Microsatellite Analysis of Museum Specimens Reveals Historical Differences in Genetic Diversity between Declining and More Stable Bombus Species

Worldwide most pollinators, e.g. bumblebees, are undergoing global declines. Loss of genetic diversity can play an essential role in these observed declines. In this paper, we investigated the level of genetic diversity of seven declining Bombus species and four more stable species with the use of microsatellite loci. Hereto we genotyped a unique collection of museum specimens. Specimens were collected between 1918 and 1926, in 6 provinces of the Netherlands which allowed us to make interspecific comparisons of genetic diversity. For the stable species B. pascuorum, we also selected populations from two additional time periods: 1949–1955 and 1975–1990. The genetic diversity and population structure in B. pascuorum remained constant over the three time periods. However, populations of declining bumblebee species showed a significantly lower genetic diversity than co-occurring stable species before their major declines. This historical difference indicates that the repeatedly observed reduced genetic diversity in recent populations of declining bumblebee species is not caused solely by the decline itself. The historically low genetic diversity in the declined species may be due to the fact that these species were already rare, making them more vulnerable to the major drivers of bumblebee decline.     

Diversity of the Schizophoria lineage (Brachiopoda: Orthida) in the Lower and Middle Devonian of Poland and adjacent areas

The nominative subgenus of Schizophoria (Brachiopoda, Orthida) is represented in the Lower and Middle Devonian of Poland and of the western Ukraine by six taxa: late Emsian Schizophoria (S.) interstrialis, late Eifelian S. (S.) schnuri biscissa, early Givetian S. (S.) schnuri schnuri, middle to late Givetian S. (S.) schnuri prohibita ssp.n., middle Givetian S. (S.) parvaepunctata and late Givetian S. (S.) sp.n. (unnamed for lack of sufficient material). Morphotypes referable to S. (S.) schnuri biscissa and to S. (S.) schnuri junkerbergiana co-occur in the same outcrop (not necessarily in the same levels), wherefore the separation of these subspecies may be maintained only if they are considered as chronosubspecies (unverifiable on the studied material) but not as geographic variants (hypothesis falsified by the studied material). A lectotype for S. (S.) interstrialis and a neotype for S. (S.) parvaepunctata are selected. Punctae diameter and density (either an important systematic character according to some authors or devoid of such value according to other ones) were investigated: they show important within-individual and within-sample variation, wherefore they cannot serve to distinguish related species. On the contrary, punctae character combinations are sufficiently constant to help differentiate subgenera or groups of species. In the studied material, three clusters are distinguished on the basis of the punctae characteristics: S. (Pachyschizophoria) and S. (S.) parvaepunctata differ between each other and from all other investigated species of S. (Schizophoria); the latter are indistinguishable on the sole basis of punctae characteristics.     

Hitting an Unintended Target: Phylogeography of Bombus brasiliensis Lepeletier, 1836 and the First New Brazilian Bumblebee Species in a Century (Hymenoptera: Apidae)

This work tested whether or not populations of Bombus brasiliensis isolated on mountain tops of southeastern Brazil belonged to the same species as populations widespread in lowland areas in the Atlantic coast and westward along the Paraná-river valley. Phylogeographic and population genetic analyses showed that those populations were all conspecific. However, they revealed a previously unrecognized, apparently rare, and potentially endangered species in one of the most threatened biodiversity hotspots of the World, the Brazilian Atlantic Forest. This species is described here as Bombus bahiensis sp. n., and included in a revised key for the identification of the bumblebee species known to occur in Brazil. Phylogenetic analyses based on two mtDNA markers suggest this new species to be sister to B. brasiliensis, from which its workers and queens can be easily distinguished by the lack of a yellow hair-band on the first metasomal tergum. The results presented here are consistent with the hypothesis that B. bahiensis sp. n. may have originated from an ancestral population isolated in an evergreen-forest refuge (the so-called Bahia refuge) during cold, dry periods of the Pleistocene. This refuge is also known as an important area of endemism for several animal taxa, including other bees. Secondary contact between B. bahiensis and B. brasiliensis may be presently prevented by a strip of semi-deciduous forest in a climate zone characterized by relatively long dry seasons. Considering the relatively limited range of this new species and the current anthropic pressure on its environment, attention should be given to its conservation status.     

Resolution of the long-standing controversy over the type species of the genus Pseudotypotherium (Notoungulata,Typotheria, Mesotheriidae)

Mesotheres (Notoungulata: Typotheria) are among the most common mammals found in upper Miocene to Pliocene deposits of central Argentina, including the classic type Monte Hermoso locality, which defines the Montehermosan South American Land Mammal “Age”. Nevertheless, the correct name for the mesothere species from this site has been shrouded in uncertainty for well over a century due to questions of taxonomic priority, specimen provenance, and ontogenetic changes in dental formula. Since the mesotheres from Monte Hermoso were named, three distinct species have been formally considered as the type species of the genus: (1) Pseudotypotherium bravardi; (2) “Pseudotypotherium” maendrum; and (3) Pseudotypotherium exiguum. However, none of these species is a nominal species of the Pseudotypotherium genus; all three were originally referred to Typotherium. Article 67.2 of the International Code of Zoological Nomenclature (ICZN, 1999) indicates that only species considered as nominal species are eligible to set the type; in the case of Pseudotypotherium, these include: P. pulchrum, P. carlesi, P. hystatum, and P. carhuense. We conclude that Pseudotypotherium pulchrum F. Ameghino, 1904 (holotype MACN A 10299, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Ameghino Collection), is the type species of the mesotheriid notoungulate genus from Monte Hermoso. According to Article 68.2, F. Ameghino fixed the type by original designation in 1904 when he described P. pulchrum and included “n. g., n. sp.”. Two of the other species previously considered species P. (= T.) bravardi and P. (= T.) exiguum are invalid as type species according to Article 70.2, since their designations overlooked the previous type fixation. The third species (M. (= T.) maendrum) represents a different mesothere genus (Mesotherium) that only occurs in younger (Pleistocene) deposits. Our analysis puts an end to a historical debate that has been ongoing for more than a century regarding the identity of this well-represented late Miocene–Pliocene mesotheriine genus (Pseudotypotherium). This study provides a solid taxonomic foundation for future studies on intraspecific and ontogenetic variation of Pseudotypotherium pulchrum.     

Alien parasite hitchhikes to Patagonia on invasive bumblebee

The worldwide trade in bumblebees can lead to the spread of diseases, which in turn has been claimed as a factor in bumblebee decline. Populations of the introduced Bombus terrestris, which invaded NW Patagonia, Argentina, in 2006, harbor the highly pathogenic protozoan Apicystis bombi. We asked whether A. bombi had been co-introduced with B. terrestris, and if so, whether spillover occurred to the two resident bumblebee species in the region: the introduced European Bombus ruderatus and the native Bombus dahlbomii. We searched for A. bombi by means of PCR in samples of B. ruderatus and B. dahlbomii collected before and after the invasion of B. terrestris and in samples of the latter. We found no A. bombi in samples of B. ruderatus and B. dahlbomii collected before B. terrestris invasion, whereas post invasion, A. bombi was present in all 3 species. The identity of the parasite was established by sequencing the 18S region, which was identical for the three bumblebee species and also matched the European sequence, confirming it to be A. bombi. This is the first report of A. bombi in B. ruderatus and B. dahlbomii. Moreover, our results suggest that Patagonia had been free of A. bombi until this parasite was co-introduced with B. terrestris, and spilled over in situ to these two previously resident species. Finally, our findings provide indirect circumstantial evidence of a potential link between the population collapse and geographic retraction of B. dahlbomii and the introduction of this novel parasite.     

Genus Triticum L. taxonomy: the present and the future

The genetic relationships and diversity within the European and Asiatic Buxus species were analysed using AFLP, genome size analysis and chromosome counts. Based on these results two major clusters could be defined. One genetic cluster contained B. sempervirens and B. balearica, European species, and B. colchica, an Asiatic species but with leaf morphology similar to B. sempervirens. Species in this cluster were characterised by a genome size between 1.38 and 1.69?pg?2C^?1 and a chromosome number of 2n?=?2x?=?28 (diploid). Only four B. sempervirens cultivars within this cluster were triploid. A second cluster contained the Asiatic Buxus species B. microphylla, B. harlandii, B. hyrcana, B. myrica, B. henryi, B. bodinieri and B. wallichiana. Within this second genetic cluster three different ploidy levels could be observed. B. harlandii, B. hyrcana and nine B. microphylla cultivars were tetraploid (2n?=?4x?=?56) with a genome size of >2.5?pg?2C^?1. Fifteen other B. microphylla cultivars were triploid (2n?=?3x?=?42). The other Asiatic Buxus species, B. henryi, B. bodinieri and eight B. microphylla cultivars, were diploid with a genome size of ca. 1.5?pg?2C^?1.     

Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Complete Genome Sequence of Borrelia afzelii K78 and Comparative Genome Analysis

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

In situ comparison of activity in two deep-sea scavenging fishes occupying different depth zones

The activity of two scavenging deep-sea fishes occupying the same niche in overlapping depth zones were compared by in situ measurements of swimming speeds, tail-beat frequencies and by arrival time at baits. At 4800 m on the Porcupine Abyssal Plain, the grenadier Coryphaenoides (Nematonurus) armatus was the dominant scavenger, arriving at baits after 30 min, and swimming at relatively slow speeds of 0.17 body lengths (BL) sec^-1. At 2500 m in the relatively food rich Porcupine Seabight both C. (N.) armatus and the blue-hake, Antimora rostrata, were attracted to bait, but A. rostrata was always the first to arrive and most of the bait was consumed before the C. (N.) armatus arrived. A. rostrata swam at mean speeds of 0.39 BL sec^-1, similar to related shallow water species at equivalent temperatures. Observations on tail-beat frequency from video sequences confirmed the greater activity of A. rostrata. The data indicate that, given sufficient food supply, high pressure and low temperature do not limit activity levels of demersal deep-sea fishes. Low activity of C. (N.) armatus is an adaptation to poor food supply in the abyss, where these fishes dominate, but prevents it competing with the more active A. rostrata in shallower depths.     

Burkholderia species in human infections in Mexico: Identification of B. cepacia,B. contaminans,B. multivorans,B. vietnamiensis,B. pseudomallei and a new Burkholderia species

BackgroundBurkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described.Methodology/Principal findingsIn this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia.Conclusions/SignificanceThe presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.     

Prevalence and diversity of small rodent-associated Bartonella species in Shangdang Basin,China

The aim of this study was to investigate the occurrence and molecular characteristics of Bartonella infections in small rodents in the Shangdang Basin, China. Small rodents were captured using snap traps, and their liver, spleen, and kidney tissues were harvested for Bartonella detection and identification using a combination of real-time PCR of the ssrA gene (296 bp) and conventional PCR and sequencing of the gltA gene (379 bp). Results showed that 55 of 147 small rodents to be positive for Bartonella, with a positivity rate of 37.41%, and 95% confidence interval of 29.50%- 45.33%. While the positivity rate across genders (42.62% in males and 33.72% in females, χ² = 1.208, P = 0.272) and tissues (28.57% in liver, 33.59% in spleen, and 36.76% in kidney, χ² = 2.197, P = 0.333) of small rodents was not statistically different, that in different habitats (5.13% in villages, 84.44% in forests, and 54.17% in farmlands, χ² = 80.105, P<0.001) was statistically different. There were 42 Bartonella sequences identified in six species, including 30 B. grahamii, three B. phoceensis, two B. japonica, two B. queenslandensis, one B. fuyuanensis and four unknown Bartonella species from Niviventer confucianus, Apodemus agrarius and Tscherskia triton. In addition to habitat, Bartonella species infection could be affected by the rodent species as well. Among the Bartonella species detected in this area, B. grahamii was the dominant epidemic species (accounting for 71.43%). B. grahamii exhibited four distinct clusters, and showed a certain host specificity. In addition, 11 haplotypes of B. grahamii were observed using DNASP 6.12.03, among which nine haplotypes were novel. Overall, high occurrence and genetic diversity of Bartonella were observed among small rodents in the Shangdang Basin; this information could potentially help the prevention and control of rodent-Bartonella species in this area.     

Three new species of Diphascon of the pingue group (Eutardigrada,Hypsibiidae) from Antarctica

This paper contributes to the knowledge of the pingue group of species within the genus Diphascon. Three new species are described, Diphascon (D.) polare, D. (D.) dastychi and D. (D.) victoriae, from Victoria Land (Antarctica); they differ from other species of the pingue group in characters of the bucco-pharyngeal apparatus. D. polare and D. victoriae differ also in the claw shape. A key is provided to the Diphascon pingue group. Accepted: 30 November 1998     

Two new chironomid species (Diptera,Chironomidae) from Wrangel Island

Two new species from Wrangel Island, Orthocladius (Eudactylocladius) tschernovi sp. n. and O. (E.) ushakovskiensis sp. n. (Orthocladiinae), are described and illustrated from on the male adults. Orthocladius (E.) tschernovi sp. n. is closely related to O. (E.) fuscimanus (Kieffer) and O. (E.) musester Sæther, but can be distinguished from both by an almost straight transverse sternapodema, a rounded triangular superior volsella, and a long phallapodema, as well as by the presence of a subapical crista dorsalis of the gonostylus and by the shape of the anal point. Orthocladius (E.) ushakovskiensis sp. n. differs from all the known species of the subgenus in the absence of superior and inferior volsellae and in the shape of both short gonostylus and the short and wide anal point.     

Biology of the New Zealand genus Neolimnia (Diptera: Sciomyzidae)

Abstract

Data are presented on the life cycles of eight species of Neolimnia, an endemic New Zealand genus of snail-killing flies. Habitats, geographical distributions, biological features of adults and immature stages, including adult and larval behaviour and feeding habits, and phenology are discussed. Larvae of subgenus Pseudolimnia live in aquatic environments; those of N. (P.) repo, N. (P.) sigma, and N. (P.) ura prey on aquatic pulmonate snails, but those of N. (P.) tranquilla prey on aquatic prosobranch snails. Larvae of Neolimnia (Neolimnia) castanea, N. (N.) irrorata, N. (N.) obscura, and N. (N.) striata live in terrestrial environments, and apparently prey overtly on terrestrial snails in nature, but will also attack aquatic snails in laboratory rearings.     

Molecular epidemiological survey of Babesia bovis,Babesia bigemina,and Babesia sp. Mymensingh infections in Mongolian cattle

Bovine babesiosis caused by Babesia species is an economically significant disease of cattle. Severe clinical babesiosis in cattle is caused by Babesia bovis, B. bigemina, and the recently discovered Babesia sp. Mymensingh. Mongolia is an agricultural country with a large cattle inventory. Although previous studies have detected active infections of B. bovis and B. bigemina in Mongolian cattle, only a few provinces were surveyed. Additionally, the endemicity of Babesia sp. Mymensingh in Mongolia remains unknown. We screened blood DNA samples from 725 cattle reared in 16 of the 21 Mongolian provinces using B. bovis-, B. bigemina-, and Babesia. sp. Mymensingh-specific PCR assays. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 27.9% (n = 202), 23.6% (n = 171), and 5.4% (n = 39), respectively. B. bovis and B. bigemina were detected in cattle in all surveyed provinces; whereas Babesia sp. Mymensingh was detected in 11 of the 16 surveyed provinces. On a per province basis, the B. bovis- B. bigemina-, and Babesia sp. Mymensingh-positive rates were 5.9–52.0%, 9.1–76.3%, and 0–35.7%, respectively. In conclusion, this is the first report of Babesia sp. Mymensingh in Mongolia. In addition, we found that species of Babesia that are capable of causing bovine clinical babesiosis, including B. bovis, B. bigemina, and Babesia sp. Mymensingh, are widespread throughout the country.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Molecular characterization of the non-costate morphotypes of buliminid foraminifers based on internal transcribed region of ribosomal DNA (ITS rDNA) sequence data

We performed molecular phylogenetic analyses of four morphotypes of the benthic foraminiferal genus Bulimina (B. aculeata, B. marginata f. marginata, B. marginata f. denudata, and B. elongata) based on sequences of internal transcribed spacer region of ribosomal DNA (ITS rDNA). Six genetically distinct phylotypes were revealed by our phylogenetic analyses. The six phylotypes basically correspond to the fundamental morphotypes: clades A + B (B. aculeata); clade C (B. elongata); clade D (B. marginata f. denudata); clade E (B. marginata f. marginata genotype 1); and clade F (B. marginata f. marginata genotype 2). All six phylotypes are well distinguished, except phylotype B, which shows only little sequence divergence compared to clade A, possibly indicating that genetic differentiation is in progress. Morphological characters including the direction, placement, and shape of spines, the angle of undercutting of chamber periphery, and the roundness of the chambers were stable among specimens of each clade. In contrast, the length and density of spines, and chamber size, were variable within each clade. These intermediate morphological characters may reflect ecophenotypic variation. Our study clearly shows that the examined B. acuelata, elongata, and denudata morphospecies are genetically separated and that B. marginata is a species complex comprising several genotypes. A novel phylotype represent different morphotype compare to B. marginata f. marginata that can be distinguished based on differences of chamber angularity, the direction, placement, and shape of spines, and test dimensions.     

New Species of Boletellus Section Boletellus (Boletaceae,Boletales) from Japan,B. aurocontextus sp. nov. and B. areolatus sp. nov.

We describe and illustrate two new species of Boletellus section Boletellus, B. aurocontextus sp. nov. and B. areolatus sp. nov., which are generally assumed to be B. emodensis. In this study, we reconstructed separate molecular phylogenetic trees of section Boletellus using the nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the largest subunit (RPB1) and the second-largest subunit (RPB2) of nuclear RNA polymerase II gene and mitochondrial cytochrome oxidase subunit 3 (cox3) gene. We also examined the morphologies of B. emodensis sensu lato (s.l.) and other related species for comparison. The molecular phylogenetic tree inferred from the sequences of nuclear DNA (ITS, and combined dataset of RPB1 and RPB2) indicated that three genetically and phylogenetically well-separated lineages were present within B. emodensis s.l. These three lineages were also distinguished on the basis of the molecular phylogenetic tree constructed using the sequences of mitochondrial DNA (cox3), suggesting distinct cytonuclear disequilibria (i.e., evidence of reproductive isolation) among these lineages. Therefore, these three lineages can be treated as independent species: B. aurocontextus, B. areolatus, and B. emodensis. Boletellus aurocontextus and B. areolatus are also distinct from B. emodensis by the macro- and microscopic morphologies. Boletellus aurocontextus is characterized by a pileus with bright yellow to lemon yellow context, which can be observed through a gap in the scales, and basidiospores with relatively large length (mean spore length, 21.4 μm; quotient of spore length and width, 2.51). In contrast, B. areolatus is characterized by a pileus with floccose to appressed thin scaly patches, a stipe with pallid or pale cream color at the upper half, and basidiospores with relatively small length (mean spore length, 16.5 μm; quotient of spore length and width, 1.80).