Non-systemic metamorphosis in male millipede appendages: long delayed, reversible effect of an early localized positional marker?

Background

The development of specialized appendages involved in sperm transfer in the males of julid millipedes is an extreme case of specialized, complex structures differentiating in a very advanced phase of post-embryonic development. Here, a non-systemic metamorphosis affects the external morphology and the internal anatomy of a trunk double segment only.

Presentation of the hypothesis

We hypothesize that during early (possibly embryonic) development a segmental marker is produced that remains unexploited throughout late embryonic and early post-embryonic development, until, activated by a systemic signal, it finally determines the release of a segmentally localized but anatomically major change.

Testing the hypothesis

Key to testing the hypothesis are (1) the identification of both the putative segmental marker involved in the localization of the legs to be eventually metamorphosed into gonopods and the systemic signal activating it, (2) the identification of the cell population from which the gonopods are built, and (3) a longitudinal study of the marker's expression throughout late embryonic and, possibly, post-embryonic development.

Implications of the hypothesis

Proving the validity of this hypothesis would demonstrate the existence of a cryptic developmental module that will be activated only months, or years, after it has been first laid down during early development. This study also opens a window onto the very poorly explored domain of late expression of developmental genes and molecular control of late developmental events.     

An analysis of segmentation dynamics throughout embryogenesis in the centipede <Emphasis Type="Italic">Strigamia maritima</Emphasis>

Background

Most segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this.

Results

Using the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning.

Conclusions

Our data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.

     

The delineation of the fourth walking leg segment is temporally linked to posterior segmentation in the mite Archegozetes longisetosus (Acari: Oribatida,Trhypochthoniidae)

Acari (mites and ticks) lack external segmentation, with the only indication of segmentation being the appendages of the prosoma (chelicerae, pedipalps, and four pairs of walking legs). Acari also have a mode of development in which the formation of the fourth walking leg is suppressed until the nymphal stages, following a hexapodal larva. To determine the number of segments in the posterior body region (opisthosoma) of mites, and to also determine when the fourth walking leg segment is delineated during embryogenesis, we followed the development of segmentation in the oribatid mite Archegozetes longisetosus using time‐lapse and scanning electron microscopy, as well as in situ hybridizations of the A. longisetosus orthologues of the segmentation genes engrailed and hedgehog. Our data show that A. longisetosus patterns only two opisthosomal segments, indicating a large degree of segmental fusion or loss. Also, we show that the formation of the fourth walking leg segment is temporally tied to opisthosomal segmentation, the first such observation in any arachnid.     

Head segmentation of trilobites

Although trilobites have provided research subjects for more than two centuries, their head segmentation has remained unresolved. Four glabellar furrows (SO and S1–S3) marking the segmental boundaries are generally present in the cephalic axis, but there are trilobites with one more pair of furrows, the so‐called S4, in the cephalic axis, causing confusion in understanding trilobite head segmentation. Recent advances in developmental biology and palaeontology have shed light on the arthropod head problem, and thus, trilobite head segmentation can be reviewed in the light of this knowledge. Based on the information from the anatomy of exceptionally preserved trilobites and artiopodans closely related to trilobites, it is inferred that trilobite head contains five segments: the anteriormost ocular segment potentially associated with the hypostome, the antennal segment and the following three segments with walking legs. When present, the S4 furrows are situated where the eye ridges meet the cephalic axis of trilobites, indicating that the furrows are incised ‘within’ the anteriormost segment in trilobites with an anteriorly enlarged frontal lobe. Trilobites of the Order Redlichiida, the most primitive stock, show variable conditions in the frontal glabellar conditions, while in other more derived groups, the condition is rather constant. The frontal glabellar condition, therefore, could provide a clue to elucidate the unresolved Cambrian trilobite phylogeny and the Cambrian roots of the post‐Cambrian trilobites.     

Complex copulatory behavior and the proximate effect of genital and body size differences on mechanical reproductive isolation in the millipede genus Parafontaria

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.     

Decoupling elongation and segmentation: Notch involvement in anostracan crustacean segmentation

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ‐secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss‐of‐function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.     

Changes in infant segment inertias during the first three months of independent walking

Background

During infancy, rapid changes in physical growth affect the size and shape of the body segments. To understand the effects of growth on movement, it is first necessary to quantify rates of development during the acquisition of important motor milestones. The goal of this longitudinal study was to quantify the physical growth of infant body segments during the initial stages of independent walking.

Methods

Ten infants (N = 10) aged between 28 and 55 weeks at the beginning of the study were tested biweekly (every two weeks) for three months. A 13-segment mathematical model of the human body was used to estimate the inertial parameters of the infant body segments at each session. An analysis of variance was used to test for significant differences in segment masses between biweekly measures. Polynomial contrasts were used to test for linear trends in the growth data.

Results

Significant differences between biweekly measures of segment mass were found only for the head/neck (F(5,45) = 3.42, p < 0.05), upper trunk (F(5,45) = 4.04, p < 0.01), and lower trunk (F(5,45) = 3.49, p < 0.01). The lower trunk demonstrated a linear increase in mass (F(1,9) = 4.56, p < 0.05). However, the upper trunk demonstrated a quadratic trend in growth (F(1,9) = 9.13, p < 0.01), while the head/neck segment showed a cubic trend in growth (F(1,9) = 3.80, p < 0.05). Significant differences in axial segment masses were also found between subjects (F(9,45) = 5.92, p < 0.001).

Conclusion

Given that postural control proceeds in a cephalocaudal manner, the lower trunk segment would be brought under control last, in terms of the axial segments. Increases in the mass of this segment could constrain the system, thereby acting as a control parameter for the onset and development of motor patterns.

     

Anamorphosis in millipedes (Diplopoda)—the present state of knowledge with some developmental and phylogenetic considerations

A review of the postembryonic development of millipedes (Diplopoda) is given, based mainly on published information. Original observations are, however, also included. Millipedes hatching from the pupoid usually have three pairs of legs; during their postembryonic growth they acquire more segments and more legs. This process is known as anamorphosis. Three types of anamorphosis are recognized. In euanamorphosis, every moult is accompanied by addition of new segments, even after the attainment of sexual maturity. In hemianamorphosis, the addition of new segments goes on until a certain stadium, and further moults take place without addition of segments. In teloanamorphosis, the addition of segments stops at a certain stadium (the adult, and ultimate, stadium) after which no further moults occur. Available information on anamorphosis in each of the millipede orders is reviewed. General patterns are emphasized, but variations are also considered in detail. It is shown that the so-called ‘law of anamorphosis’ is valid only for the ‘ring-forming’ millipedes (Merocheta and Juliformia) in which tergites, pleurites, and sternites of each diplosegment are firmly fused into a complete ‘ring’, and for some other forms (Polyzoniida, Chordeumatida), where there is a constant relationship between rings and legs. The chapter on the order Julida is particularly detailed and includes discussions of patterns in the variation and a section on periodomorphosis. The general chapter on developmental patterns includes inter alia an interpretation of the variations in millipede anamorphosis in terms of the ‘biometabolic modi’ of Remane. The hypothetical ancestral millipede is shown to have developed by hemianamorphosis. Euanamorphosis was acquired by the ancestral species of Helminthomorpha. Within this clade, Chordeumatida and Merocheta have secondarily become teloanamorphic, whereas some Juliformia seem to have returned to hemianamorphosis. The contrasting principles of elongation and contraction, subject of much debate among diplopodologists, are shown both to have played a role in the course of millipede evolution.     

The influence of mistletoes on the litter-layer arthropod abundance and diversity in a semi-arid savanna,Southwest Zimbabwe

Aims

The below-canopy soil moisture content and litter-layer arthropod abundance and diversity of Acacia karroo trees parasitized by each of three mistletoe species (Erianthemum ngamicum, Plicosepalus kalachariensis, and Viscum verrucosum) and uninfected A. karroo trees were investigated in semi-arid savanna, southwest Zimbabwe.

Results

The soils below the canopies of mistletoe-infected trees were significantly low in moisture content compared to those beneath uninfected A. karroo trees. Nevertheless, arthropod species diversity was greater by up to 34 % below the canopies of mistletoe-infected trees than beneath uninfected A. karroo trees, with greater abundances beneath trees infected by E. ngamicum and P. kalachariensis. In addition, the majority of the arthropod species associated with mistletoe-infected trees had litter as their dominant foraging substrate.

Conclusions

Our findings show that mistletoes increase the abundance and diversity of litter-dwelling and –foraging arthropods due to increase in the quality and quantity of litterfall beneath mistletoe-infected trees. By altering the below-canopy arthropod communities and soil moisture content, mistletoes have potential to modify ecosystem processes such as decomposition, soil process rates, and nutrient cycling. Therefore, we suggest that the resulting increase in resource heterogeneity plays an important role in determining the structure and functioning of semi-arid savanna ecosystems.     

The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues

The centipede Strigamia maritima forms all of its segments during embryogenesis. Trunk segments form sequentially from an apparently undifferentiated disk of cells at the posterior of the germ band. We have previously described periodic patterns of gene expression in this posterior disc that precede overt differentiation of segments, and suggested that a segmentation oscillator may be operating in the posterior disc. We now show that genes of the Notch signalling pathway, including the ligand Delta, and homologues of the Drosophila pair-rule genes even-skipped and hairy, show periodic expression in the posterior disc, consistent with their involvement in, or regulation by, such an oscillator. These genes are expressed in a pattern of apparently expanding concentric rings around the proctodeum, which become stripes at the base of the germ band where segments are emerging. In this transition zone, these primary stripes define a double segment periodicity: segmental stripes of engrailed expression, which mark the posterior of each segment, arise at two different phases of the primary pattern. Delta and even-skipped are also activated in secondary stripes that intercalate between primary stripes in this region, further defining the single segment repeat. These data, together with observations that Notch mediated signalling is required for segment pattern formation in other arthropods, suggest that the ancestral arthropod segmentation cascade may have involved a segmentation oscillator that utilised Notch signalling.     

Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus

Background

Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome.

Results

Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses.

Conclusion

Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.     

Mesothelioma mortality in Europe: impact of asbestos consumption and simian virus 40

Background

Hypophosphatasia (HP) is an inborn error of bone metabolism characterized by a genetic defect in the gene encoding the tissue-nonspecific alkaline phosphatase (TNSALP). There is a lack of knowledge as to how the variability and clinical severity of the HP phenotype (especially pain and walking impairment) are related to metabolic disturbances or impairments, subsequent to the molecular defect.

Methods

We analyzed the changes in clinical symptoms and the prostaglandin (PG) metabolism in response to treatment with non-steroidal anti-inflammatory drugs (NSAIDs) in six children affected by childhood HP. In addition, by exposing HP fibroblasts to pyridoxal phosphate and/or calcium pyrophosphate in vitro, we analyzed whether the alterations in PG levels are sequelae related to the metabolic defect.

Results

Childhood HP patients, who often complain about pain in the lower limbs without evident fractures, have systemic hyperprostaglandinism. Symptomatic anti-inflammatory treatment with NSAIDs significantly improved pain-associated physical impairment. Calcium pyrophosphate, but not pyridoxal phosphate, induced cyclooxygenase-2 (COX-2) gene expression and PG production in HP and normal fibroblasts in vitro.

Conclusion

Clinical features of childhood HP related to pain in the lower legs may be, at least in part, sequelae related to elevated PG levels, secondary to the primary metabolic defect. Consequently, NSAID treatment does improve the clinical features of childhood HP.     

Neural Markers Reveal a One-Segmented Head in Tardigrades (Water Bears)

Background

While recent neuroanatomical and gene expression studies have clarified the alignment of cephalic segments in arthropods and onychophorans, the identity of head segments in tardigrades remains controversial. In particular, it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure. To clarify this, we applied a variety of histochemical and immunocytochemical markers to specimens of the tardigrade Macrobiotus cf. harmsworthi and the onychophoran Euperipatoides rowelli.

Methodology/Principal Findings

Our immunolabelling against serotonin, FMRFamide and α-tubulin reveals that the tardigrade brain is a dorsal, bilaterally symmetric structure that resembles the brain of onychophorans and arthropods rather than a circumoesophageal ring typical of cycloneuralians (nematodes and allies). A suboesophageal ganglion is clearly lacking. Our data further reveal a hitherto unknown, unpaired stomatogastric ganglion in Macrobiotus cf. harmsworthi, which innervates the ectodermal oesophagus and the endodermal midgut and is associated with the second leg-bearing segment. In contrast, the oesophagus of the onychophoran E. rowelli possesses no immunoreactive neurons, whereas scattered bipolar, serotonin-like immunoreactive cell bodies are found in the midgut wall. Furthermore, our results show that the onychophoran pharynx is innervated by a medullary loop nerve accompanied by monopolar, serotonin-like immunoreactive cell bodies.

Conclusions/Significance

A comparison of the nervous system innervating the foregut and midgut structures in tardigrades and onychophorans to that of arthropods indicates that the stomatogastric ganglion is a potential synapomorphy of Tardigrada and Arthropoda. Its association with the second leg-bearing segment in tardigrades suggests that the second trunk ganglion is a homologue of the arthropod tritocerebrum, whereas the first ganglion corresponds to the deutocerebrum. We therefore conclude that the tardigrade brain consists of a single segmental region corresponding to the arthropod protocerebrum and, accordingly, that the tardigrade head is a non-composite, one-segmented structure.     

Sap flow-based quantitative indication of progression of Dutch elm disease after inoculation with Ophiostoma novo-ulmi

Key message

The rate of progression of Dutch elm disease can be continuously and quantitatively estimated from sap flow measurements.

Abstract

Response of sap flow to inoculation with Ophiostoma novo-ulmi, a causal agent which causes vascular mycosis called Dutch elm disease, was studied in a field experiment comprised of 4-year-old wych elm trees (Ulmus glabra). Sap flow was measured on inoculated trees using the trunk heat balance method with external heating (EMS 62, Czech Republic) throughout the experiment. The first detectable symptoms of reduction in sap flow occurred 6 days after inoculation and all inoculated trees died within 16 days. Our experiment confirmed the ability of O. novo-ulmi to quickly kill young elm trees. The disease progressed faster than in previous experiments utilizing O. ulmi. To the best of our knowledge, this is the first experiment using sap flow measurements on trees inoculated by O. novo-ulmi. The trunk heat balance sap flow method is an effective non-invasive tool for continuous quantitative monitoring of the progression of vascular tree diseases, and show increased potential for field and greenhouse studies on changes in xylem hydraulic conductivity in a wide range of broadleaved and coniferous tree species.     

Association between walking ability and trunk and lower-limb muscle atrophy in institutionalized elderly women: a longitudinal pilot study

Background

The aim of this study was to investigate the association between walking ability and muscle atrophy in the trunk and lower limbs.

Methods

Subjects in this longitudinal study were 21 elderly women who resided in nursing homes. The thicknesses of the following trunk and lower-limb muscles were measured using B-mode ultrasound: rectus abdominis, external oblique, internal oblique, transversus abdominis, erector spinae, lumbar multifidus, psoas major, gluteus maximus, gluteus medius, gluteus minimus, rectus femoris, vastus lateralis, vastus intermedius, biceps femoris, gastrocnemius, soleus, and tibialis anterior. Maximum walking speed was used to represent walking ability. Maximum walking speed and muscle thickness were assessed before and after a 12-month period.

Results

Of the 17 measured muscles of the trunk and lower limbs, age-related muscle atrophy in elderly women was greatest in the erector spinae, rectus femoris, vastus lateralis, vastus intermedius, and tibialis anterior muscles. Correlation coefficient analyses showed that only the rate of thinning of the vastus lateralis was significantly associated with the rate of decline in maximum walking speed (r = 0.518, p < 0.05).

Conclusions

This longitudinal study suggests that reduced walking ability may be associated with muscle atrophy in the trunk and lower limbs, especially in the vastus lateralis muscle, among frail elderly women.     

Exclusive Male Egg Care and Determinants of Brooding Success in a Millipede

The evolution of exclusive male parental care is a major and controversial issue in behavioural ecology. Although arthropods practicing paternal care are thought to be key taxa for investigating this issue, few studies have attempted to clarify the selection factors associated with male behaviour and fitness consequences in arthropods. In the millipede Brachycybe nodulosa, males curl their bodies around egg masses on the undersides of decaying logs. Male‐removal experiments in the laboratory strongly suggest that males defend the eggs against fungal infection. Orphaned eggs were soon covered by hyphae and no eggs hatched, whereas almost all eggs brooded by males successfully hatched. The egg‐brooding males showed no aggressive responses when disturbed. Only some mature males bred in the field. Furthermore, the number of eggs brooded varied greatly among the males. Selected generalized linear models revealed that males with a wide seventh body segment, which possesses gonopods (genital legs), tended to succeed in brooding; and males with a wider body also obtained more eggs. Colony attributes had no significant effects on male brooding. We discuss the possible sexual selection mechanisms that could accomplish this pattern of brooding success among male B. nodulosa.     

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.