Aflatoxin B1 is produced by four species: A. astellatus, A. miraensis, A. olivicola, and A. venezuelensis.

Taxonomy and biogeography ofBunopus spatalurus(Reptilia; Gekkonidae) from the Arabian Peninsula

In the last decade, taxonomic studies have drastically increased the number of species known to inhabit the Arabian deserts. While ongoing phylogenetic studies continue to identify new species and high levels of intraspecific genetic diversity, few studies have yet explored the biogeographic patterns in this arid region using an integrative approach. In the present work, we apply different phylogenetic methods to infer relationships within the Palearctic naked-toed geckos. We specifically address for the first time the taxonomy and biogeography of Bunopus spatalurus Anderson, 1901, from Arabia using multilocus concatenated and species tree phylogenies, haplotype networks and morphology. We also use species distribution modelling and phylogeographic interpolation to explore the phylogeographic structure of Bunopus spatalurus hajarensis in the Hajar Mountains and the roles of climatic stability and possible biogeographic barriers on lineage occurrence and contact zones in this arid mountain endemism hot spot. According to the inferred topology recovered using concatenated and species tree methods, the genus ‘Bunopus’ is polyphyletic. Bunopus tuberculatus and B. blanfordii form a highly supported clade closely related to Crossobamon orientalis, while the two subspecies of ‘Bunopus’ spatalurus branch together as an independent highly supported clade that diverged during the Miocene according to our estimations. Within B. s. hajarensis, three geographically structured clades can be recognized that according to our estimations diverged during the Late Miocene to Pliocene. The paleodistribution models indicate climatic stability during the Late Pleistocene and the lineage occurrence, and predicted contact zones obtained from phylogeographic interpolation therefore probably result from the older splits of the groups when these lineages originated in allopatry. As demonstrated by the results of the multilocus molecular phylogenetic analyses and the topological test carried out in this study, the genus ‘Bunopus’ is not monophyletic. To resolve this, we resurrect the genus Trachydactylus Haas and Battersby, 1959; for the species formerly referred to as Bunopus spatalurus. Considering the morphological differences, the high level of genetic differentiation in the 12S mitochondrial gene and the results of the phylogenetic and the cmos haplotype network analysis, we elevate Trachydactylus spatalurus hajarensis to the species level Trachydactylus hajarensis (Arnold, 1980).

First Report of Mango Malformation Disease Caused by Fusarium pseudocircinatum in Mexico.

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, causing severe economic loss due to reduction of yield. After the first report in India in 1891 (3), MMD has spread worldwide to most mango-growing regions. Several species of Fusarium cause the disease, including F. mangiferae in India, Israel, the USA (Florida), Egypt, South Africa, Oman, and elsewhere; F. sterilihyphosum in South Africa and Brazil; F. proliferatum in China; F. mexicanum in Mexico; and recently, F. tupiense in Brazil (1,2,3,4). Besides F. mexicanum, F. pseudocircinatum, not yet reported as a causal agent of MMD, was isolated in Mexico from affected inflorescences and vegetative malformed tissues (4). Symptoms of vegetative malformation caused by F. pseudocircinatum included hypertrophied, tightly bunched young shoots, with swollen apical and lateral buds producing misshapen terminals with shortened internodes and dwarfed leaves. Infected inflorescences of primary or secondary axes on affected panicles were shortened, thickened, and highly branched, while the peduncles became thick, remained green and fleshy, and branches profusely resembled a cauliflower in shape and size (3). Ten isolates of F. pseudocircinatum were recovered from cultivars Ataulfo, Criollo, Haden, and Tommy Atkins in Guerrero, Campeche, and Chiapas states and characterized. Isolates produced mostly 0-septate but occasionally 1- to 3-septate oval, obovoid, or elliptical aerial conidia (0-septate: 4 to 19 [avg. 8.7] × 1.5 to 4 [avg. 2.6] μm) in false heads in the dark and in short false chains under black light, unbranched or sympodially branched prostrate aerial conidiophores producing mono- and polyphialides, and sporodochia with straight or falcate conidia that were mostly 3- to 5-septate, but sometimes up to 7-septate (3-septate: 25 to 58 [avg. 41] × 2 to 3.3 [avg. 2.9] μm; 5-septate: 33.5 to 76.5 [avg. 56.7] × 2.5 to 6 [avg. 3.5] μm). Circinate sterile hyphae were rarely formed. Two representative isolates, NRRL 53570 and 53573, were subjected to multilocus molecular phylogenetic analyses of portions of five genes: nuclear large subunit 28S ribosomal RNA, β-tubulin, calmodulin, histone H3, and translation elongation factor (TEF)-1α (GenBank GU737456, GU737457, GU737290, GU737291, GU737371, GU737372, GU737425, GU737426, GU737398, and GU737399). Two pathogenicity tests were conducted with NRRL 53570 and 53573 on healthy 2-year-old nucellar seedlings of polyembryonic Criollo; 20 μl conidial suspensions (5 × 106 conidia/ml) of each isolate and water controls were inoculated separately on 15 buds on 3 different trees, as described previously (1). The following conditions were used in experiment 1: 24 to 27°C with light intensity of 16.2 to 19.8 •Mol m-2s-1 in the range of 400 to 700 nm, and photoperiods of 14 h light and 10 h dark. Typical vegetative disease symptoms were discernible in plants inoculated with NRRL 53570 (20%) and 53573 (7%) after 8 months. In experiment 2, after 3 months growth under the above conditions, seedlings were transferred to an outdoor nursery in Iguala, Guerrero. Typical vegetative symptoms of MMD were observed in 86.7 and 13.3% of the buds inoculated with F. pseudocircinatum NRRL 53570 and 53573, respectively, after 9 months. Isolates from typical symptomatic vegetative buds were confirmed as F. pseudocircinatum by sequencing a portion of their TEF-1α gene, thus fulfilling Koch's postulates. This is the first report of F. pseudocircinatum as a causal agent of MMD.