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Three new species of the genus Longior Travassos & Kloss, 1958 are described and illustrated, namely L. surieli n. sp. in Antillanax dominicanus (Doesburg, 1953) from the Dominican Republic, L. lamothei n. sp. in Passalus punctiger Le Peletier & Serville, 1825 from Mexico and Colombia and L. zumpimito n. sp. in P. punctatostriatus Percheron, 1835 from Mexico. These constitute the first records of the genus Longior for the aforementioned countries, rising to nine species in the genus. The new species can be differentiated mainly by the length of their body, oesophagus and tail in both sexes, the extension of the lateral alae in the females and the morphology of the cephalic and posterior end in the males. The molecular phylogeny of the new taxa is inferred by the 28S and 18S rDNA and they form a monophyletic clade with other Longior species. The phylogeny of Longior and that of their passalid hosts reveal coevolutionary relationships. These patterns suggest that the phylogeny of Longior species is probably strongly influenced by the evolutionary trajectories of their passalid hosts.

Climate changes are likely to have major influences on the distribution and abundance of Antarctic terrestrial biota. To assess arthropod distribution and diversity within the Ross Sea region, we examined mitochondrial DNA (COI) sequences for three currently recognized species of springtail (Collembola) collected from sites in the vicinity, and to the north of, the Mackay Glacier (77°S). This area acts as a transition between two biogeographic regions (northern and southern Victoria Land). We found populations of highly divergent individuals (5%–11.3% intraspecific sequence divergence) for each of the three putative springtail species, suggesting the possibility of cryptic diversity. Based on molecular clock estimates, these divergent lineages are likely to have been isolated for 3–5 million years. It was during this time that the Western Antarctic Ice Sheet (WAIS) was likely to have completely collapsed, potentially facilitating springtail dispersal via rafting on running waters and open seaways. The reformation of the WAIS would have isolated newly established populations, with subsequent dispersal restricted by glaciers and ice-covered areas. Given the currently limited distributions for these genetically divergent populations, any future changes in species’ distributions can be easily tracked through the DNA barcoding of springtails from within the Mackay Glacier ecotone.

Coynema poeyi (Coy, García & Álvarez, 1993) (Oxyuridomorpha: Hystrignathidae) is redescribed and illustrated with the aid of SEM. New features of the cephalic end of both sexes and copulatory papillae pattern of the males were observed and the generic diagnosis is emended in order to include such features. New locality records are given. The phylogenetic position of the species is inferred on the basis of the D2-D3 segment of the LSU rDNA. C. poeyi is located basal in a monophyletic clade formed by other hystrignathids: two species of Longior Travassos & Kloss, 1958 and Hystrignathus sp.

The status of Longior alius García & Coy, 1994 (Nematoda: Thelastomatoidea: Hystrignathidae) is discussed on the basis of new material obtained from its type host Antillanax pertyi (Kaup, 1869) (Coleoptera: Passalidae) from Limonar, Guantánamo province, Cuba, near the type locality. Based on morphological and molecular studies L. alius is considered synonymous with L. longior Morffe & García, 2011. L. longior and L. similis Morffe, García & Ventosa, 2009 are redescribed and illustrated with the aid of SEM. New locality records for both species are given. The conspecificity of females and males of both species is supported by comparison of the D2-D3 segment of the 28S LSU rDNA. The inter-specific differences and phylogenetic position of L. longior and L. similis are discussed.

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.

Photorhabdus is a genus of Gram-negative bacteria belonging to the Enterobacteriaceae family. In addition to forming a mutualistic relationship with the Heterorhabditidae family of nematodes, these bacteria are the causal agent of insect mortality during nematode infection, and are commonly used as biocontrol agents against pest insects in managed ecosystems. There are three described species of Photorhabdus; Photorhabdus luminescens and Photorhabdus temperata, which are strictly entomopathogens, and Photorhabdus asymbiotica, which has been isolated from wound infections in humans. While there has been extensive research on its virulence mechanisms, the evolution of virulence in Photorhabdus has not previously been investigated within a phylogenetic context. To investigate how virulence has evolved in this genus, we first reconstructed the phylogenetic relationships among 18 strains representing each of the main taxonomic lineages in the genus. Bacterial cells were injected into Galleria mellonella and Tenebrio molitor larvae, and the LT50 was calculated for each strain. These values were mapped onto the phylogeny using ancestral character reconstruction methods. With few exceptions, we found that the general trend of Photorhabdus evolution is one of increasing virulence. We also explored the relationship between virulence and Photorhabdus cell types and growth rates. Although we found no correlation between cell type and virulence, there was a strong correlation between virulence and growth rates in T. molitor. A better understanding of the origin and maintenance of virulence in this bacterium will aid in unraveling the mechanisms of the Heterorhabditis-Photorhabdus complex, resulting in the selection of more effective nematode-bacterium complexes for biocontrol.

Background Parasitism is a major ecological niche for a variety of nematodes. Multiple nematode lineages have specialized as pathogens, including deadly parasites of insects that are used in biological control. We have sequenced and analyzed the draft genomes and transcriptomes of the entomopathogenic nematode Steinernema carpocapsae and four congeners (S. scapterisci, S. monticolum, S. feltiae, and S. glaseri). Results We used these genomes to establish phylogenetic relationships, explore gene conservation across species, and identify genes uniquely expanded in insect parasites. Protein domain analysis in Steinernema revealed a striking expansion of numerous putative parasitism genes, including certain protease and protease inhibitor families, as well as fatty acid- and retinol-binding proteins. Stage-specific gene expression of some of these expanded families further supports the notion that they are involved in insect parasitism by Steinernema. We show that sets of novel conserved non-coding regulatory motifs are associated with orthologous genes in Steinernema and Caenorhabditis. Conclusions We have identified a set of expanded gene families that are likely to be involved in parasitism. We have also identified a set of non-coding motifs associated with groups of orthologous genes in Steinernema and Caenorhabditis involved in neurogenesis and embryonic development that are likely part of conserved protein–DNA relationships shared between these two genera. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0746-6) contains supplementary material, which is available to authorized users.

Francisella tularensis has been the focus of much research over the last two decades mainly because of its potential use as an agent of bioterrorism. F. tularensis is the causative agent of zoonotic tularemia and has a worldwide distribution. The different subspecies of F. tularensis vary in their biogeography and virulence, making early detection and diagnosis important in both the biodefense and public health sectors. Recent genome sequencing efforts reveal aspects of genetic diversity, evolution and phylogeography previously unknown for this relatively small organism, and highlight a role for detection by various PCR assays. This review explores the advances made in understanding the evolution and genetic diversity of F. tularensis and how these advances have led to better PCR assays for detection and identification of the subspecies.

Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30–35% amino acid identity to ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in a Mg2+-dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5–10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniae RNase III.

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipidinduced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

The complete mitochondrial cytochrome oxidase II gene was sequenced from 17 black flies, representing 13 putative species, and used to infer phylogenetic relationships. A midge (Paratanytarsus sp.) and three mosquitoes (Aedes aegypti, Anopheles quadrimaculatus, and Culex quinquefasciatus) were used as outgroup taxa. All outgroup taxa were highly divergent from black flies. Phylogenetic trees based on weighted parsimony (a priori and a posteriori), maximum likelihood, and neighbor-joining (log-determinant distances) differed topologically, with deeper nodes being the least well-supported. All analyses supported current classification into species groups but relationships among those groups were poorly resolved. The majority of phylogenetic signal came from closely related sister taxa. The CO-II gene may be useful for exploring relationships at or below the subgeneric level, but is of questionable value at higher taxonomic levels. The weighting method employed gave phylogenetic results similar to those reported by other authors for other insect CO-II data sets. A best estimate of phylogenetic relationships based on the CO-II gene is presented and discussed in relation to current black fly classification.

The nucleic acid of a serologically distinct potyvirus, originally isolated out of sugar cane from Pakistan, was reverse transcribed and the 3' terminal 2000 bp was PCR amplified, cloned, and sequenced. Phylogenetic comparisons of viruses representing each genus of the Potyviridae show that the Pakistani isolate is most closely related to the rymoviruses wheat streak mosaic virus (WSMV) and brome streak mosaic virus. We therefore propose that this new virus species be named sugar cane streak mosaic virus to reflect its similarity to WSMV. The phylogenetic data also show that the genus Rymovirus contains at least two unique evolutionary lineages. Thus the current taxonomy, based on transmission vector, is paraphyletic. We present an analysis of the taxonomic relationships among members of the family and propose a classification that both resolves the paraphyly and more accurately represents the evolutionary history of the Potyviridae.

Photorhabdus spp., the only known bioluminescent terrestrial bacteria are well known for their symbiotic association with heterorhabditid nematodes. This association, along with their ability to kill insects, has aroused interest in the evolutionary relationships within this bacterial group. Currently, three species are recognized within the genus Photorhabdus; P. temperata and P. luminescens, which are endosymbionts of Heterorhabditis spp., and P. asymbiotica, which has been isolated from human wounds and has recently been shown to also have a heterorhabditid nematode vector. To examine phylogenetic relationships among these taxa, we utilize total evidence Bayesian, likelihood, and parsimony based analyses of three genetic loci (16S rRNA gene, gyrB, and glnA) to construct a robust evolutionary hypothesis for the genus Photorhabdus. Here we use this phylogeny to evaluate existing specific and sub-specific taxonomic statements within the genus, identify previously undescribed Photorhabdus strains, test the utility of 16S rRNA gene, gyrB, and glnA in resolving various levels of relationships within the genus, and, finally, to investigate the evolution of bioluminescence. The genes examined produced the most robust phylogenetic hypothesis to date for the genus Photorhabdus, as indicated by strong bootstrap and posterior probability values at previously unresolved or poorly resolved nodes. We show that glnA is particularly useful in resolving specific and intra-specific relationships poorly resolved in other studies. We conclude that P. asymbiotica is the sister group to P. luminescens and that the new strains HIT and JUN should be given a new group designation within P. asymbiotica. Furthermore, we reveal a pattern of decline in bioluminescent intensity through the evolution of Photorhabdus, suggesting that this may be a trait acquired and maintained under previous ecological (aquatic) selection pressures that is now gradually being lost in its terrestrial environment.

Background The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms. Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers. Results The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism. Conclusion The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.

In this study, we used a recently developed supertrees method to test for shared phylogeographical signal in partially overlapping geographical ranges of lizards of the genus Liolaemus from the Andean Range in south-central Chile. We reconstruct mtDNA gene trees for three partially codistributed species (Liolaemus tenuis, L. lemniscatus and L. pictus), and our sampling effort is sufficient to allow statistical tests of shared signal between the combinations L. tenuis-L. pictus, and L. tenuis-L. lemniscatus. For both combinations, standardized maximum agreement subtrees scores showed statistically significant signal for shared pattern in regions of overlap, as evaluated by randomization tests (P0.001 and0.05, respectively). The matrix representation with parsimony tree obtained from the combination of the three different gene trees revealed concordant phylogeographical associations of all species, and was consistent with the geographical association of intraspecific haploclades with three Chilean bioclimatic zones. A multidimensional scaling analysis of several climate variables showed highly significant differences among these zones, which further suggests that they may have contributed to similar patterns of intraspecific divergence across all three species. In the mesomorphic zone in Central Chile, the species L. tenuis and L. lemniscatus may have codiverged in response to shared orogenic vicariant events, which likely predominated over climatic events associated with cycles of glacial advance and retreat. In the hygromorphic zone in southern Chile, however, glacial cycles likely predominated in structuring the phylogeographical histories of L. tenuis and L. pictus, although important ecological differences between these two caution against broad generalizations at this point.

Seventy-two full genomes corresponding to nine mammalian (67 strains) and two avian (5 strains) polyomavirus species were analyzed using maximum likelihood and Bayesian methods of phylogenetic inference. Our fully resolved and well-supported (bootstrap proportions > 90%; posterior probabilities = 1.0) trees separate the bird polyomaviruses (avian polyomavirus and goose hemorrhagic polyomavirus) from the mammalian polyomaviruses, which supports the idea of spitting the genus into two subgenera. Such a split is also consistent with the different viral life strategies of each group. Simian (simian virus 40, simian agent 12 [Sa12], and lymphotropic polyomavirus) and rodent (hamster polyomavirus, mouse polyomavirus, and murine pneumotropic polyomavirus [MPtV]) polyomaviruses did not form monophyletic groups. Using our best hypothesis of polyomavirus evolutionary relationships and established host phylogenies, we performed a cophylogenetic reconciliation analysis of codivergence. Our analyses generated six optimal cophylogenetic scenarios of coevolution, including 12 codivergence events ( P < 0.01), suggesting that Polyomaviridae coevolved with their avian and mammal hosts. As individual lineages, our analyses showed evidence of host switching in four terminal branches leading to MPtV, bovine polyomavirus, Sa12, and BK virus, suggesting a combination of vertical and horizontal transfer in the evolutionary history of the polyomaviruses.

Nematodes of the suborder Cephalobina include an ecologically and morphologically diverse array of species that range from soil-dwelling microbivores to parasites of vertebrates and invertebrates. Despite a long history of study, certain of these microbivores (Cephaloboidea) present some of the most intractable problems in nematode systematics; the lack of an evolutionary framework for these taxa has prevented the identification of natural groups and inhibited understanding of soil biodiversity and nematode ecology. Phylogenetic analyses of ribosomal (LSU) sequence data from 53 taxa revealed strong support for monophyly of taxa representing the Cephaloboidea, but do not support the monophyly of most genera within this superfamily. Historically these genera have primarily been recognized based on variation in labial morphology, but molecular phylogenies show the same general labial (probolae) morphotype often results from recurrent similarity, a result consistent with the phenotypic plasticity of probolae previously observed for some species in ecological time. Phylogenetic analyses of LSU rDNA also recovered strong support for some other groups of cephalobs, including taxa representing most (but not all) Panagrolaimoidea. In addition to revealing homoplasy of probolae, molecular trees also imply other unexpected patterns of character evolution or polarity, including recurrent similarity of offset spermatheca presence, and representation of complex probolae as the ancestral condition within Cephaloboidea. For Cephalobidae, molecular trees do not support traditional genera as natural groups, but it remains untested if deconstructing probolae morphotypes or other structural features into finer component characters may reveal homologies that help delimit evolutionary lineages.

Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is recognized as the vector of citrus leprosis virus that is a significant problem in several South American countries. Citrus leprosis has been reported from Florida in the past but no longer occurs on citrus in North America. The disease was recently reported in Central America, suggesting that B. phoenicis constitutes a potential threat to the citrus industries of North America and the Caribbean. Besides B. phoenicis, B. obovatus Donnadieu, and B. californicus (Banks) have been incriminated as vectors of citrus leprosis virus and each species has hundreds of host plants. In this study, Brevipalpus mite specimens were collected from different plants, especially citrus, in the States of Florida (USA) and Sao Paulo (Brazil), and reared on citrus fruit under standard laboratory conditions. Mites were taken from these colonies for DNA extraction and for morphological species identification. One hundred and two Random Amplified Polymorphic DNA (RAPD) markers were scored along with amplification and sequencing of a mitochondrial cytochrome oxidase subunit I gene fragment (374 bp). Variability among the colonies was detected with consistent congruence between both molecular data sets. The mites from the Florida and Brazilian colonies were morphologically identified as belonging to B. phoenicis, and comprise a monophyletic group. These colonies could be further diagnosed and subdivided geographically by mitochondrial DNA analysis.

The Helicosporidia are invertebrate pathogens that have recently been identified as non-photosynthetic green algae (Chlorophyta). In order to confirm the algal nature of the genus Helicosporidium, the presence of a retained chloroplast genome in Helicosporidia cells was investigated. Fragments homologous to plastid 16S rRNA (rrn16) genes were amplified successfully from cellular DNA extracted from two different Helicosporidium isolates. The fragment sequences are 1269 and 1266 bp long, are very AT-rich (60.7 %) and are similar to homologous genes sequenced from non-photosynthetic green algae. Maximum-parsimony, maximum-likelihood and neighbour-joining methods were used to infer phylogenetic trees from an rrn16 sequence alignment. All trees depicted the Helicosporidia as sister taxa to the non-photosynthetic, pathogenic alga Prototheca zopfii. Moreover, the trees identified Helicosporidium spp. as members of a clade that included the heterotrophic species Prototheca spp. and the mesotrophic species Chlorella protothecoides. The clade is always strongly supported by bootstrap values, suggesting that all these organisms share a most recent common ancestor. Phylogenetic analyses inferred from plastid 16S rRNA genes confirmed that the Helicosporidia are non-photosynthetic green algae, close relatives of the genus Prototheca (Chlorophyta, Trebouxiophyceae). Such phylogenetic affinities suggest that Helicosporidium spp. are likely to possess Prototheca-like organelles and organelle genomes.

Phylogenetic relationships among six isolates of Halicephalobus gingivalis (Stefanski, 1954), a species with pathogenic potential in horses and humans, were evaluated using DNA sequences from the nuclear large-subunit ribosomal RNA (LSU rDNA) gene. Sequences from nematodes obtained from in vitro cultures (soil or clinical sources), or isolated from infected horse tissues, were compared. Gene sequences from a fatal equine clinical case from southern California and a free-living isolate recovered from southern California soil showed no fixed differences. Sequences from isolates representing two fatal equine cases from North America, one from Ontario, Canada and another from Tennessee also showed no fixed differences. In contrast, two equine cases from Tennessee had 18 fixed differences for this LSU region, the greatest observed among isolates from horses. Phylogenetic analysis of six Halicephalobus sequences and four outgroup taxa by maximum parsimony yielded one tree with five well-supported clades. This phylogeny did not group isolates of Halicephalobus strictly by region of geographic isolation or source of sample, and depicted one clinical and one soil isolate as sister taxa. These results confirm that free-living environmental isolates are potential sources of infection for horses. The phylogeny also reveals that diverse isolates can cause infections in horses within a relatively limited geographic region, and conversely that genetically similar sister taxa can be recovered from geographically distant localities. PCR primers that selectively amplify Halicephalobus DNA were designed and tested based on comparison of closely related nematodes as inferred from phylogenetic analysis.

Sequence analysis of the 330-kb chlorella virus PBCV-1 genome revealed an open-reading frame, A94L, that encodes a protein with significant amino acid identity to Glycoside Hydrolase Family 16 beta-1,3-glucanases. The a94l gene was cloned and the protein was expressed as a GST-A94L fusion protein in Escherichia coli. The recombinant A94L protein hydrolyzed the beta-1,3-glucose polymer laminarin and had slightly less hydrolytic activity on beta-1,3-1, 4-glucose polymers, lichenan and barley beta-glucan. The recombinant enzyme had the highest activity at 65 degrees C and pH 8. We predicted that the a94l-encoded beta-1,3-glucanase is involved in degrading the host cell wall either during virus release and/or is packaged in the virion particle and involved in virus entry. Therefore, we expected a94l to be expressed late in virus infection. However, contrary to expectations, both the a94l mRNA and the A94L protein appeared 15 min after PBCV-1 infection and disappeared 60- and 120-min p.i. postinfection, respectively, indicating that a94l is an early gene. Twenty-seven of 42 chlorella viruses contained the a94l gene. To our knowledge, this is the first report of a virus-encoded beta-1,3-glucanase.

INTRODUCTIONThe genus Plectus is one of the most widely distributed and common nematode taxa of freshwater and terrestrial habitats in the world, and is of particular interest because of its phylogenetic position relative to the origin of the Secernentean radiation. Plectus murrayi, a bacteria-feeding nematode, inhabits both semi-aquatic and terrestrial biotopes in the Antarctic McMurdo Dry Valleys (MCM), where its distribution is limited by organic carbon and soil moisture. Plectus nematodes from the MCM can survive extreme desiccation, freezing conditions, and other types of stress. Ongoing investigations of the physiological and molecular aspects of the stress biology of P. murrayi, along with the availability of genomic resources, will likely establish this nematode as an excellent invertebrate model system for studies of extreme environmental survival, and may provide a valuable source of genomic resources for comparative studies in other organisms. Moreover, because P. murrayi and Caenorhabditis elegans share a most recent common ancestor with the rest of the Secernentea, and given the ability of P. murrayi to be cultured at lower temperatures compared to C. elegans, P. murrayi could also be an emerging model system for the study of the evolution of environment-sensitive (stress response) alleles in nematodes.

Previously we reported that 19 of 42 viruses that infect Chlorella strain NC64A (NC64A viruses) contain a short, nuclear-located, spliceosomal-processed intron in a pyrimidine dimer-specific glycosylase/apyrimidine lyase (pdg) gene. Surprisingly, the nucleotide sequence of the intron region is more conserved than the exon regions of the gene (L. Sun et al., 2000, J. Mol. Evol. 50, 82–92). For comparative purposes, we determined the nucleotide sequence of a similar intron type and its flanking coding regions in the DNA polymerase (dnapol) gene from the same 42 NC64A viruses and also 5 viruses that infect Chlorella strain Pbi. Thirty-eight of the 42 NC64A viruses contained a 101-nucleotide intron and the remaining 4 had an 86-nucleotide intron located in the same position in dnapol. The 4 viruses with the smaller intron in dnapol also have a smaller intron in their pdg gene. There was no intron in the dnapol gene of the 5 Pbi viruses. Phylogenetic analyses indicate that the dnapol genes containing the 86-nucleotide intron represent the ancestral condition among the NC64A viruses. The intron in the dnapol gene is phase 0 (keeps codons intact), which differs from the phase 1 intron in the pdg gene. The intron in the dnapol gene, unlike the pdg intron, was conserved (83 to 100% identical) to about the same extent as the coding regions of the gene (78 to 100% identical).

Chlorella virus PBCV-1 encodes two putative chitinase genes, a181/182r and a260r, and one chitosanase gene, a292l. The three genes were cloned and expressed in Escherichia coli. The recombinant A181/182R protein has endochitinase activity, recombinant A260R has both endochitinase and exochitinase activities, and recombinant A292L has chitosanase activity. Transcription of a181/182r, a260r, and a292l genes begins at 30, 60, and 60 min p.i., respectively; transcription of all three genes continues until the cells lyse. A181/182R, A260R, and A292L proteins are first detected by Western blots at 60, 90, and 120 min p.i., respectively. Therefore, a181/182r is an early gene and a260r and a292l are late genes. All three genes are widespread in chlorella viruses. Phylogenetic analyses indicate that the ancestral condition of the a181/182r gene arose from the most recent common ancestor of a gene found in tobacco, whereas the genealogical position of the a260r gene could not be unambiguously resolved.

Historically, the invertebrate pathogens of the genus Helicosporidium were considered to be either protozoa or fungi, but the taxonomic position of this group has not been considered since 1931. Recently, a Helicosporidium sp., isolated from the blackfly Simulium jonesi Stone & Snoddy (Diptera: Simuliidae), has been amplified in the heterologous host Helicoverpa zea. Genomic DNA has been extracted from gradient-purified cysts. The 185, 28S and 5.8S regions of the Helicosporidium rDNA, as well as partial sequences of the actin and beta-tubulin genes, were amplified by PCR and sequenced. Comparative analysis of these nucleotide sequences was performed using neighbour-joining and maximum-parsimony methods. All inferred phylogenetic trees placed Helicosporidium sp. among the green algae (Chlorophyta), and this association was supported by bootstrap and parsimony jackknife values. Phylogenetic analysis focused on the green algae depicted Helicosporidium sp. as a close relative of Prototheca wickerhamii and Prototheca zopfii (Chlorophyta, Trebouxiophyceae), two achlorophylous, pathogenic green algae. On the basis of this phylogenetic analysis, Helicosporidium sp. is clearly neither a protist nor a fungus, but appears to be the first described algal invertebrate pathogen. These conclusions lead us to propose the transfer of the genus Helicosporidium to Chlorophyta, Trebouxiophyceae.

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences.