Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.     

The red-tailed hawk, Buteo jamaicensis, a native definitive host of Frenkelia microti (Apicomplexa) in North America.

Oral inoculation of prairie voles, Microtus ochrogaster, with coccidian sporocysts isolated from the feces of a red-tailed hawk, Buteo jamaicensis, in Kansas, USA, resulted in formation of Frenkelia microti (Apicomplexa: Sarcocystidae) tissue cysts in the brains of the voles. Five additional isolates of morphologically similar sporocysts collected from red-tailed hawks or other Buteo spp. in Kansas failed to result in detectable infections in rodents. These results are the first to verify that red-tailed hawks are natural definitive host in North America for F. microti.     

Resolving the evolutionary history of Campanula (Campanulaceae) in western North America

Recent phylogenetic works have begun to address long-standing questions regarding the systematics of Campanula (Campanulaceae). Yet, aspects of the evolutionary history, particularly in northwestern North America, remain unresolved. Thus, our primary goal in this study was to infer the phylogenetic positions of northwestern Campanula species within the greater Campanuloideae tree. We combined new sequence data from 5 markers (atpB, rbcL, matK, and trnL-F regions of the chloroplast and the nuclear ITS) representing 12 species of Campanula with previously published datasets for worldwide campanuloids, allowing us to include approximately 75% of North American Campanuleae in a phylogenetic analysis of the Campanuloideae. Because all but one of North American Campanula species are nested within a single campanuloid subclade (the Rapunculus clade), we conducted a separate set of analyses focused specifically on this group. Our findings show that i) the campanuloids have colonized North America at least 6 times, 4 of which led to radiations, ii) all but one North American campanuloid are nested within the Rapunculus clade, iii) in northwestern North America, a C. piperi-C. lasiocarpa ancestor gave rise to a monophyletic Cordilleran clade that is sister to a clade containing C. rotundifolia, iv) within the Cordilleran clade, C. parryi var. parryi and C. parryi var. idahoensis exhibit a deep, species-level genetic divergence, and v) C. rotundifolia is genetically diverse across its range and polyphyletic. Potential causes of diversification and endemism in northwestern North America are discussed.     

Splenomegaly and Reticulocytosis Caused by Babesia microti Infections in Natural Populations of the Montane Vole, Microtus montanus

ABSTRACT. A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus , 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Splenomegaly and reticulocytosis caused by Babesia microti infections in natural populations of the montane vole, Microtus montanus.

A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus, 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Community structure of benthic invertebrates in interior Alaskan (USA) streams and rivers

Taxonomic composition of benthic invertebrates in interior Alaskan streams and rivers is summarized from published and unpublished data. Diptera dominate the Alaskan stream fauna and constitute a larger proportion of the benthos in Alaskan streams than in streams of temperate North America. Plecoptera and Ephemeroptera are the next most abundant in Alaskan streams with Trichoptera generally very scarce. Several orders that occur regularly in streams of temperate North America are absent (or in very low abundance) in interior Alaskan streams: Hemiptera, Odonata, Megaloptera, Coleoptera. Net-spinning caddisflies, burrowing mayflies, and several families of stoneflies (Pteronarcyidae, Peltoperlidae and Perlidae) are conspicuous by their absence or extreme scarcity. Taxonomic composition varies significantly among hydrologic regions (major watersheds) and among stream types (springs, headwater streams, small rivers, and large rivers). Only two taxa (Chironomidae and Nemouridae) significantly increase in proportional contribution from south to north while many taxa decrease.     

Primary infections with Babesia microti are not prolonged by concurrent Heligmosomoides polygyrus

Seven experiments were carried out to test the hypothesis that concurrent infection with the chronic and immunomodulatory intestinal nematode parasites, Heligmosomoides polygyrus, and the piroplasm, Babesia microti, would result in more intense and long-lasting infections with the hemoprotozoan. However, despite variations in the experimental protocols (different mouse strains, varying levels of infection and different intervals between infection with the two species) a significantly higher B. microti parasitaemia was detected on only one occasion, and a significantly lower parasitaemia on two occasions, relative to control mice. In none of our experiments was the duration of infection prolonged. We conclude that the presence of H. polygyrus does not interfere markedly with the host's ability to mount a protective response against B. microti and hence season-dependent peaks of abundance of H. polygyrus in wild rodents are unlikely to present a particular threat to human communities by providing a greater reservoir of infection with B. microti in wild rodents than at other times of the year.     

Chloroplast phylogenomics of the New World grape species (Vitis,Vitaceae)

Vitis L. (the grape genus) is the economically most important fruit crop, as the source of grapes and wine. Phylogenetic relationships within the genus have been highly controversial. Herein, we employ sequence data from whole plastomes to attempt to enhance Vitis phylogenetic resolution. The results support the New World Vitis subgenus Vitis as monophyletic. Within the clade, V. californica is sister to the remaining New World Vitis subgenus Vitis. Furthermore, within subgenus Vitis, a Eurasian clade is robustly supported and is sister to the New World clade. The clade of Vitis vinifera ssp. vinifera and V. vinifera ssp. sylvestris is sister to the core Asian clade of Vitis. Several widespread species in North America are found to be non‐monophyletic in the plastome tree, for example, the broadly defined Vitis cinerea and V. aestivalis each needs to be split into several species. The non‐monophyly of some species may also be due to common occurrences of hybridizations in North American Vitis. The classification of North American Vitis by Munson into nine series is discussed based on the phylogenetic results. Analyses of divergence times and lineage diversification support a rapid radiation of Vitis in North America beginning in the Neogene.     

Japanese Babesia microti cytologically detected in salivary glands of naturally infected tick Ixodes ovatus

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.     

Interactions within and between clades shaped the diversification of terrestrial carnivores

A longstanding debate in evolutionary biology and paleontology is whether ecological interactions such as competition impose diversity dependence on speciation and extinction rates. Here, we analyze the fossil record of terrestrial mammalian carnivores in North America and Eurasia using a Bayesian framework to assess whether their diversity dynamics were affected by diversity dependence within and between families (12 in Eurasia, 10 in North America). We found eight instances of within‐clade diversity dependence suppressing speciation rates and detected between‐clade effects increasing extinction rates in six instances. Diversity dependence often involved lineages that migrated between continents and we found that speciation was more responsive to diversity changes within the clade, whereas extinction responded to diversity of taxa in other clades. The analysis of the fossil record of Carnivora suggests that interactions within and between clades are associated with different speciation and extinction regimes, opening room for a broader theory of diversity dependence.     

中国与北美大陆的高卢蜜环菌系统发育分析

在中国和北美大陆分别收集53和15株高卢蜜环菌Armillaria gallica菌株,并用ISSR（inter-simple sequence repeat）分子标记对这些菌株进行了亲缘关系及系统发育分析。结果表明：中国和北美大陆的A. gallica因地理隔离产生明显的遗传分异,在系统发育树上分别形成了各自的进化分支;北美大陆的分支内部遗传分化尚不明显,而中国的进化分支遗传分化程度相对较大且更为古老,中国菌株可能是两个大陆A. gallica的祖先株系。     

Phylogeny and taxonomy of the North American clade of the Ceratocystis fimbriata complex

Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.     

Extinct Beringian wolf morphotype found in the continental U.S. has implications for wolf migration and evolution

Pleistocene diversity was much higher than today, for example there were three distinct wolf morphotypes (dire, gray, Beringian) in North America versus one today (gray). Previous fossil evidence suggested that these three groups overlapped ecologically, but split the landscape geographically. The Natural Trap Cave (NTC) fossil site in Wyoming, USA is an ideally placed late Pleistocene site to study the geographical movement of species from northern to middle North America before, during, and after the last glacial maximum. Until now, it has been unclear what type of wolf was present at NTC. We analyzed morphometrics of three wolf groups (dire, extant North American gray, Alaskan Beringian) to determine which wolves were present at NTC and what this indicates about wolf diversity and migration in Pleistocene North America. Results show NTC wolves group with Alaskan Beringian wolves. This provides the first morphological evidence for Beringian wolves in mid‐continental North America. Their location at NTC and their radiocarbon ages suggest that they followed a temporary channel through the glaciers. Results suggest high levels of competition and diversity in Pleistocene North American wolves. The presence of mid‐continental Beringian morphotypes adds important data for untangling the history of immigration and evolution of Canis in North America.     

Genetic Population Structure of Chum Salmon in the Pacific Rim Inferred from Mitochondrial DNA Sequence Variation

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5′ end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency χ² tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.     

Molecular evidence for an Asian origin and a unique westward migration of species in the genus Castanea via Europe to North America

The genus Castanea (Fagaceae) is widely distributed in the deciduous forests of the Northern Hemisphere. The striking similarity between the floras of eastern Asia and those of eastern North America and the difference in chestnut blight resistance among species has been of interest to botanists for a century. To infer the biogeographical history of the genus, the phylogeny of Castanea was estimated using DNA sequence data from different regions of the chloroplast genome. Sequencing results support the genus Castanea as a monophyletic group with Castanea crenata as basal. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene followed by intercontinental dispersion and divergence between the Chinese and the European/North American species during the middle Eocene and a split between the European and the North American species in the late Eocene. The differentiation within North America and China might have occurred in early or late Miocene. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and Castanea dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.     

Phylogeography of the North American red fox: vicariance in Pleistocene forest refugia

Fossil, archaeological, and morphometric data suggest that indigenous red foxes in North America were derived from vicariance in two disjunct refugia during the last glaciation: one in Beringia and one in the contiguous USA. To test this hypothesis, we conducted a phylogeographical analysis of the North American red fox within its presettlement range. We sequenced portions of the mitochondrial cytochrome b (354 bp) gene and D-loop (342 bp) from 220 historical red fox specimens. Phylogenetic analysis of the cytochrome b gene produced two clades that diverged c . 400 000 years before present ( bp ): a Holarctic and a Nearctic clade. D-loop analyses of the Nearctic clade indicated three distinct subclades (≥ 99% Bayesian posterior probability); two that were more recently derived (rho estimate c . 20 000 bp ) and were restricted to the southwestern mountains and the eastern portion of North America, and one that was older (rho estimate c . 45 000 bp ) and more widespread in North America. Populations that migrated north from the southern refugium following deglaciation were derived from the colonization of North America during or prior to the Illinoian glaciation (300 000–130 000 bp ), whereas populations that migrated south from the northern refugium represent a more recent colonization event during the Wisconsin glaciation (100 000–10 000 bp ). Our findings indicate that Nearctic clade red foxes are phylogenetically distinct from their Holarctic counterparts, and reflect long-term isolation in two disjunct forest refugia during the Pleistocene. The montane lineage, which includes endangered populations, may be ecologically and evolutionarily distinct.     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

Delimitation and phylogeny of Aletris (Nartheciaceae) with implication for perianth evolution

Aletris, containing approximately 21 species, is the largest genus in Nartheciaceae, and is disjunctively distributed in eastern Asia and eastern North America. Its delimitation has been controversial because it is uncertain whether Metanarthecium should be included in the genus. Although there are a few molecular phylogenetic studies on Aletris, the interspecific relationships within the genus have never been evaluated in a phylogenetic context. Here we used two cpDNA loci, matK and trnL-F, to delimitate Aletris and discuss the phylogeny within the genus. Phylogenetic analyses showed Metanarthecium might be distantly related to Aletris. This is also supported by morphological, palynological, cytological, and phytochemical data. Therefore, Metanarthecium should be excluded from Aletris. Within Aletris, there are two major clades: A. farinosa and A. lutea of eastern North America and A. glabra of eastern Asia form clade A; and the remaining Asian species form clade B. The Asian clade includes three subclades: subclade I (two varieties of A. pauciflora, and A. glandulifera and A. megalantha), subclade II (three samples of A. laxiflora), and subclade III (all other sampled Asian species). Based on phylogenetic relationships, A. pauciflora var. khasiana deserves a specific status, and A. gracilipes, formerly a synonym of A. laxiflora, should be reinstated. The reconstruction of the perianth evolution indicates that perianth connate halfway and glabrous on abaxial surface are plesiomorphic for Aletris and Nartheciaceae. Farinose-glutinous perianth is a diagnostic character for clade A.