Development and characterization of 24 chloroplast microsatellite markers for two species of <Emphasis Type="Italic">Eranthis</Emphasis> (Ranunculaceae)

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

Molecular Systematic Study of <Emphasis Type="Italic">Chrysosplenium</Emphasis> Series <Emphasis Type="Italic">Pilosa</Emphasis> (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.     

Species delimitation tests of endemic <Emphasis Type="Italic">Lepidium papilliferum</Emphasis> and identification of other possible evolutionarily significant units in the <Emphasis Type="Italic">Lepidium montanum</Emphasis> complex (Brassicaceae) of western North America

Lepidium papilliferum of southwest Idaho was previously treated as an infraspecific variety of Lepidium montanum. Chloroplast (cpDNA) sequences, nuclear ribosomal internal transcribed spacer (ITS) sequences, and AFLPs were used to test species delimitations and other possible evolutionarily significant units (ESU) based on genetic differentiation, isolation by distance (IBD), and genetic admixture among 32 L. montanum and 21 L. papilliferum collections from the western US. The L. papilliferum AFLP genotypes formed a monophyletic clade. However, the AFLP genotypes of L. montanum samples from eight western sites were more similar to L. papilliferum, which together comprise a regionally significant West clade showing significant differentiation from eastern L. montanum collections (East clade). Bayesian analysis of AFLP genotypes detected possible admixture between L. papilliferum and related western L. montanum collections. Neither taxa nor regionally significant AFLP clades displayed reciprocally monophyletic cpDNA or ITS sequences, but the AFLP clades showed stronger cpDNA differentiation and unique ITS alleles. The East and West clades fit models of speciation with relatively strong IBD within groups and weak IBD between groups, based on correlations between the average number of AFLP differences and geographic distances among collection sites, but comparisons between taxa did not fit this model. Conversely, relatively strong partial correlations between AFLP and taxonomic differences, controlling for geography, support taxonomic delimitations. Results suggest that L. papilliferum is a distinct subgroup of L. montanum influenced by speciation. However, gene flow or common ancestry between L. papilliferum and western forms of L. montanum provide a basis for other possible ESUs.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Phylogeny and taxonomy of <Emphasis Type="Italic">Cladosporium</Emphasis>-like hyphomycetes,including <Emphasis Type="Italic">Davidiella</Emphasis> gen. nov., the teleomorph of <Emphasis Type="Italic">Cladosporium s. str.</Emphasis>

A phylogenetic study employing sequence data from the internal transcribed spacers (ITS1, ITS2) and 5.8S gene, as well as the 18S rRNA gene of various Cladosporium-like hyphomycetes revealed Cladosporium s. lat. to be heterogeneous. The genus Cladosporium s. str. was shown to represent a sister clade to Mycosphaerella s. str., for which the teleomorph genus Davidiella is proposed. The morphology, phylogeny and taxonomy of the cladosporioid fungi are discussed on the basis of this phylogeny, which consists of several clades representing Cladosporium-like genera. Cladosporium is confined to Davidiella (Mycosphaerellaceae) anamorphs with coronate conidiogenous loci and conidial hila. Pseudocladosporium is confined to anamorphs of Caproventuria (Venturiaceae). Cladosporium-like anamorphs of the Venturia (conidia catenate) are referred to Fusicladium. Human-pathogenic Cladosporium species belong in Cladophialophora (Capronia, Herpotrichiellaceae) and Cladosporium fulvum is representative of the Mycosphaerella/Passalora clade (Mycosphaerellaceae). Cladosporium malorum proved to provide the correct epithet for Pseudocladosporium kellermanianum (syn. Phaeoramularia kellermaniana, Cladophialophora kellermaniana) as well as Cladosporium porophorum. Based on differences in conidiogenesis and the structure of the conidiogenous loci, further supported by molecular data, C. malorum is allocated to Alternaria.     

Phylogenetic relationships among subgenera,species, and varieties of Japanese <Emphasis Type="Italic">Salvia</Emphasis> L. (Lamiaceae)

To determine evolutionary relationships among all Japanese members of the genus Salvia (Lamiaceae), we conducted molecular phylogenetic analyses of two chloroplast DNA (cpDNA) regions (rbcL and the intergenic spacer region of trnL−trnF:trnL−trnF) and one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS). In cpDNA, nrDNA, and cpDNA+nrDNA trees, we found evidence that all Japanese and two Taiwanese Salvia species are included in a clade with other Asian Salvia, and Japanese Salvia species were distributed among three subclades: (1) S. plebeia (subgenus Sclarea), (2) species belonging to subg. Salvia, and (3) species belonging to subg. Allagospadonopsis. At the specific level our findings suggest: a close relationship between S. nipponica and S. glabrescens, no support for monophyly of S. lutescens and its varieties in cpDNA, nrDNA and cpDNA+nrDNA trees, and that S. pygmaea var. simplicior may be more closely related to S. japonica than to other varieties of S. pygmaea.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Maternal origin,genome constitution and evolutionary relationships of polyploid <Emphasis Type="Italic">Elymus</Emphasis> species and <Emphasis Type="Italic">Hordelymus europaeus</Emphasis>

The trnS/psbC region of chloroplast DNA (cpDNA) was sequenced for 18 Elymus polyploid species, Hordelymus europaeus and their putative diploid ancestors. The objective was to determine the maternal origin and evolutionary relationships of these polyploid taxa. Phylogenetic analysis showed that Elymus and Pseudoroegneria species formed a highly supported monophyletic group (100 % bootstrap values), suggesting that Pseudoroegneria is the maternal genome donor to polyploid Elymus species studied here. The phylogenetic tree based on cpDNA sequence data indicates that E. submuticus contains a St-genome. Taking into consideration of our previously published RPB2 data, we can conclude that hexaploid E. submuticus contains at least one copy of St and Y genomes. Our Neighor-joining analysis of cpDNA data put Psathyrostachys juncea, Hordeum bogdanii and Hordelymus europaeus into one group, suggesting a close relationship among them.     

The unique genome of two-chromosome grasses <Emphasis Type="Italic">Zingeria</Emphasis> and <Emphasis Type="Italic">Colpodium</Emphasis>, its origin,and evolution

Chromosome C-banding and two-color fluorescent in situ hybridization (FISH) were used to compare the chromosomes, to identify the chromosomal localization of the 45S and 5S rRNA genes, and to analyze the sequences of internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the 45S rRNA genes in the genomes of grasses Zingeria biebersteiniana (2n = 4), Z. pisidica, Z. trichopoda (2n = 8), Colpodium versicolor (2n = 4), and Catabrosella variegata (syn. Colpodium variegatum) (2 n = 10). Differences in C-banding pattern were observed for two Z. biebersteiniana accessions from different localities. Similar C-banding patterns of chromosomes 1 and 2 were demonstrated for the Z. pisidica and Z. biebersteininana karyotypes. Chromosome C banding and localization of the 45S and 5S rRNA genes on the chromosomes of the two Zingeria species confirmed the assumption that Z. pisidica is an allotetraploid with one of the subgenomes similar to the Z. biebersteiniana genome. ITS comparisons showed that the unique two-chromosome grasses (x = 2)—Z. biebersteiniana (2n = 4), Z. trichopoda (2n = 8), Z. pisidica (2n = 8), and C. versicolor (2n = 4), which were earlier assigned to different tribes of subtribes of the family Poaceae—represent two closely related genera, the genetic distance (p-distance) between their ITSs being only 1.2–4.4%. The Zingeria species and C. versicolor formed a common clade with Catabrosella araratica (2n = 42, x = 7) on a molecular phylogenetic tree. Thus, the karyotypes of Zingeria and Colpodium, which have the lowest known basic chromosome number (x = 2), proved to be monophyletic, rather than originating from different phylogenetic lineages.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Two new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from Yunnan,China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.     

Incongruence among mitochondrial,chloroplast and nuclear gene trees in <Emphasis Type="Italic">Pinus</Emphasis> subgenus <Emphasis Type="Italic">Strobus</Emphasis> (Pinaceae)

Introgression has been considered to be one of main factors leading to phylogenetic incongruence among different datasets at lower taxonomic levels. In the plants of Pinaceae, the mtDNA, cpDNA, and nuclear DNA (nrDNA) may have different evolutionary histories through introgression because they are inherited maternally, paternally and biparentally, respectively. We compared mtDNA, cpDNA, and two low-copy nrDNA phylogenetic trees in the genus Pinus subgenus Strobus, in order to detect unknown past introgression events in this group. nrDNA trees were mostly congruent with the cpDNA tree, and supported the recent sectional and subsectional classification system. In contrast, mtDNA trees split the members of sect. Quinquefoliae into two groups that were not observed in the other gene trees. The factors constituting incongruence may be divided into the following two categories: the different splits within subsect. Strobus, and the non-monophyly of subsect. Gerardianae. The former was hypothesized to have been caused by the past introgression of cpDNA, mtDNA or both between Eurasian and North American species through Beringia. The latter was likely caused by the chimeric structure of the mtDNA sequence of P. bungeana, which might have originated through past hybridization, or through a horizontal transfer event and subsequent recombination. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Olea europaea</Emphasis> (Oleaceae) phylogeography based on chloroplast DNA polymorphism

Chloroplast DNA diversity in the olive (Olea europaea L.) complex was studied using PCR-RFLP and microsatellite markers. Fifteen chlorotypes were distinguished. We constructed a cpDNA phylogenetic tree in which five clades were recognised and located in distinct geographic areas: clade A in Central and Southern Africa, clade C in Asia, clade M in North-West Africa, clade E1 in the Mediterranean Basin and Sahara, and clade E2 in West Mediterranea. Cultivated olive clustered with Mediterranean and Saharan wild forms (clades E1 and E2). Strong genetic differentiation for cpDNA markers was observed between eastern and western Mediterranean olives, suggesting that these areas have represented different glacial refugia. Humans most likely spread one eastern chlorotype, preponderant in cultivars, across the western Mediterranean Basin. Its presence in O. e. subsp. laperrinei from the Sahara suggests a possible Mediterranean olive origin in an African population, which may have overlapped in the Southern Mediterranean during the Quaternary.     

Morphology and phylogeny of <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis> causing fruit rot of olives

The taxonomic position of the causal agent of fruit rot of olives was determined from fresh collections of the fungus from central Greece. In culture it formed two types of conidia, namely fusiform, hyaline, aseptate conidia typical of the genus Fusicoccum, and dark-walled, ovoid, ellipsoid or fusiform, 1–2 septate conidia that are not typically observed in Fusicoccum. A phylogenetic analysis based on ITS and EF1- sequences placed the fungus within the same clade as Fusicoccum aesculi, which is the anamorph of Botryosphaeria dothidea, and the type of the genus Fusicoccum.