BACKGROUND
the nematode caenorhabditis elegans has long served as an exceptional model system for understanding diverse biological phenomena such as development, neurobiology and genome structure  <cit> .
over the last decade nematodes in the caenorhabditis genus have come to serve as increasingly powerful evolutionary models for studying diverse processes ranging from the mutation rate  <cit>  to the evolution of reproductive mode transitions  <cit>  to speciation mechanisms  <cit> .
although c. elegans and c. briggsae were originally thought to share a common origin of hermaphroditic reproduction, more recent molecular phylogenetic approaches have shown independent origins of hermaphroditism for these two caenorhabditis species  <cit> .
there exists over one million genetically diverse nematode species outside of caenorhabditis  <cit>  that are also coming to serve roles as models for other evolutionary phenomena.
for example, nematodes in the genera strongyloides and rhabditophanes are growing models for understanding the evolution of parasitism  <cit> ; oscheius and pristionchus nematodes offer strong models for investigating the evolution of developmental processes  <cit> .
multiple independent evolutionary transitions from gonochoristic to hermaphroditic reproduction have also been observed in the latter two nematode genera  <cit>  and a broad review of numerous rhabditid nematodes showed up to ten independent transitions to hermaphroditism  <cit> .
panagrolaimus nematodes have also played roles in comparative evolution of development studies  <cit> , and panagrolaimus constitutes the only nematode genus in which three different reproductive modes  have been reported.
well-studied parthenogenetic strains of panagrolaimus include a number of isolates from north america  as well as p. davidi from antarctica; the latter has been shown to reproduce through meiotic parthenogenesis  <cit> .
more recently this group has emerged as a model for understanding the physiological and molecular mechanisms underlying anhydrobiosis and extreme cold tolerance, due in part to the discovery of p. davidi in antarctica.
this species has evolved multiple strategies to deal with extreme cold including cryoprotective dehydration and the prevention and tolerance of intracellular freezing in adults and eggs  <cit> .
a recent comparative analysis of extreme cold tolerance showed that the majority of individuals in experimental lab populations of p. davidi survived -15°c treatments  whereas much lower survival rates  were observed in five other nematode species surveyed, including p. davidi's congener p. rigidus  <cit> .
panagrolaimus strain ps <dig> survived over eight years in a state of anhydrobiosis  <cit> .
a previous phylogenetic analysis of panagrolaimus species and strain relationships based on ribosomal rna  internal transcribed spacer regions showed that a number of strains in the same monophyletic clade as p. davidi were able to survive desiccation treatments at high rates whereas others outside that clade were much more sensitive to desiccation  <cit> .
although this initial study provided important insights into the evolutionary relationships of different panagrolaimus strains and species, it was limited by inclusion of only eleven panagrolaimus strains and the use of only one very distantly related outgroup .
the evolutionary origins of varying reproductive modes across different panagrolaimus lineages remain unknown, as do the origins of the lineage leading to the antarctic species p. davidi.
here we provide an extensive evolutionary analysis of thirty-one panagrolaimus species and strains that utilizes both slowly-evolving nuclear loci and a faster-evolving mitochondrial locus to provide insights into the phylogenetic relationships of different lineages and to map the origins of parthenogenetic and hermaphroditic reproductive modes.
strain-specific reproductive modes were analyzed by assaying for the presence of sperm using microscopy techniques.
to provide a rough estimate of the general timeline for p. davidi's arrival in antarctica, we extended a molecular clock approach previously applied to caenorhabditis  <cit>  to approximate the divergence time between p. davidi and its closest known relative.
RESULTS
panagrolaimus strains and reproductive mode analysis
we initiated our study by requesting available panagrolaimus strains from the caenorhabditis genetics center and other helpful colleagues .
further, in the summer of  <dig> we sampled soils from diverse locations around oregon and discovered seven additional panagrolaimus isolate strains from which laboratory cultures were generated and frozen stocks were made.
a total of thirty-one strains were collected from our colleagues and the field that were successfully established in laboratory culture .
four additional nematode strains isolated in monroe, oregon, usa  were used for molecular analyses but were not kept in long-term laboratory culture.
we are able to maintain the thirty-one panagrolaimus strains listed in table  <dig> at 25°c using standard op <dig> escherichia coli-seeded ngm plates.
we are also able to cryogenically preserve all strains in glycerol solutions using standard c. elegans techniques  <cit> , and to developmentally synchronize lab populations using the hypochlorite treatment method  <cit> .
we discovered that multiple larval and adult developmental stages of panagrolaimus nematodes survived cryogenic storage whereas in c. elegans only l1-stage larvae generally survive.
we kept track of generation times and brood sizes for two panagrolaimus strains  and found that they experience generation times of approximately seven days and brood sizes of 300– <dig> eggs under the aforementioned laboratory culturing conditions.
thus, panagrolaimus offers many of the same basic attractive features that have contributed to the outstanding success of the c. elegans system.
furthermore, rna interference-mediated gene silencing has recently been documented to be effective in p. superbus and the parthenogenetic panagrolaimus strain ps <dig>  <cit> .
strains sources are: jb, james g. baldwin; ab, ann burnell; dl, denver lab; maf, marie-anne félix; es, einhard schierenberg; ps, paul w. sternberg; dw, david wharton.
modes of reproduction  are: g, gonochoristic; h, hermaphroditic; p parthenogenetic.
we initially distinguished between gonochoristic and self-fertile panagrolaimus strains by carrying out self-fertility tests whereby early larval stage nematodes were individually transferred to plates and then allowed to develop in isolation.
strains repeatedly unable to carry out self-fertilization were defined as gonochoristic – in all cases, mass populations of strains found to be gonochoristic by this criterion were also observed to contain large numbers of mating males.
our definitions of gonochoristic strains were also consistent with previous independent observations  <cit> .
for all self-fertile strains, we tested for the presence or absence of sperm using dic/epifluorescence microscopy techniques .
three self-fertile panagrolaimus species/strains  were observed to produce sperm  – we consider these to most likely be hermaphroditic, though we cannot rule out the possibility that the sperm is only used for egg activation and does not contribute genetic material.
for all other self-fertile panagrolaimus strains examined, no sperm was detected and we consider them to be parthenogenetic .
in mass laboratory populations, males are frequently observed for p. detritophagus and occasionally observed for ju <dig> and ps <dig>  we have not observed males in any of the parthenogenetic strains, though a focused study on male frequencies has not yet been carried out.
males were observed to be abundant in field populations of the antarctic species p. davidi whereas parthenogenetic females were observed to dominate long-term lab cultures  <cit> .
panagrolaimus evolutionary relationships
we applied a molecular phylogenetic approach to understand the evolutionary relationships of panagrolaimus species and strains, and to map the origins of reproductive mode transitions in this nematode genus.
we pcr-amplified and directly sequenced segments of the 18s and 28s rrna genes from genomic dna samples derived from each of the thirty-one panagrolaimus strains .
amplified sequences were subjected to multiple alignment using clustalw in the program mega <dig>  <cit> ; subsequently numerous orthologous outgroup sequences representing diverse nematode taxa of varying evolutionary distances from panagrolaimus were also included in the alignment .
after making minor corrections to the alignment based on visual inspection, we performed phylogenetic analyses in mega <dig> using both neighbor-joining  and maximum parsimony  approaches .
a total of  <dig> aligned nucleotide sites were analyzed for 18s rrna;  <dig> aligned nucleotide sites were analyzed for 28s rrna.
concatenated 18s and 28s sequences  were used for phylogenetic analyses.
additional analyses involved a segment  of the more rapidly evolving mitochondrial nadh dehydrogenase subunit  <dig>  gene to independently test the results of nuclear rrna analyses and further analyze molecular diversification patterns.
nd <dig> sequences were successfully obtained for all panagrolaimus except dl <dig>  and es <dig> .
as with the nuclear loci, aligned nd <dig> sequences were subject to mp and nj phylogenetic analyses in mega <dig> .one thousand bootstrap replicates were performed in each analysis to estimate confidence in nodes resulting from the analyses.
nj and mp analyses of the aligned nuclear rrna data yielded highly congruent bootstrap consensus trees .
although there was a single well-supported monophyletic clade  that exclusively contained 25/ <dig> panagrolaimus sequences analyzed, four other panagrolaimus sequences  were included with a separate monophyletic clade, referred to herein as clade pii, also containing sequences from halicephalobus gingivalis  <cit>  and related isolates.
the sequence from strain af <dig> was grouped with the rhabditid oscheius tipulae and the sequence from strain bw <dig> was grouped with c. elegans and its congener c. briggsae.
a nearly complete mitochondrial genome sequence was previously generated for bw <dig> which confirmed that it is a strain of c. briggsae  <cit> .
these two strains historically identified as members of this genus  are clearly members of the very distantly related rhabditid group of nematodes and likely constitute simple accidental cases of misidentification or laboratory contamination.
our results suggest that a taxonomic revision is required for panagrolaimus since those strains historically identified as members of the genus do not compose a monophyletic group.
there are two approaches to solving this dilemma given the phylogeny shown in figure 3: one would be to remove the four pii panagrolaimus strains from the genus rendering pi the monophyletic panagrolaimus genus; the second would be to reassign halicephalobus gingivalis and related nematodes to the genus panagrolaimus rendering the monophyletic clade containing pi and pii the panagrolaimus genus.
proper systematic revision of this nematode group and its relatives, though clearly necessary, will require a more broad-based analysis involving additional nematode genera , more genetic markers and reconsideration of morphological features  <cit> .
rather than proposing specific taxonomic revisions for the present study whose focus is outside of these specific systematic issues, we will maintain the historical panagraimus identifiers listed in table  <dig> and refer to clades pi and pii  to distinguish between different panagrolaimus subgroups.
evolution of reproductive mode transitions
we next mapped reproductive modes onto the rrna consensus phylogeny to identify the evolutionary origins of reproductive mode transitions.
within clade pi we identified a monophyletic subclade  that exclusively contained all of the parthenogenetic pangrolaimus strains and species .
this observation indicates a single evolutionary origin of parthenogenetic reproduction in panagrolaimus.
the remaining clade pi species and strains outside of subclade pip were all gonochoristic – additional file  <dig> provides a summary of knowledge on species boundaries among the pi gonochoristic strains.
the three hermaphroditic panagrolaimus species and strains  were all placed outside of clade pi and were included in a separate monophyletic clade  containing one gonochoristic panagrolaimus species  along with the opportunistic equine  pathogen halicephalobus gingivalis  <cit>  and related nematodes  that we did not maintain in long-term culture.
the nuclear phylogeny suggests a single origin of hermaphroditism in pii, although the small number of pii panagrolaimus strains available for analysis precludes any strong conclusions.
in the pi clade there appears to have been a single evolutionary transition to parthenogenesis; among the pii panagrolaimus strains the phylogeny suggests a single transition to hermaphroditism.
by contrast, there is evidence for at least five independent transitions to hermaphroditism in pristionchus  <cit> , three in oscheius  <cit> , and three in caenorhabditis .
although this might superficially suggest a reduced plasticity in sex determination mechanisms regulating hermaphroditic versus female development in panagrolaimus versus other well-studied nematode genera, panagrolaimus likely suffers more than the other genera in terms of taxon undersampling – our observations in pii are based on only four panagrolaimus strains.
although a more rigorous sampling of nematodes in the pii clade is required for a robust understanding of reproductive mode transitions in this group, it is interesting to note that h. gingivalis in pii reproduces parthenogenetically in infected horses  <cit> .
pii thus might represent a nematode group of exceptional sex determination plasticity able to explore multiple reproductive mode strategies.
molecular diversification in the parthenogens
in addition to illuminating the broad-based evolutionary relationships of all nematodes historically placed in the panagrolaimus genus, our rrna analyses also provided evidence for subdivision of the pip clade of parthenogens into three subclades .
to further investigate divergence in the pip clade, we analyzed a segment of the more rapidly evolving nd <dig> gene from a subset of panagrolaimus strains to independently test the results of nuclear rrna analyses and further analyze diversification within the pip clade of parthenogens.
sequences were successfully obtained for 13/ <dig> of the pip parthenogens – we were repeatedly unable to pcr amplify the nd <dig> fragment from dl <dig> .
the mitochondrial nd <dig> gene nj phylogeny  was highly congruent with the nuclear rrna phylogeny .
the pi, hi and pip clades were each supported with strong  boostrap support in nj analysis of mtdna sequence.
clade pip was also monophyletic in mp analysis of mtdna, though with low boostrap support; similarly clade pii was monophyletic in mp mtdna analysis with low bootstrap support .
clade pi, however, was strongly supported in both nj and mp analyses of the nd <dig> sequences; furthermore, topological arrangement of all strain analyzed was identical for nj and mp analyses of nd <dig> .
the distribution of parthenogenetic strains in clade pip subclades in nd <dig> phylogenetic analyses was the same for that of nuclear rrna analyses.
among the thirteen pip panagrolaimus parthenogenetic strains considered in mtdna analysis, seven unique nd <dig> haplotypes were observed.
one haplotype was shared by six strains – three were from california, usa, the fourth was from north carolina, usa, the fifth was from baja california, mexico and the sixth was from senegal.
the sharing of a mtdna haplotype between strains from north america with one from the west coast of africa might indicate that panagrolaimus strains are able to very rapidly disperse across large geographic distances, as has been proposed for c. elegans where identical mitochondrial genome haplotypes have also been observed for geographically disparate isolates  <cit> .
like caenorhabditis and pristionchus  <cit> , panagrolaimus has recently been described to associate with arthropod hosts that might aid in its geographic dispersal  <cit> .
the three pip oregon strains analyzed formed a well-supported monophyletic group and all had highly similar nd <dig> haplotypes.
strain ps <dig> from china had a unique nd <dig> haplotype.
ps <dig> and ps <dig> were found to share a haplotype.
p. davidi cb <dig> and these two california strains  were again found to form a well-supported monophyletic clade and share highly similar nd <dig> sequences , suggesting that the antarctic strain p. davidi cb <dig> and these strains from california shared a common ancestor in the not-too-distant past.
arrival and evolution of p. davidi in antarctica
two independent molecular phylogenies presented here  show very close evolutionary relationships between two pip parthenogens from southern california  and p. davidi cb <dig> from antarctica.
to extend upon the phylogenetic results, we applied a molecular clock approach to estimate the number of nematode generations that separate ps <dig> and p. davidi cb <dig>  under the neutral theory of molecular evolution  <cit> , the rate of mutation is expected to be equal to the rate of substitution at neutral sites, and the time to the most recent common ancestor  for any two taxa for which there is relevant dna sequence data can be estimated by the equation t = k/ where t is the time to the most recent common ancestor, k is a measure of the neutral molecular divergence between the compared taxa and μ is the mutation rate  <cit> .
for the nematode c. elegans, there are direct estimates of the per-generation nuclear and mtdna mutation rates based on mutation-accumulation line systems  <cit> .
the nuclear rate estimate was recently employed in an analysis of divergence times among caenorhabditis species utilizing an internally calibrated molecular clock approach  <cit> .
we applied a similar  approach here, assuming that the panagrolaimus mtdna μ is the same as that estimated for c. elegans, to estimate the tmrca for ps <dig> from california and p. davidi from antarctica.
to provide a broader mtdna data set for evolutionary rate analysis, we collected additional mtdna protein-coding gene sequences for ps <dig> and p. davidi cb1; data examined includes sequences from cytochrome b, cytochrome oxidase i , nd <dig> and nd5;  <dig>  bp,  <dig> codons total.
we employed dnasp v <dig>   <cit>  to calculate the rate of silent-site substitution , a value often used to estimate neutral evolutionary rates, in the concatenated protein-coding gene sequence pairwise alignment – a ds value of  <dig>  was estimated from the ps1579-p. davidi alignment.
employing the mtdna ds  estimate suggests a rough tmrca value of  <dig>  nematode generations for ps <dig> and p. davidi.
translating the number of nematode generations into actual  time is problematic due to uncertainties in the numbers of generations experienced by panagrolaimus in the wild – a general problem for nematodes  <cit> .
although ps <dig> was observed to experience generation times of seven days in the lab at 25°c under food-rich conditions, suggesting approximately fifty-two generations per year, massive environmental differences between the lab and nature  almost certainly affect the number of panagrolaimus generations per year in nature.
in particular, p. davidi's native antarctic environment likely provides conditions where nematodes are able to reproduce for a few months  per year  <cit> .
by contrast, the much warmer climate of southern california experienced by ps <dig> likely enables a much greater number of generations per year.
additional file  <dig> shows the relationship between tmrca estimates  for ps <dig> and p. davidi and assumed numbers of nematode generations per year.
if ps <dig> and p. davidi have both experienced as few as ten generations per year since their divergence, our analyses based on ds roughly approximate a tmrca of only ~ <dig>  years.
even if these two taxa have each experienced only one generation per year since their divergence, a tmrca of only ~ <dig>  years is approximated.
these tmrca measures should be interpreted with great caution, however, given the untested assumptions underlying these particular molecular clock analyses – for example, that mtdna mutation rates are the same for c. elegans and panagrolaimus and that the rate of mutation equals the rate of silent-site substitution.
nonetheless, the very close genetic distances between ps <dig>  ps <dig> and p. davidi cb <dig> along with the phylogenies presented in figures  <dig> and  <dig> also generally suggest that p. davidi colonized antarctica in the very recent past.
did p. davidi adaptively evolve extreme cold tolerance after its arrival in antarctica?
although a current comparative cryotolerance study involving p. davidi showed that this species was unique in its extreme cold tolerance as compared to other nematode species examined, the other nematodes used for comparison in the study were very distantly related to p. davidi – the closest relative was p. rigidus that our phylogenetic and genetic distance analyses show is very distantly related to p. davidi.
future comparative cryotolerance studies involving p. davidi's closely-related parthenogen relatives  would shed light on whether extreme cold tolerance is a feature unique to p. davidi, or if it is shared by its closely-related parthenogenetic relatives.
if the latter scenario is true, it would suggest that p. davidi arrived in antarctica in a "pre-adapted", cryotolerant state.
if the latter scenario is not true, it would suggest that p. davidi was able to very rapidly adapt to the harsh antarctic environment.
CONCLUSIONS
our study sheds light on the evolutionary relationships of panagrolaimus nematodes, showing that the traditional genus is paraphyletic.
our analyses suggest a single origin of parthenogenesis in panagrolaimus and provide evidence that p. davidi arrived in antarctica in the very recent evolutionary past.
this nematode genus offers an outstanding model for analyzing the evolutionary and biological causes of transitions from gonochoristic to parthenogenetic reproduction, and the adaptive evolution of extreme cold tolerance.
we also note here that a brief soil survey effort in northwest oregon performed by a single undergraduate student  resulted in the discovery two probable new gonochoristic species of panagrolaimus  and a new major clade of pip parthenogens.
the worldwide diversity of panagrolaimus and other nematode species in the soil and other ecologies likely remains vastly undersurveyed, being a key limiting factor in our understanding of evolutionary processes in this diverse animal phylum.

 the nematode caenorhabditis elegans has long served as an exceptional model system for understanding diverse biological phenomena such as development, neurobiology and genome structure. over the last decade nematodes in the caenorhabditis genus have come to serve as increasingly powerful evolutionary models for studying diverse processes ranging from the mutation rate to the evolution of reproductive mode transitions to speciation mechanisms. although c. elegans and c. briggsae were originally thought to share a common origin of hermaphroditic reproduction, more recent molecular phylogenetic approaches have shown independent origins of hermaphroditism for these two caenorhabditis species. there exists over one million genetically diverse nematode species outside of caenorhabditis that are also coming to serve roles as models for other evolutionary phenomena. for example, nematodes in the genera strongyloides and rhabditophanes are growing models for understanding the evolution of parasitism; oscheius and pristionchus nematodes offer strong models for investigating the evolution of developmental processes. multiple independent evolutionary transitions from gonochoristic to hermaphroditic reproduction have also been observed in the latter two nematode genera and a broad review of numerous rhabditid nematodes showed up to ten independent transitions to hermaphroditism. panagrolaimus nematodes have also played roles in comparative evolution of development studies, and panagrolaimus constitutes the only nematode genus in which three different reproductive modes have been reported. well-studied parthenogenetic strains of panagrolaimus include a number of isolates from north america as well as p. davidi from antarctica; the latter has been shown to reproduce through meiotic parthenogenesis. more recently this group has emerged as a model for understanding the physiological and molecular mechanisms underlying anhydrobiosis and extreme cold tolerance, due in part to the discovery of p. davidi in antarctica. this species has evolved multiple strategies to deal with extreme cold including cryoprotective dehydration and the prevention and tolerance of intracellular freezing in adults and eggs. a recent comparative analysis of extreme cold tolerance showed that the majority of individuals in experimental lab populations of p. davidi survived -15°c treatments whereas much lower survival rates were observed in five other nematode species surveyed, including p. davidi's congener p. rigidus. panagrolaimus strain ps survived over eight years in a state of anhydrobiosis. a previous phylogenetic analysis of panagrolaimus species and strain relationships based on ribosomal rna internal transcribed spacer regions showed that a number of strains in the same monophyletic clade as p. davidi were able to survive desiccation treatments at high rates whereas others outside that clade were much more sensitive to desiccation. although this initial study provided important insights into the evolutionary relationships of different panagrolaimus strains and species, it was limited by inclusion of only eleven panagrolaimus strains and the use of only one very distantly related outgroup. the evolutionary origins of varying reproductive modes across different panagrolaimus lineages remain unknown, as do the origins of the lineage leading to the antarctic species p. davidi. here we provide an extensive evolutionary analysis of thirty-one panagrolaimus species and strains that utilizes both slowly-evolving nuclear loci and a faster-evolving mitochondrial locus to provide insights into the phylogenetic relationships of different lineages and to map the origins of parthenogenetic and hermaphroditic reproductive modes. strain-specific reproductive modes were analyzed by assaying for the presence of sperm using microscopy techniques. to provide a rough estimate of the general timeline for p. davidi's arrival in antarctica, we extended a molecular clock approach previously applied to caenorhabditis to approximate the divergence time between p. davidi and its closest known relative.  panagrolaimus strains and reproductive mode analysis we initiated our study by requesting available panagrolaimus strains from the caenorhabditis genetics center and other helpful colleagues. further, in the summer of we sampled soils from diverse locations around oregon and discovered seven additional panagrolaimus isolate strains from which laboratory cultures were generated and frozen stocks were made. a total of thirty-one strains were collected from our colleagues and the field that were successfully established in laboratory culture. four additional nematode strains isolated in monroe, oregon, usa were used for molecular analyses but were not kept in long-term laboratory culture. we are able to maintain the thirty-one panagrolaimus strains listed in table at 25°c using standard op escherichia coli-seeded ngm plates. we are also able to cryogenically preserve all strains in glycerol solutions using standard c. elegans techniques, and to developmentally synchronize lab populations using the hypochlorite treatment method. we discovered that multiple larval and adult developmental stages of panagrolaimus nematodes survived cryogenic storage whereas in c. elegans only l1-stage larvae generally survive. we kept track of generation times and brood sizes for two panagrolaimus strains and found that they experience generation times of approximately seven days and brood sizes of 300– eggs under the aforementioned laboratory culturing conditions. thus, panagrolaimus offers many of the same basic attractive features that have contributed to the outstanding success of the c. elegans system. furthermore, rna interference-mediated gene silencing has recently been documented to be effective in p. superbus and the parthenogenetic panagrolaimus strain ps. strains sources are: jb, james g. baldwin; ab, ann burnell; dl, denver lab; maf, marie-anne félix; es, einhard schierenberg; ps, paul w. sternberg; dw, david wharton. modes of reproduction are: g, gonochoristic; h, hermaphroditic; p parthenogenetic. we initially distinguished between gonochoristic and self-fertile panagrolaimus strains by carrying out self-fertility tests whereby early larval stage nematodes were individually transferred to plates and then allowed to develop in isolation. strains repeatedly unable to carry out self-fertilization were defined as gonochoristic – in all cases, mass populations of strains found to be gonochoristic by this criterion were also observed to contain large numbers of mating males. our definitions of gonochoristic strains were also consistent with previous independent observations. for all self-fertile strains, we tested for the presence or absence of sperm using dic/epifluorescence microscopy techniques. three self-fertile panagrolaimus species/strains were observed to produce sperm – we consider these to most likely be hermaphroditic, though we cannot rule out the possibility that the sperm is only used for egg activation and does not contribute genetic material. for all other self-fertile panagrolaimus strains examined, no sperm was detected and we consider them to be parthenogenetic. in mass laboratory populations, males are frequently observed for p. detritophagus and occasionally observed for ju and ps we have not observed males in any of the parthenogenetic strains, though a focused study on male frequencies has not yet been carried out. males were observed to be abundant in field populations of the antarctic species p. davidi whereas parthenogenetic females were observed to dominate long-term lab cultures. panagrolaimus evolutionary relationships we applied a molecular phylogenetic approach to understand the evolutionary relationships of panagrolaimus species and strains, and to map the origins of reproductive mode transitions in this nematode genus. we pcr-amplified and directly sequenced segments of the 18s and 28s rrna genes from genomic dna samples derived from each of the thirty-one panagrolaimus strains. amplified sequences were subjected to multiple alignment using clustalw in the program mega; subsequently numerous orthologous outgroup sequences representing diverse nematode taxa of varying evolutionary distances from panagrolaimus were also included in the alignment. after making minor corrections to the alignment based on visual inspection, we performed phylogenetic analyses in mega using both neighbor-joining and maximum parsimony approaches. a total of aligned nucleotide sites were analyzed for 18s rrna; aligned nucleotide sites were analyzed for 28s rrna. concatenated 18s and 28s sequences were used for phylogenetic analyses. additional analyses involved a segment of the more rapidly evolving mitochondrial nadh dehydrogenase subunit gene to independently test the results of nuclear rrna analyses and further analyze molecular diversification patterns. nd sequences were successfully obtained for all panagrolaimus except dl and es. as with the nuclear loci, aligned nd sequences were subject to mp and nj phylogenetic analyses in mega.one thousand bootstrap replicates were performed in each analysis to estimate confidence in nodes resulting from the analyses. nj and mp analyses of the aligned nuclear rrna data yielded highly congruent bootstrap consensus trees. although there was a single well-supported monophyletic clade that exclusively contained 25/ panagrolaimus sequences analyzed, four other panagrolaimus sequences were included with a separate monophyletic clade, referred to herein as clade pii, also containing sequences from halicephalobus gingivalis and related isolates. the sequence from strain af was grouped with the rhabditid oscheius tipulae and the sequence from strain bw was grouped with c. elegans and its congener c. briggsae. a nearly complete mitochondrial genome sequence was previously generated for bw which confirmed that it is a strain of c. briggsae. these two strains historically identified as members of this genus are clearly members of the very distantly related rhabditid group of nematodes and likely constitute simple accidental cases of misidentification or laboratory contamination. our results suggest that a taxonomic revision is required for panagrolaimus since those strains historically identified as members of the genus do not compose a monophyletic group. there are two approaches to solving this dilemma given the phylogeny shown in figure 3: one would be to remove the four pii panagrolaimus strains from the genus rendering pi the monophyletic panagrolaimus genus; the second would be to reassign halicephalobus gingivalis and related nematodes to the genus panagrolaimus rendering the monophyletic clade containing pi and pii the panagrolaimus genus. proper systematic revision of this nematode group and its relatives, though clearly necessary, will require a more broad-based analysis involving additional nematode genera, more genetic markers and reconsideration of morphological features. rather than proposing specific taxonomic revisions for the present study whose focus is outside of these specific systematic issues, we will maintain the historical panagraimus identifiers listed in table and refer to clades pi and pii to distinguish between different panagrolaimus subgroups. evolution of reproductive mode transitions we next mapped reproductive modes onto the rrna consensus phylogeny to identify the evolutionary origins of reproductive mode transitions. within clade pi we identified a monophyletic subclade that exclusively contained all of the parthenogenetic pangrolaimus strains and species. this observation indicates a single evolutionary origin of parthenogenetic reproduction in panagrolaimus. the remaining clade pi species and strains outside of subclade pip were all gonochoristic – additional file provides a summary of knowledge on species boundaries among the pi gonochoristic strains. the three hermaphroditic panagrolaimus species and strains were all placed outside of clade pi and were included in a separate monophyletic clade containing one gonochoristic panagrolaimus species along with the opportunistic equine pathogen halicephalobus gingivalis and related nematodes that we did not maintain in long-term culture. the nuclear phylogeny suggests a single origin of hermaphroditism in pii, although the small number of pii panagrolaimus strains available for analysis precludes any strong conclusions. in the pi clade there appears to have been a single evolutionary transition to parthenogenesis; among the pii panagrolaimus strains the phylogeny suggests a single transition to hermaphroditism. by contrast, there is evidence for at least five independent transitions to hermaphroditism in pristionchus, three in oscheius, and three in caenorhabditis. although this might superficially suggest a reduced plasticity in sex determination mechanisms regulating hermaphroditic versus female development in panagrolaimus versus other well-studied nematode genera, panagrolaimus likely suffers more than the other genera in terms of taxon undersampling – our observations in pii are based on only four panagrolaimus strains. although a more rigorous sampling of nematodes in the pii clade is required for a robust understanding of reproductive mode transitions in this group, it is interesting to note that h. gingivalis in pii reproduces parthenogenetically in infected horses. pii thus might represent a nematode group of exceptional sex determination plasticity able to explore multiple reproductive mode strategies. molecular diversification in the parthenogens in addition to illuminating the broad-based evolutionary relationships of all nematodes historically placed in the panagrolaimus genus, our rrna analyses also provided evidence for subdivision of the pip clade of parthenogens into three subclades. to further investigate divergence in the pip clade, we analyzed a segment of the more rapidly evolving nd gene from a subset of panagrolaimus strains to independently test the results of nuclear rrna analyses and further analyze diversification within the pip clade of parthenogens. sequences were successfully obtained for 13/ of the pip parthenogens – we were repeatedly unable to pcr amplify the nd fragment from dl. the mitochondrial nd gene nj phylogeny was highly congruent with the nuclear rrna phylogeny. the pi, hi and pip clades were each supported with strong boostrap support in nj analysis of mtdna sequence. clade pip was also monophyletic in mp analysis of mtdna, though with low boostrap support; similarly clade pii was monophyletic in mp mtdna analysis with low bootstrap support. clade pi, however, was strongly supported in both nj and mp analyses of the nd sequences; furthermore, topological arrangement of all strain analyzed was identical for nj and mp analyses of nd. the distribution of parthenogenetic strains in clade pip subclades in nd phylogenetic analyses was the same for that of nuclear rrna analyses. among the thirteen pip panagrolaimus parthenogenetic strains considered in mtdna analysis, seven unique nd haplotypes were observed. one haplotype was shared by six strains – three were from california, usa, the fourth was from north carolina, usa, the fifth was from baja california, mexico and the sixth was from senegal. the sharing of a mtdna haplotype between strains from north america with one from the west coast of africa might indicate that panagrolaimus strains are able to very rapidly disperse across large geographic distances, as has been proposed for c. elegans where identical mitochondrial genome haplotypes have also been observed for geographically disparate isolates. like caenorhabditis and pristionchus, panagrolaimus has recently been described to associate with arthropod hosts that might aid in its geographic dispersal. the three pip oregon strains analyzed formed a well-supported monophyletic group and all had highly similar nd haplotypes. strain ps from china had a unique nd haplotype. ps and ps were found to share a haplotype. p. davidi cb and these two california strains were again found to form a well-supported monophyletic clade and share highly similar nd sequences, suggesting that the antarctic strain p. davidi cb and these strains from california shared a common ancestor in the not-too-distant past. arrival and evolution of p. davidi in antarctica two independent molecular phylogenies presented here show very close evolutionary relationships between two pip parthenogens from southern california and p. davidi cb from antarctica. to extend upon the phylogenetic results, we applied a molecular clock approach to estimate the number of nematode generations that separate ps and p. davidi cb under the neutral theory of molecular evolution, the rate of mutation is expected to be equal to the rate of substitution at neutral sites, and the time to the most recent common ancestor for any two taxa for which there is relevant dna sequence data can be estimated by the equation t = k/ where t is the time to the most recent common ancestor, k is a measure of the neutral molecular divergence between the compared taxa and μ is the mutation rate. for the nematode c. elegans, there are direct estimates of the per-generation nuclear and mtdna mutation rates based on mutation-accumulation line systems. the nuclear rate estimate was recently employed in an analysis of divergence times among caenorhabditis species utilizing an internally calibrated molecular clock approach. we applied a similar approach here, assuming that the panagrolaimus mtdna μ is the same as that estimated for c. elegans, to estimate the tmrca for ps from california and p. davidi from antarctica. to provide a broader mtdna data set for evolutionary rate analysis, we collected additional mtdna protein-coding gene sequences for ps and p. davidi cb1; data examined includes sequences from cytochrome b, cytochrome oxidase i, nd and nd5; bp, codons total. we employed dnasp v to calculate the rate of silent-site substitution, a value often used to estimate neutral evolutionary rates, in the concatenated protein-coding gene sequence pairwise alignment – a ds value of was estimated from the ps1579-p. davidi alignment. employing the mtdna ds estimate suggests a rough tmrca value of nematode generations for ps and p. davidi. translating the number of nematode generations into actual time is problematic due to uncertainties in the numbers of generations experienced by panagrolaimus in the wild – a general problem for nematodes. although ps was observed to experience generation times of seven days in the lab at 25°c under food-rich conditions, suggesting approximately fifty-two generations per year, massive environmental differences between the lab and nature almost certainly affect the number of panagrolaimus generations per year in nature. in particular, p. davidi's native antarctic environment likely provides conditions where nematodes are able to reproduce for a few months per year. by contrast, the much warmer climate of southern california experienced by ps likely enables a much greater number of generations per year. additional file shows the relationship between tmrca estimates for ps and p. davidi and assumed numbers of nematode generations per year. if ps and p. davidi have both experienced as few as ten generations per year since their divergence, our analyses based on ds roughly approximate a tmrca of only ~ years. even if these two taxa have each experienced only one generation per year since their divergence, a tmrca of only ~ years is approximated. these tmrca measures should be interpreted with great caution, however, given the untested assumptions underlying these particular molecular clock analyses – for example, that mtdna mutation rates are the same for c. elegans and panagrolaimus and that the rate of mutation equals the rate of silent-site substitution. nonetheless, the very close genetic distances between ps ps and p. davidi cb along with the phylogenies presented in figures and also generally suggest that p. davidi colonized antarctica in the very recent past. did p. davidi adaptively evolve extreme cold tolerance after its arrival in antarctica? although a current comparative cryotolerance study involving p. davidi showed that this species was unique in its extreme cold tolerance as compared to other nematode species examined, the other nematodes used for comparison in the study were very distantly related to p. davidi – the closest relative was p. rigidus that our phylogenetic and genetic distance analyses show is very distantly related to p. davidi. future comparative cryotolerance studies involving p. davidi's closely-related parthenogen relatives would shed light on whether extreme cold tolerance is a feature unique to p. davidi, or if it is shared by its closely-related parthenogenetic relatives. if the latter scenario is true, it would suggest that p. davidi arrived in antarctica in a "pre-adapted", cryotolerant state. if the latter scenario is not true, it would suggest that p. davidi was able to very rapidly adapt to the harsh antarctic environment.  our study sheds light on the evolutionary relationships of panagrolaimus nematodes, showing that the traditional genus is paraphyletic. our analyses suggest a single origin of parthenogenesis in panagrolaimus and provide evidence that p. davidi arrived in antarctica in the very recent evolutionary past. this nematode genus offers an outstanding model for analyzing the evolutionary and biological causes of transitions from gonochoristic to parthenogenetic reproduction, and the adaptive evolution of extreme cold tolerance. we also note here that a brief soil survey effort in northwest oregon performed by a single undergraduate student resulted in the discovery two probable new gonochoristic species of panagrolaimus and a new major clade of pip parthenogens. the worldwide diversity of panagrolaimus and other nematode species in the soil and other ecologies likely remains vastly undersurveyed, being a key limiting factor in our understanding of evolutionary processes in this diverse animal phylum. 
panagrolaimus nematodes are also unique in that examples of gonochoristic, hermaphroditic and parthenogenetic reproductive modes have been reported for members of this genus. the evolutionary origins of these varying reproductive modes and the antarctic species p. davidi, however, remain enigmatic. strain-specific reproductive modes were mapped onto the molecular phylogeny to show a single origin of parthenogenesis from a presumably gonochoristic ancestor. our study provides a phylogenetic framework for understanding the evolutionary origins and diversification patterns of varying reproductive modes within panagrolaimus and important insights into the origin of the antarctic species p. davidi. we collected nuclear ribosomal rna gene and mitochondrial protein-coding gene sequences from diverse panagrolaimus species and strains, including newly discovered isolates from oregon, to investigate phylogenetic relationships in this nematode genus. nematodes in the genus panagrolaimus have served in comparative development and anhydrobiosis studies, and the antarctic species p. davidi offers a powerful paradigm for understanding the biological mechanisms of extreme cold tolerance. panagrolaimus offers a powerful nematode model for understanding diverse evolutionary phenomena including the evolution of asexuality and the adaptive evolution of extreme cold tolerance. the antarctic species p. davidi was found to be very closely related to two panagrolaimus strains from southern california. phylogenetic and molecular clock analyses suggested that p. davidi and the california strain ps shared a common ancestor in the very recent evolutionary past. the hermaphroditic strains were all placed outside a major monophyletic clade that contained the majority of other panagrolaimus nematodes. phylogenetic analyses of mitochondrial sequences showed that substantial molecular and geographic diversity exists within the clade of parthenogenetic strains. as exemplified by the famously successful model organism caenorhabditis elegans, nematodes offer outstanding animal systems for investigating diverse biological phenomena due to their small genome sizes, short generation times and ease of laboratory maintenance. nuclear phylogenies showed that the species and strains historically identified as members of panagrolaimus constitute a paraphyletic group, suggesting that taxonomic revision is required for panagrolaimus and related nematode lineages.
 as exemplified by the famously successful model organism caenorhabditis elegans, nematodes offer outstanding animal systems for investigating diverse biological phenomena due to their small genome sizes, short generation times and ease of laboratory maintenance. nematodes in the genus panagrolaimus have served in comparative development and anhydrobiosis studies, and the antarctic species p. davidi offers a powerful paradigm for understanding the biological mechanisms of extreme cold tolerance. panagrolaimus nematodes are also unique in that examples of gonochoristic, hermaphroditic and parthenogenetic reproductive modes have been reported for members of this genus. the evolutionary origins of these varying reproductive modes and the antarctic species p. davidi, however, remain enigmatic.  we collected nuclear ribosomal rna gene and mitochondrial protein-coding gene sequences from diverse panagrolaimus species and strains, including newly discovered isolates from oregon, to investigate phylogenetic relationships in this nematode genus. nuclear phylogenies showed that the species and strains historically identified as members of panagrolaimus constitute a paraphyletic group, suggesting that taxonomic revision is required for panagrolaimus and related nematode lineages. strain-specific reproductive modes were mapped onto the molecular phylogeny to show a single origin of parthenogenesis from a presumably gonochoristic ancestor. the hermaphroditic strains were all placed outside a major monophyletic clade that contained the majority of other panagrolaimus nematodes. phylogenetic analyses of mitochondrial sequences showed that substantial molecular and geographic diversity exists within the clade of parthenogenetic strains. the antarctic species p. davidi was found to be very closely related to two panagrolaimus strains from southern california. phylogenetic and molecular clock analyses suggested that p. davidi and the california strain ps shared a common ancestor in the very recent evolutionary past.  our study provides a phylogenetic framework for understanding the evolutionary origins and diversification patterns of varying reproductive modes within panagrolaimus and important insights into the origin of the antarctic species p. davidi. panagrolaimus offers a powerful nematode model for understanding diverse evolutionary phenomena including the evolution of asexuality and the adaptive evolution of extreme cold tolerance. 