Nuclear ITS Research Articles

The generic placement of Grimmia teretinervis Limpr.

Kiebacher, T. & Meier, M. 2020. The generic placement of Grimmia teretinervis Limpr. – Herzogia 33: 544 –547.Grimmia teretinervis is a dioicous moss of the Grimmiaceae that has never been found with sporophytes. Consequently, its generic placement is uncertain, and although it is mostly referred to Grimmia, some authors assign it to Schistidium. The phylogenetic analysis of the nuclear ITS region indicates that it is a species of Schistidium and that it is related to the likewise dioicous S. tenerum.

Molecular Phylogenetics and Micromorphology of Australasian Stipeae (Poaceae, Subfamily Pooideae), and the Interrelation of Whole-Genome Duplication and Evolutionary Radiations in This Grass Tribe.

The mainly Australian grass genus Austrostipa (tribe Stipeae) comprising approximately 64 species represents a remarkable example of an evolutionary radiation. To investigate aspects of diversification, macro- and micromorphological variation in this genus, we conducted molecular phylogenetic and scanning electron microscopy (SEM) analyses including representatives from most of Austrostipa’s currently accepted subgenera. Because of its taxonomic significance in Stipeae, we studied the lemma epidermal pattern (LEP) in 34 representatives of Austrostipa. Plastid DNA variation within Austrostipa was low and only few lineages were resolved. Nuclear ITS and Acc1 yielded comparable groupings of taxa and resolved subgenera Arbuscula, Petaurista, and Bambusina in a common clade and as monophyletic. In most of the Austrostipa species studied, the LEP was relatively uniform (typical maize-like), but six species had a modified cellular structure. The species representing subgenera Lobatae, Petaurista, Bambusina as well as A. muelleri from subg. Tuberculatae were well-separated from all the other species included in the analysis. We suggest recognizing nine subgenera in Austrostipa (with number of species): Arbuscula (4), Aulax (2), Austrostipa (36), Bambusina (2), Falcatae (10), Lobatae (5), Longiaristatae (2), Petaurista (2) and the new subgenus Paucispiculatae (1) encompassing A. muelleri. Two paralogous sequence copies of Acc1, forming two distinct clades, were found in polyploid Austrostipa and Anemanthele. We found analogous patterns for our samples of Stipa s.str. with their Acc1 clades strongly separated from those of Austrostipa and Anemanthele. This underlines a previous hypothesis of Tzvelev (1977) that most extant Stipeae are of hybrid origin. We also prepared an up-to-date survey and reviewed the chromosome number variation for our molecularly studied taxa and the whole tribe Stipeae. The chromosome base number patterns as well as dysploidy and whole-genome duplication events were interpreted in a phylogenetic framework. The rather coherent picture of chromosome number variation underlines the enormous phylogenetic and evolutionary significance of this frequently ignored character.

Caulerpa wysorii sp. nov., a denuded Caulerpa (Chlorophyta) resembling C. sertularioides when ‘dressed’

ABSTRACT Routine DNA barcoding with the chloroplast gene tufA identified novel records of an unknown and denuded (i.e. lacking branchlets) Caulerpa species previously reported from the Florida Middle Grounds. tufA barcoding not only confirmed the matching molecular identity of this taxon with several newly collected denuded specimens from shallow habitats of the Yucatán shelf, but also with a set of plumose specimens previously collected from mesophotic depths of the West Florida shelf and recorded as Caulerpa sertularioides. Sequencing of two additional, faster-evolving DNA markers, i.e. nuclear ITS and the newly tested chloroplast-encoded rpoA, each confirmed conspecificity of the above morphotypes and the molecular separation of the new taxon from C. sertularioides and filiform species found in the region, namely C. cupressoides f. denudata and C. fastigiata. In light of our sequencing results and review of the early taxonomic history of filiform Caulerpa, we propose the description of Caulerpa wysorii sp. nov. for this new species that exhibits two distinct morphotypes in currently available collections, i.e. filiform or plumose assimilators. We also subsume C. tongaensis within the broadly distributed C. fastigiata, whose lectotype is also designated herein. In addition to Caulerpa wysorii sp. nov., we discovered yet another unknown denuded species from the Campeche Banks represented by a single specimen thus far.

Metabarcoding of Soil Fungal Communities Associated with Alpine Field-Grown Saffron (Crocus sativus L.) Inoculated with AM Fungi.

Soil fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological services as decomposers, plant mutualists and pathogens. Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbiotic associations with plant roots and act as biofertilizers by enhancing plant nutrients and water uptake. Information about the AMF association with Crocus sativus L. (saffron) and their impact on crop performances and spice quality has been increasing in recent years. Instead, there is still little data on the biodiversity of soil microbial communities associated with this crop in the Alpine environments. The aims of this study were to investigate the fungal communities of two Alpine experimental sites cultivated with saffron, and to rank the relative impact of two AMF inocula, applied to soil as single species (R = Rhizophagus intraradices, C. Walker & A. Schüßler) or a mixture of two species (M = R. intraradices and Funneliformis mosseae, C. Walker & A. Schüßler), on the resident fungal communities which might be influenced in their diversity and composition. We used Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region to characterize the fungal communities associated to Crocus sativus cultivation in two fields, located in the municipalities of Saint Christophe (SC) and Morgex (MG), (Aosta Valley, Italy), treated or not with AMF inocula and sampled for two consecutive years (Y1; Y2). Data analyses consistently indicated that Basidiomycota were particularly abundant in both sites and sampling years (Y1 and Y2). Significant differences in the distribution of fungal taxa assemblages at phylum and class levels between the two sites were also found. The main compositional differences consisted in significant abundance changes of OTUs belonging to Dothideomycetes and Leotiomycetes (Ascomycota), Agaricomycetes and Tremellomycetes (Basidiomycota), Mortierellomycetes and Mucoromycetes. Further differences concerned OTUs, of other classes, significantly represented only in the first or second year of sampling. Concerning Glomeromycota, the most represented genus was Claroideoglomus always detected in both sites and years. Other AMF genera such as Funneliformis, Septoglomus and Microdominikia, were retrieved only in MG site. Results highlighted that neither sites nor inoculation significantly impacted Alpine saffron-field fungal communities; instead, the year of sampling had the most appreciable influence on the resident communities.

Spore viability and germination of some ectomycorrhizal fungi from New Zealand and implications for forest restoration

ABSTRACT Ectomycorrhizal fungi (EMF) are critical to the establishment of many plant species and an important consideration in ecological restoration of southern beech (Nothofagaceae) forest. However, information on spore viability and storage is lacking for EMF taxa associated with southern beech. This study tested the viability and germination of spores from fruiting bodies collected during autumn and spring from mountain beech forest, Otago, New Zealand. Nuclear ribosomal ITS sequences matched autumn collections to the EMF genera Cortinarius, Inocybe and Russula, but all spring collections were an undescribed hypogeous taxon resembling the native EMF genus Hysterangium, referred to here as Hysterangiaceae ‘Motatapu’. We investigated propidium iodide as a spore viability test, and compared spore germination in water vs glucose, in different temperature and light regimes, and between freshly collected spores and spores from fruiting bodies dried at 30°C or frozen at −20°C. Propidium iodide did not stain all devitalised spores, so may be unreliable for quantifying viability of fresh spores. Spore germination in water was <6% for all genera and <2% for most taxon × treatment combinations. Fresh spores of Hysterangiaceae ‘Motatapu’ suspended in 4°C water showed higher germination than at 20°C; excluding light had no significant effect. Spores of the same taxon suspended in 10% and 20% glucose showed lower germination in comparison to water and showed deformations. Germination percentages of all taxa decreased with storage, but spores of some taxa retained viability for 120 days in 4°C water suspensions. Spores of Hysterangiaceae ‘Motatapu’ showed the least loss of viability with storage of all taxa investigated. For the purposes of storing EMF material for use as inoculum, we recommend drying fruiting bodies at 30°C for storage, then preparing fresh spore suspensions in water when inoculum is required.

A new species of Neoheterobothrium Price, 1943 (Monogenea, Diclidophoridae) from Syacium papillosum (Linnaeus) (Pleuronectiformes, Paralichthyidae) in the Yucatan Shelf, with notes on the validity of the subfamilies in the Diclidophoridae

Neoheterobothrium papillosum n. sp. (Monogenea: Diclidophoridae) is described based on specimens collected from the gills of the dusky flounder Syacium papillosum (Linnaeus) (Paralichthyidae) in the Yucatan Shelf. The new species can be differentiated from other congeneric species by the following characteristics: (1) number of hooks in the genital atrium (8 hooks), (2) number of testes (20-34), (3) pharynx size, (4) peduncle length, and (5) egg size. Neoheterobothrium papillosum n. sp. resembles Neoheterobothrium syacii; however, detailed morphological analysis allowed for their separation into two different species. Sequences of the nuclear ribosomal DNA partial 28S and ITS1 were obtained and compared with available sequences of other species and genera of the family Diclidophoridae from GenBank. A phylogenetic analysis was conducted, including 33 sequences, 18 of which represented 17 species and 11 genera of the family Diclidophoridae. The phylogenetic tree showed that Diclidophoridae is a monophyletic family and species of Diclidophora were grouped as the sister group of Neoheterobothrium papillosum n. sp. Our phylogenetic analyses were consistent with Mamaev's hypothesis, who divided Diclidophoridae into Diclidophorinae and Choricotylinae and concluded that Neoheterobothrium was a genus belonging to Diclidophorinae.

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Gyrocheilos taishanense G.T. Wang, Yu Q. Chen &amp; R.J. Wang (Gesneriaceae) from south China is described and illustrated with photographs. It is morphologically similar to G. microtrichum W.T.Wang, but it is distinguished by its glabrous or sparsely hairy pedicels, zygomorphic calyx with hairs in unequal length, and slightly bilobed adaxial corolla lips. Molecular phylogenetic analysis based on nuclear ITS and plastid trnL-F sequences strongly supported this new species as sister to a clade comprising the previously described Gyrocheilos species (100% posterior probability and bootstrap support). The new species is evaluated here as Least Concern (LC) according to the IUCN Red List Categories and Criteria.

Rediscovery of Mazus lanceifolius reveals a new genus and a new species in Mazaceae.

Mazus lanceifolius (Mazaceae) is a perennial herb with opposite leaves and endemic to central China that has not been collected for 130 years. Rediscovery of this enigmatic species in the wild allows for determination of its phylogenetic position within Mazaceae. Phylogenetic reconstruction of Mazaceae based on DNA sequences from four plastid markers (matK, rbcL, rps16 and trnL-trnF) and nuclear ribosome ITS consistently showed that Mazus was not monophyletic. Mazus lanceifolius is in the most basal clade within Mazaceae, as sister to the remaining species of three recognized genera Dodartia, Lancea and Mazus. These results support the separation of M. lanceifolius from Mazus as a new genus, which was established here as Puchiumazus Bo Li, D.G. Zhang & C.L. Xiang. Meanwhile, a collection from Shennongjia Forestry District of Hubei Province, China, misidentified as “M. lanceifolius” in previous molecular study, is here revealed to represent an undescribed species of Mazus, i.e., M. fruticosus Bo Li, D.G. Zhang & C.L. Xiang, sp. nov. Morphologically, Puchiumazus is clearly distinct from the other three genera by having quadrangular to somewhat ribbed stems, and obviously opposite leaves. In addition, we provide a taxonomic key to the four genera of Mazaceae.

The genetic diversity ofPaeonia anomala(Paeoniaceae), as indicated by nuclear ITS and plastid ycf1 molecular markers

Variability of nuclear ITS rDNA and plastid ycf1 gene was studied to elucidate genetic diversity ofPaeonia anomala(Paeoniaceae), which possesses the most extensive range among all species of the genus. Sixteen populations were sampled across the entire distribution range of the species. The principal coordinate analysis suggested the existence of three ycf1 haplotype groups distributed in distinct geographical locations. For ITS region, no variability throughout the range was found; only samples ofP. anomalafrom its north-western range margin (the Kola Peninsula) are characterized by unique nucleotide substitutions.

Molecular, Morphological, and Spatial Study of Galba schirazensis (Pulmonata, Lymnaeidae) from Southeastern Iran.

Background:Snails of the genus Galba are the intermediate hosts of Fasciola species, the etiological agents of liver fluke disease, fascioliasis. A genetically different but morphologically very similar species in the genus, G. schirazensis, is sympatrically distributed with G. truncatula in some regions of the world. We aimed to investigate the occurrence of G. schirazensis in Kerman province, Iran and to characterize genetically G. schirazensis specimens from southeast Iran.Methods:Field-collected snails from four localities in Jiroft, Bam and Faryab, Kerman province, southeastern Iran were studied. Hydrological variables including temperature and pH were recorded for each habitat. Each specimen was identified using morphological as well as conchological characteristics. Genetic characterization was performed using PCR-sequencing followed by phylogenetic analyses on nuclear ITS2 as well as mitochondrial cox1 gene fragments. MaxEnt software was used to predict the most appropriate ecological niches for the targeted species.Results:G. schirazensis was found in 4 out of 28 locations. One ITS2 and two cox1 haplotypes were detected among G. schirazensis populations from the four localities. Habitat study showed that G. schirazensis thrives in habitats with alkaline pH. G.schirazensis from South America were clustered with specimens from Bam, Kerman, Iran; however, north Iranian isolates of G. schirazensis were strongly correlated with specimens from Jiroft and Faryab. MaxEnt model for the most appropriate ecological niches of the targeted species predicted environmental suitability for this species in western Africa as well as coastal areas in north and southwestern Africa.Conclusion:G. schirazensis is frequently present in southern areas of Kerman Province. At least two genetically different haplotypes are present in southeastern Iran.

Crittendenia gen. nov., a new lichenicolous lineage in the Agaricostilbomycetes (Pucciniomycotina), and a review of the biology, phylogeny and classification of lichenicolous heterobasidiomycetes

AbstractThe lichenicolous ‘heterobasidiomycetes’ belong in the Tremellomycetes (Agaricomycotina) and in the Pucciniomycotina. In this paper, we provide an introduction and review of these lichenicolous taxa, focusing on recent studies and novelties of their classification, phylogeny and evolution. Lichen-inhabiting fungi in the Pucciniomycotina are represented by only a small number of species included in the genera Chionosphaera, Cyphobasidium and Lichenozyma. The phylogenetic position of the lichenicolous representatives of Chionosphaera has, however, never been investigated by molecular methods. Phylogenetic analyses using the nuclear SSU, ITS, and LSU ribosomal DNA markers reveal that the lichenicolous members of Chionosphaera form a monophyletic group in the Pucciniomycotina, distinct from Chionosphaera and outside the Chionosphaeraceae. The new genus Crittendenia is described to accommodate these lichen-inhabiting species. Crittendenia is characterized by minute synnemata-like basidiomata, the presence of clamp connections and aseptate tubular basidia from which 4–7 spores discharge passively, often in groups. Crittendenia, Cyphobasidium and Lichenozyma are the only lichenicolous lineages known so far in the Pucciniomycotina, whereas Chionosphaera does not include any lichenicolous taxa.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI’s nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

English

Abstract The crayfish plague disease agent, Aphanomyces astaci, has coevolved with the native European crayfish since its arrival to mainland Europe in the 1860s. There are indications that some of the A. astaci strains are of reduced virulence, while the resistance against A. astaci infection varies among native European crayfish stocks. In Lake Venesjärvi, Finland, a potential case of latent crayfish plague infection was observed in the early 2000s. We have isolated an oomycete from live Astacus astacus originating from Lake Venesjärvi and identified it as A. a staci of haplogroup A by specific qPCR, nuclear ITS and mitochondrial LSU and SSU sequencies, and named the isolate as UEF_VEN5/14. The A. astaci was isolated from a potentially latently infected A. astacus, subsequently identified, genotyped and proven to be of reduced virulence in a separate infection challenge experiment under laboratory conditions. These findings add to speculations of rather rapid coevolution of A. astaci and European native crayfish and support the reports on latent crayfish plague infections among wild native European crayfish.

1431. Occurrence of Sporadic Human Ascariasis in Non-Endemic Regions: The Importance of Zoonotic Transmission from Swine

BackgroundAscariasis in developed countries occurs only sporadically, and usually in travelers or in children in rural settings with exposure to Ascaris suum from swine. Reciprocal transmission between humans and swine is possible given A. suum and A. lumbricoides are considered conspecific based on published mtDNA and nuclear ribosomal ITS-1 studies with recognized phenotypic/genotypic differences reflecting host-specific adaptive changes. Here we evaluated 15 cases of human ascariasis detected over 6 a year period in a non-endemic region of the Upper Midwest USA.MethodsHelminth specimens (n=15) spontaneously passed per rectum were submitted for laboratory identification during 2013-19 and identified morphologically as A. lumbricoides/suum (undifferentiated). All patients attended local clinics and brought specimens in for identification. Clinical records were available for 13 patients.ResultsAges ranged from 14 months to 41 years with 13 cases (87%) occurring in children < =12 years and 2 (13%) >30 years; 9 patients (60%) were female. Thirteen (87%) of the A. lumbricoides/suum specimens were adults and 2 (13%) were juveniles. Individuals with records available either lived on or had visited a farm (5) or hobby farm (2) where pigs were currently or likely historically present; lived at a rural address (4); used animal manure for gardening (1); or lacked discernable farm connections though was active outdoors (1). International travel history was lacking in all cases. One 2-year old child from a rural address had passed 2 worms 6 months apart. All 13 patients were treated with albendazole per guideline without complication.ConclusionAscariasis attributable to poor sanitation has been largely eradicated from the USA since the early 1980s. Sporadic infections in non-travelers have continued to be recognized and likely represent zoonotic transmission from domesticated swine. While human and pig Ascaris have long been considered distinct species, recently published molecular and cross-transmission experiments point to conspecificity. This case series is a reminder of the zoonotic disease risks posed by swine-origin Ascaris, especially in young children, and reinforces the need for proper herd management and attention to personal hygiene for at-risk individuals.Disclosures All Authors: No reported disclosures

Integrative Description of Pseudechiniscus lalitae sp. nov. (Tardigrada: Heterotardigrada: Echiniscidae) from the Azores Archipelago (Portugal)

We applied an integrative taxonomy approach to describe a new tardigrade species from Pico Island (the Azores Archipelago). Analysis was based on morphological and morphometrical data (using both light and scanning electron microscopy) and combined with multilocus molecular data (nuclear barcode sequence ITS-2 and mitochondrial COI sequence). Molecular data allowed us to determine that genetically (based on COI sequences) Pseudechiniscus lalitaesp. nov. is most similar to Pse. aff. facettalis and the least similar to Pse. indistinctus. The new species differs morphologically from other Pseudechiniscus species, mostly, by the ventral sculpture. Moreover, it differs from other species from suillus-facettalis complex also by some other more subtle morphological and morphometrical characters.

New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa.

Sixteen species of Latrunculiidae Topsent, 1922, belonging to the genera Latrunculia du Bocage, 1869, Strongylodesma Lévi, 1969, Cyclacanthia Samaai Kelly, 2004, Samaai Kelly, 2002, are currently known from the temperate waters of South Africa. Extensive new sponge collections from the Amathole region of South Africa revealed the existence of three new species of Tsitsikamma, T. amatholensis sp. nov., T. madiba sp. nov., and T. beukesi sp. nov., and a new species of the endemic South African genus Cyclacanthia, C. rethahofmeyri sp. nov. With the recent addition of two new species of Tsitsikamma from Algoa Bay and Tsitsikamma National Park (T. michaeli Parker-Nance, 2019; T. nguni Parker-Nance, 2019) the total number of known South African Latrunculiidae is now 20 species in four genera. Here we propose two new subgenera of Tsitsikamma, Tsitsikamma Samaai Kelly, 2002 and Clavicaulis subgen. nov., based on the morphological groups "favus" and "pedunculata" hypothesized by Parker-Nance et al. (2019). Species in the nominotypical subgenus Tsitsikamma, containing the type species, are thick encrusting to hemispherical with a rigid honeycombed choanosome, while species in the new subgenus Clavicaulis subgen. nov. have a purse or sac-like morphology with little choanosomal structure. Despite the obvious species-level differences in morphology, multivariate analysis based on spicule measurements (anisostyle length, discorhabd length, shaft and whorl length) was not able to distinguish between the proposed Tsitsikamma species, but separated known species T. favus Samaai Kelly, 2002, T. pedunculata Samaai Kelly, 2003, and T. scurra Samaai Kelly, 2003, from each other. Similarly, DNA barcoding of the mitochondrial COI and the nuclear ITS of Tsitsikamma specimens failed to clearly differentiate between species, but was able to differentiate sister taxon relationships within the Latrunculiidae.

Molecular Phylogeny and Phylogeography of Potentilla multifida L. agg. (Rosaceae) in Northern Eurasia with Special Focus on Two Rare and Critically Endangered Endemic Species, P. volgarica and P. eversmanniana.

The results of a molecular genetic study of Potentilla multifida agg. using two plastid markers (ndhC-trnV and psbA-trnH) and a nuclear ITS marker suggested that this group comprises a number of relatively young and incompletely differentiated species widely distributed in Northern Eurasia. The sequences were analyzed using tree-based (maximum likelihood) and network-based (statistical parsimony network) approaches. The plastid data suggested incomplete lineage sorting, characteristic of the group as a whole. The nuclear ITS results demonstrated quite a different pattern, with mostly conspecific accessions shaping monophyletic clades. The majority of the Potentilla sect. Multifidae species studied possess few, usually closely related plastid haplotypes, or are even monomorphic. In contrast, P. volgarica, a narrow endemic from the Volga River valley, presents plastid haplotypes belonging to two distantly related groups. Such a pattern of genetic diversity in P. volgarica may be explained by a long persistence of the species within an extremely small distribution range, on the right bank of the Volga River, most likely representing a contemporary refugium. The genealogy of plastid markers in P. volgarica suggests that this species is ancestral to P. eversmanniana, another narrow endemic from the S Urals.

A phylogeny of Emilia (Senecioneae, Asteraceae) – implications for generic and sectional circumscription

AbstractEmilia is a widely distributed, mainly African, palaeotropical genus in the tribe Senecioneae (Asteraceae). It comprises 117 species, most of which are annual herbs. Bayesian and parsimony phylogenetic analyses were performed on 51 Emilia species along with closely related genera in the Senecioneae using nuclear ITS and plastid trnL‐trnF sequence data to address questions around the generic circumscription of Emilia, including the status of the similar genera Emiliella and Bafutia, assess Jeffrey's sectional classification of Emilia, and evaluate the distinctness of the morphologically similar species in the large‐headed Emilia coccinea complex. Both nuclear and plastid phylogenies reveal Emilia to be paraphyletic and polyphyletic, with Bafutia and Emiliella nested within Emilia, and Jeffrey's sectional classification is not supported. The phylogenies provide additional evidence that Emilia coccinea and E. caespitosa should be synonymised, as shown by published phenetic cluster and multivariate analyses. The necessary taxonomic changes are made in this manuscript.

Plant DNA Barcoding Principles and Limits: A Case Study in the Genus Vanilla.

Powerful DNA barcodes have been much more difficult to define in plants than in animals. In 2009, the international Consortium for the Barcoding Of Life (CBOL) chose the combination of the chloroplast genes (rbcL + matK) as the proposed official barcode for plants. However, this system has got important limits. First, any barcode system will only be useful if there is a clear barcode gap and if species are monophyletic. Second, chloroplast and mitochondrial (COI gene used for animals) barcodes will not be usable for discriminating hybrid species. Moreover, it was also shown that, using chloroplast regions, maximum species discrimination would be around 70% and very variable among plant groups. This is why many authors have more recently advocated for the addition of the nuclear ITS region to this barcode because it reveals more variations and allows the resolution of hybrid or closely related species. We tested different chloroplast genes (rbcL, matK, psaB, psbC) and the nuclear ITS region in the genus Vanilla, a taxonomically complex group and therefore a good model to test for the efficiency of different barcode systems. We found that the CBOL official barcode system performed relatively poorly in Vanilla (76% species discrimination), and we demonstrate that adding ITS to this barcode system allows to increase resolution (for closely related species and to the subspecies level) and to identify hybrid species. The best species discrimination attained was 96.2% because of one paraphyletic species that could not be resolved.

Molecular Identification and Phylogenetic Placement of Rosa arabica Crép. (Rosaceae), a Critically Endangered Plant Species.

The Egyptian narrowly endemic and critically endangered plant species Rosa arabica Crép. was studied employing a taxonomic and molecular approach. Morphological investigations, distance analysis, and phylogenetic reconstruction revealed that R. arabica is a distinct species with great affinity to R. canina and differentiated from R. rubiginosa. Molecular identification based on the sequences of multiple markers single or in combination ITS, matK, rbcL, and trnL-F succeeded in identifying R. arabica at genus and species levels. We evaluated the potential of each marker and a combination of the nuclear ITS -Internal Transcribed Spacer- with one of the plastid markers, matK, rbcL, or trnL-F, to accurately identify Rosa species. All of them were successful in identifying R. arabica. Classification based on DNA sequences shows that R. arabica is placed within section Caninae in a clade comprising R. canina and R. rubiginosa. Moreover, R. arabica is closely related to other European Rosa species. In conclusion, our results indicate that the four DNA markers can provide species resolution in the context of the genus Rosa and relatives, aiming to characterize morphology and genetic diversity in the ecological and economically important genus Rosa.