Discovery of <Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> on <Emphasis Type="Italic">Eucalyptus</Emphasis> wood chips in China

Ophiostoma species such as O. quercus are the most frequent causal agents of sapstain of freshly felled hardwood timber and pulpwood. Many species are regarded as economically important agents of wood degradation. The aim of this study was to identify a collection of Ophiostoma isolates, resembling O. quercus, found on stained Eucalyptus pulpwood chips in China. DNA sequences of the internal transcribed spacer regions, including the 5.8S region, of the ribosomal DNA, and parts of the β-tubulin and elongation factor-1α genes, revealed that the isolates were not O. quercus. Surprisingly, they represented O. tsotsi, a wound-infesting fungus recently described from hardwoods in Africa. In addition, sequence data from an isolate from agarwood in Vietnam, identified in a previous study as belonging to an unknown Pesotum species, were also shown to represent O. tsotsi. A high level of genetic variability was observed among isolates of both O. quercus and O. tsotsi. This was unexpected and suggests that both species have been present in Asia for a significant amount of time.     

A new <Emphasis Type="Italic">Ophiostoma</Emphasis> species from loblolly pine roots in the southeastern United States

During the course of a survey of fungi in loblolly pine (Pinus taeda) roots in Georgia, USA, a species of Ophiostoma morphologically similar to O. pluriannulatum, was isolated. Morphological characteristics and DNA sequence comparisons were used to identify the fungus. The isolates produced perithecia with unusually long necks similar to those of O. pluriannulatum but they had few or no annuli. DNA sequences for the ribosomal internal transcribed spacer regions 1 and 2 were identical to those of O. pluriannulatum. Sequence data of the β-tubulin gene region revealed the absence of intron 4 and presence of intron 5, distinguishing the isolates from O. pluriannulatum, which has intron 4 but not intron 5. Phylogenetic analyses of the β-tubulin sequences showed that the isolates from loblolly pine roots grouped together in a lineage distinct from O. multiannulatum and O. subannulatum, both of which lack intron 4 and have intron 5. The fungus is consequently described as O. sparsiannulatum sp. nov., a novel taxon in the O. pluriannulatum complex.     

A diverse assemblage of <Emphasis Type="Italic">Ophiostoma</Emphasis> species,including two new taxa on eucalypt trees in South Africa

Fungi in the Ophiostomatales include important pathogens of trees as well as agents of wood stain, reducing the economic value of timber. They rely on insects, such as bark beetles, for dispersal and are commonly associated with wounds on trees. Although Ophiostoma spp. have been reported from eucalypt wood chips in South Africa, very little is known about the diversity of the Ophiostomatales, or their insect associates, on plantation-grown Eucalyptus spp. The aim of this study was to consider the diversity and distribution of the Ophiostomatales infecting fresh wounds on Eucalyptus trees in the country. Additionally, knowledge regarding their association with nitidulid beetles, which have previously been shown to carry Ophiostoma spp., was sought. Surveys were conducted in five provinces where Eucalyptus spp. are commonly grown, and the fungi collected were identified using morphological comparisons and multigene sequence phylogenies. Of the 139 isolates collected, five Ophiostoma spp. were identified including O. quercus, O. tsotsi and O. tasmaniense. These were from cut stumps as well as from the nitidulid beetles Brachypeplus depressus and Carpophilus spp. In addition, two new taxa in the O. stenoceras–Sporothrix schenckii complex were identified from Eucalyptus trees infested by Phoracantha semipunctata. The two new taxa are described as O. candidum sp. nov., and O. fumeum sp. nov., respectively. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on Eucalyptus trees in South Africa is poorly understood and that further studies are required to determine the possible economic relevance of these fungi.     

Genetic diversity and domestication history of African rice (<Emphasis Type="Italic">Oryza glaberrima</Emphasis>) as inferred from multiple gene sequences

Nucleotide variation in 14 unlinked nuclear genes was investigated in species-wide samples of African rice (Oryza glaberrima) and its wild progenitor (O. barthii). Average estimates of nucleotide diversity were extremely low in both species (θ _sil = 0.0007 for O. glaberrima; θ _sil = 0.0024 for O. barthii). About 70% less diversity was found in O. glaberrima than in its progenitor O. barthii. Coalescent simulation indicated that such dramatic reduction of nucleotide diversity in African rice could be explained mainly by a severe bottleneck during its domestication. The progenitor of African rice maintained also low genetic diversity, which may be attributed to small effective population size in O. barthii. Self-pollinating would be another factor leading to the unusually low diversity in both species. Genealogical analyses showed that all O. glaberrima accessions formed a strongly supported cluster with seven O. barthii individuals that were sampled exclusively from the proposed domestication centers of African rice. Population structure and principal component analyses found that the O. glaberrima group was homogeneous with no obvious genetic subdivision, in contrast to the heterogeneous O. barthii cluster. These findings support a single domestication origin of African rice in areas of the Upper Niger and Sahelian Rivers.     

Phylogeny and species delimitation of the C‐genome diploid species in Oryza

Abstract The diploid Oryza species with C‐genome type possesses abundant genes useful for rice improvement and provides parental donors of many tetraploid species with the C‐genome (BBCC, CCDD). Despite extensive studies, the phylogenetic relationship among the C‐genome species and the taxonomic status of some taxa remain controversial. In this study, we reconstructed the phylogeny of three diploid species with C‐genome (Oryza officinalis, O. rhizomatis, and O. eichingeri) based on sequences of 68 nuclear single‐copy genes. We obtained a fully resolved phylogenetic tree, clearly indicating the sister relationship of O. officinalis and O. rhizomatis, with O. eichingeri being the more divergent lineage. Incongruent phylogenies of the C‐genome species found in previous studies might result from lineage sorting, introgression/hybridization and limited number of genetic markers used. We further applied a recently developed Bayesian species delimitation method to investigate the species status of the Sri Lankan and African O. eichingeri. Analyses of two datasets (68 genes with a single sample, and 10 genes with multiple samples) support the distinct species status of the Sri Lankan and African O. eichingeri. In addition, we evaluated the impact of the number of sampled individuals and loci on species delimitation. Our simulation suggests that sampling multiple individuals is critically important for species delimitation, particularly for closely related species.     

A reappraisal of the systematic affinities of Socotran,Arabian and East African scops owls (Otus,Strigidae) using a combination of molecular,biometric and acoustic data

We investigated phylogenetic relationships among Otus scops owls from Socotra Island, the Arabian Peninsula and East Africa using molecular, vocalization and biometric data. The Socotra Scops Owl Otus senegalensis socotranus, currently treated as a subspecies of the African Scops Owl Otus senegalensis, is more closely related to the Oriental Scops Owl Otus sunia and to the endemic Seychelles Scops Owl Otus insularis. Considerable mitochondrial genetic distance and significant morphological differentiation from its two closest relatives, as well as its distinctive vocalizations compared with O. insularis, strongly support recognition of Socotra Scops Owl as a full species. Unexpectedly, two taxa from the Arabian Peninsula, Pallid Scops Owl Otus brucei and African Scops Owl Otus senegalensis pamelae, represent very distinct lineages; O. brucei is basal to a clade that includes taxa found in the Indo‐Malayan region and on Indian Ocean islands. In contrast, O. s. pamelae occupies a well‐supported basal position within a clade of continental Afro‐Palaearctic taxa. The uncorrected‐p genetic distance between O. s. pamelae and its closest relatives (other populations of senegalensis from mainland Africa) is c. 4%. As O. s. pamelae is also well differentiated phylogenetically, morphologically and vocally from O. s. senegalensis, we recommend its elevation to species status, as Otus pamelae. Among mainland African O. senegalensis subspecies, Ethiopian populations appear to represent the most divergent lineage, whereas other lineages from Somalia, Kenya and South Africa are poorly differentiated. The large genetic distance between the Ethiopian haplotype and other African haplotypes (3.2%) suggests that the Ethiopian Otus may represent a cryptic taxon, and we recommend that more individuals be sampled to assess the taxonomic status of this population.     

Genetic variation of <Emphasis Type="Italic">Phoma sorghina</Emphasis> isolates from Southern Africa and Texas

Genetic variability of Phoma sorghina, a ubiquitous facultative phytopathogen, was investigated on 41 isolates cultivated from surface-sterilized sorghum grains originating from South Africa and Texas; pearl millet isolates from Namibia were also included. Most of the isolates from Texas produced intense red pigments, especially on Czapek-Dox agar plates. Many African isolates formed conspicuous dark radial substrate hyphae with intercalated chlamydospores on oatmeal plates. Conidial dimensions and shape were very variable (mean lengths 4.5–5.7 μm). Haplotypes were defined based on 53 markers from banding patterns obtained with rep-PCR (primers: M13core, ERIC IR). The shared geographic origin was partially reflected in the clades of the haplotype phylogram. The values of G _ST were intermediate; 16–37 % of the variation was found between the populations. Nm values of gene flow were 0.84–1.15. Average gene diversity H _E was moderate (0.256). Sequences of ITS-rDNA were obtained from 21 isolates. Allele 1 was found in 9 isolates scattered throughout the clades, allele 2 occurred in 6 isolates (5 of them from the same clade), alleles 3 and 4 were shared by two isolates each and two isolates were unique. Alleles 1 and 2 were also found among highly related sequences from GenBank. All shared an 8-bp deletion near the 5′ end of ITS2 that was not found in any other Phoma/Didymella species and which may be a typical marker for P. sorghina. Among related species, members of legume-associated Ascochyta/Didymella complex, Epicoccum spp., D. applanata and P. glomerata were found.     

Pliocene isolation of a north‐west Saharan cichlid fish

The evolutionary origin of the only north‐west Saharan haplochromine cichlid, Astatotilapia desfontainii, was explored using mitochondrial DNA sequences. Phylogenetic analyses revealed that this species belonged to the main East African–Nilotic haplochromine clade, but was a distinct lineage that diverged from modern haplochromines in the Pliocene. The results suggest that A. desfontainii is a relict haplochromine lineage that has endured major climate fluctuations in North Africa.     

Olive genetic diversity assessed using amplified fragment length polymorphisms

 Amplified fragment length polymorphism (AFLP) analysis was used to study the genetic variation within and among populations of genus Olea. A group of genotypes, all of them cultivated varieties of a single species, Olea europaea, was compared with wild olives and with a group of individuals belonging to different Olea species. Five primer combinations were used which produced about 290 polymorphic bands. The data obtained were elaborated with the Nei’s genetic similarity coefficient, applying different clustering methods and the Principal Coordinate Analysis. Cultivars, wild olives and North-West African species formed groups clustering together at a similarity level of 0.56, while the Olea species from East Africa and Asia grouped separately. Species from the Indian Ocean and Australia showed the highest diversity. We hypothesize that cultivars and wild plants are different forms of the same O. europaea species. The Olea from East Africa and Asia may be assigned to a different species, while the role of O. laperrini as well as that of O. maroccana as an intermediary form is confirmed. Received: 30 April 1998 / Accepted: 13 August 1998     

Genetic differentiation of the African dwarf crocodile Osteolaemus tetraspis Cope, 1861 (Crocodylia: Crocodylidae) and consequences for European zoos

The endangered African dwarf crocodile Osteolaemus tetraspis is distributed in Central and Western Africa. Conventionally, two subspecies were distinguished: Osteolaemus tetraspis tetraspis and Osteolaemus tetraspis osborni. The taxonomic significance of diagnostic morphological characters is still being discussed and the existence of additional species in the Osteolaemus group remains unclear. Recent molecular studies suggest the existence of three allopatric species in the genus Osteolaemus. These results supported a division of the dwarf crocodile into a Congo Basin form (O. osborni), an Ogooué Basin form (O. tetraspis), and a third separate evolutionary lineage from Western Africa. Several European zoos host African dwarf crocodiles. For reasons of conservation and possible reintroduction, it is important to clarify provenance of these zoo animals. Therefore, we conducted molecular and phylogenetic analyses of three mitochondrial and two nuclear gene sequences with all available samples from European zoos and museums. We also estimated the origin of the zoo animals by comparing sequences of wild animals and museum samples of known provenance. Our study strongly supports three distinct lineages of Osteolaemus as recently postulated, but also reveals a fourth evolutionary lineage. We demonstrate that, of the European zoo animals sampled, only one dwarf crocodile corresponds to the Congo Basin form (O. osborni) whereas the majority of individuals correspond to the three other forms. Four zoo animals belong to the new fourth group; but their provenance is still unresolved. The origin of these animals is probably located in an African region from which no wild animal samples are currently available. Further investigations and sampling of other regions should be completed to clarify the identity of this fourth lineage. We found potential hybrids from European zoological gardens using nuclear DNA sequences. The European Studbook will use these results for further breeding programmes to keep genetically suitable ex-situ populations as reassurance colonies for prospective reintroduction into African countries.     

Morphological and phylogenetic analyses of Pythium species in South Africa

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363–1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.     

Fungal associates of the lodgepole pine beetle, <Emphasis Type="Italic">Dendroctonus murrayanae</Emphasis>

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74–100% of all beetles) followed closely by Ophiostoma abietinum (29–75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0–13%), Ophiostoma ips (0–15%), Ophiostoma piliferum (0–11%), a Pesotum sp. (0–11%) and Ophiostoma floccosum (0–1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.     

Salmo macrostigma (Teleostei,Salmonidae): Nothing more than a brown trout (S. trutta) lineage?

We examined specimens of the macrostigma trout Salmo macrostigma, which refers to big black spots on the flanks, to assess whether it is an example of taxonomic inflation within the brown trout Salmo trutta complex. Using new specimens, publicly available data and a mitogenomic protocol to amplify the control and cytochrome b regions of the mitochondrial genome from degraded museum samples, including one syntype specimen, the present study shows that the macrostigma trout is not a valid species. Our results suggest the occurrence of a distinct evolutionary lineage of S. trutta in North Africa and Sicily. The name of the North African lineage is proposed for this lineage, which was found to be sister to the Atlantic lineage of brown trout, S. trutta.     

Redheadia quercus gen. et sp. nov., the teleomorph of Mycopappus quercus, the frosty mildew fungus in Quercus acutissima

The teleomorph of Mycopappus quercus causing frosty mildew in Quercus acutissima is described as a new genus and species, Redheadia quercus, in the Sclerotiniaceae. Apothecia sprout from sclerotia on the fallen infected leaves kept for 10 months at 5°C and subsequent incubation at 15°C under diffused room light. Typical zonate lesions and multicellular propagules of M. quercus are produced on Q. acutissima, by mycelial inoculation using an isolate from a single ascospore, confirming the teleomorph–anamorphic connection. No significant differences are observed between cultured colonies of isolates from the ascospore and those from the propagule. Sclerotia and microconidia of the fungus are produced on culture media.     

<Emphasis Type="Italic">Ochroconis calidifluminalis</Emphasis>, a Sibling of the Neurotropic Pathogen <Emphasis Type="Italic">O. gallopava</Emphasis>, Isolated from Hot Spring

Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42°C, while the maximum growth temperature was 49°C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.     

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid

We isolated Rhizoctonia-like fungi from populations of the threatened orchid Cypripedium macranthos. In ultrastructural observations of the septa, the isolates had a flattened imperforate parenthesome consisting of two electron-dense membranes bordered by an internal electron-lucent zone, identical to the septal ultrastructure of Rhizoctonia repens (teleomorph Tulasnella), a mycorrhizal fungus of many orchid species. However, hyphae of the isolates did not fuse with those of known tester strains of R. repens and grew less than half as fast as those of R. repens. In phylogenetic analyses, sequences for rDNA and internal transcribed spacer (ITS) regions of the isolates were distinct from those of the taxonomically identified species of Tulasnella. On the basis of the ITS sequences, the isolates clustered into two groups that corresponded exactly with the clades demonstrated for other Cypripedium spp. from Eurasia and North America despite the geographical separation, suggesting high specificity in the Cypripedium–fungus association. In addition, the two phylogenetic groups corresponded to two different plant clones at different developmental stages. The fungi from one clone constituted one group and did not belong to the other fungal group isolated from the other clone. The possibility of switching to a new mycorrhizal partner during the orchid’s lifetime is discussed.     

Chloroplast phylogeography of threatened aquatic <Emphasis Type="Italic">Oxalis</Emphasis> (Oxalidaceae): significant inter-population structure,divergent haplotypes and conservation implications

The aquatic lineage consisting of the sister taxa Oxalis dines and O. disticha is confined to a few small vernal pools in the semi-arid Greater Cape Floristic Region of South Africa. All known populations are at risk of extinction due to anthropogenically induced disturbance. To identify priority sites for focused conservation management, the chloroplast intergenic spacer regions psbA-trnH and trnS-trnG were used to determine population structure and genetic diversity in this lineage across its distribution range. Population viability was assessed using flower morph ratios as surrogate for sexual health. Fourteen and four haplotypes were identified from O. disticha and O. dines populations, respectively. Analyses of Molecular Variance indicated an extremely high level of interpopulation differentiation across the entire aquatic lineage as well as within O. disticha and O. dines. Fifty percent of the eighteen haplotypes were confined to single pools, and 84% of populations contained only a single haplotype, even though pool interspacing distance for O. disticha was often less than 5 km. Almost half of O. disticha haplotype diversity was restricted to very small populations. Two O. dines haplotypes were restricted to small populations, with one of these presenting a divergent haplotype sister to the remainder of the aquatic lineage. Flower morph frequency ratio analyses suggested that most populations were reproductively healthy. Low haplotypic diversity within local populations and differentiation between populations are consistent with very low seed-level gene flow and sporadic founder effects. Conservation efforts should be focussed on preserving as many pools as possible with small, genetically distinct populations representing a main concern.     

Phylogeography and genetic relationships of North African <Emphasis Type="Italic">Bufo mauritanicus</Emphasis> Schlegel, 1841 estimated from mitochondrial DNA sequences

Genetic variation within the North African toad Bufo mauritanicus was estimated by sequencing partial 12S rRNA and 16S rRNA mitochondrial regions from widespread populations in Morocco, Algeria and Tunisia. Unlike many other wide ranging species from this area, B. mauritanicus demonstrated very low levels of intraspecific variation. The minimal intraspecific genetic variation may be due to a relatively recent, possibly post-glacial, expansion into its current range. Further phylogeographic studies of other North African species are needed to assess if this is a common biogeographical phenomenon. Phylogenetic analyses support immunological data that B. mauritanicus is part of a clade of predominantly sub-Saharan Bufo, recently assigned to a new genus Amietophrynus. Two different lineages within this clade, B. mauritanicus and the B. pardalis group, appear to have reverted from 20 chromosomes to the more typical 22 chromosomes found in most other Bufonids. However, the alternative hypothesis that the Bufo species with 20 chromosomes form a monophyletic lineage cannot be rejected.