Thirty years of telemetry-based data acquisition for cardiovascular drug safety evaluation: Applications and optimization.

Conducting safety evaluations of new drugs using conscious animals has been a specialty of our working group for thirty years. In this article, we review the various technical challenges and solutions dealt with over the years to improve both the data quality and the well being of our animal subjects. Of particular interest for us has been the use of telemetry-based data acquisition for conducting studies on cardiovascular (CV) function. This includes the evolving technical aspects of the studies, as well as the development of new applications that take advantage of this technical approach.

Description of three Pristionchus species (Nematoda: Diplogastridae) from Japan that form a cryptic species complex with the model organism P. pacificus.

Three new species of Pristionchus (P. exspectatus, P. arcanus, and P. japonicus) are described from Japan. They are morphologically similar, with P. exspectatus and P. arcanus being almost indistinguishable from the model organism P. pacificus. Reproductive isolation, namely the inability to produce interfertile F1 hybrids, separates all species pairs in the species complex. Additionally, all three new species are distinguished from P. pacificus Sommer, Carta, Kim, and Sternberg, 1996 by having a gonochoristic instead of hermaphroditic mode of reproduction. In addition to its reproductive isolation, P. japonicus is distinct from other Pristionchus species by its arrangement of genital papillae. All species in the complex are separated from each other by molecular sequence divergence, as indicated by analysis of 27 nuclear protein-coding genes and unique sequences of the small subunit ribosomal RNA gene. The identification of a species complex that includes P. pacificus is invaluable for studies of population genetics, speciation, and macroevolution, particularly the evolution of hermaphroditism in the genus.

Horizontal gene transfer of microbial cellulases into nematode genomes is associated with functional assimilation and gene turnover.

BACKGROUND: Natural acquisition of novel genes from other organisms by horizontal or lateral gene transfer is well established for microorganisms. There is now growing evidence that horizontal gene transfer also plays important roles in the evolution of eukaryotes. Genome-sequencing and EST projects of plant and animal associated nematodes such as Brugia, Meloidogyne, Bursaphelenchus and Pristionchus indicate horizontal gene transfer as a key adaptation towards parasitism and pathogenicity. However, little is known about the functional activity and evolutionary longevity of genes acquired by horizontal gene transfer and the mechanisms favoring such processes. RESULTS: We examine the transfer of cellulase genes to the free-living and beetle-associated nematode Pristionchus pacificus, for which detailed phylogenetic knowledge is available, to address predictions by evolutionary theory for successful gene transfer. We used transcriptomics in seven Pristionchus species and three other related diplogastrid nematodes with a well-defined phylogenetic framework to study the evolution of ancestral cellulase genes acquired by horizontal gene transfer. We performed intra-species, inter-species and inter-genic analysis by comparing the transcriptomes of these ten species and tested for cellulase activity in each species. Species with cellulase genes in their transcriptome always exhibited cellulase activity indicating functional integration into the host's genome and biology. The phylogenetic profile of cellulase genes was congruent with the species phylogeny demonstrating gene longevity. Cellulase genes show notable turnover with elevated birth and death rates. Comparison by sequencing of three selected cellulase genes in 24 natural isolates of Pristionchus pacificus suggests these high evolutionary dynamics to be associated with copy number variations and positive selection. CONCLUSION: We could demonstrate functional integration of acquired cellulase genes into the nematode's biology as predicted by theory. Thus, functional assimilation, remarkable gene turnover and selection might represent key features of horizontal gene transfer events in nematodes.

Molecular phylogeny of beetle associated diplogastrid nematodes suggests host switching rather than nematode-beetle coevolution.

BACKGROUND: Nematodes are putatively the most species-rich animal phylum. They have various life styles and occur in a variety of habitats, ranging from free-living nematodes in aquatic or terrestrial environments to parasites of animals and plants. The rhabditid nematode Caenorhabditis elegans is one of the most important model organisms in modern biology. Pristionchus pacificus of the family of the Diplogastridae has been developed as a satellite model for comparison to C. elegans. The Diplogastridae, a monophyletic clade within the rhabditid nematodes, are frequently associated with beetles. How this beetle-association evolved and whether beetle-nematode coevolution occurred is still elusive. As a prerequisite to answering this question a robust phylogeny of beetle-associated Diplogastridae is needed. RESULTS: Sequences for the nuclear small subunit ribosomal RNA and for 12 ribosomal protein encoding nucleotide sequences were collected for 14 diplogastrid taxa yielding a dataset of 5996 bp of concatenated aligned sequences. A molecular phylogeny of beetle-associated diplogastrid nematodes was established by various algorithms. Robust subclades could be demonstrated embedded in a phylogenetic tree topology with short internal branches, indicating rapid ancestral divergences. Comparison of the diplogastrid phylogeny to a comprehensive beetle phylogeny revealed no major congruence and thus no evidence for a long-term coevolution. CONCLUSION: Reconstruction of the phylogenetic history of beetle-associated Diplogastridae yields four distinct subclades, whose deep phylogenetic divergence, as indicated by short internal branch lengths, shows evidence for evolution by successions of ancient rapid radiation events. The stem species of the Diplogastridae existed at the same time period when the major radiations of the beetles occurred. Comparison of nematode and beetle phylogenies provides, however, no evidence for long-term coevolution of diplogastrid nematodes and their beetle hosts. Instead, frequent host switching is observed. The molecular phylogeny of the Diplogastridae provides a framework for further examinations of the evolution of these associations, for the study of interactions within the ecosystems, and for investigations of diplogastrid genome evolution.

Fast radiation of the subfamily Lacertinae (Reptilia: Lacertidae): history or methodical artefact?

Lacertinae is one of the three lacertid lizard subfamilies with a geographical distribution confined to the Palaearctic. Several past attempts to reconstruct its phylogeny resulted in unresolved bush-like topologies. We address the question of whether the lack of resolution is due to insufficient data or whether this lack reflects a rapid succession of speciation events. We analyzed four partial and one complete gene sequences from mitochondrial and nuclear genomes, totalling roughly 3600 bp. We included 29 species representing all 19 genera suggested in recent revision of Lacertinae [Arnold, E.N., Arribas, O., Carranza, S., 2007. Systematics of the palaearctic and oriental lizard tribe Lacertini (Squamata: Lacertidae: Lacertinae), with descriptions of eight new genera. Zootaxa 1430, 1-86]. The resulting phylogeny, first, corroborates monophyly at the genus level for the suggested genera, as well as the finding that Atlantolacerta andreanskyi, until recently part of Lacertinae, belongs to the subfamily Eremiadinae. Second, we find that increasing the sequence length and combining multiple nuclear and mitochondrial sequences did not resolve the polytomy, suggesting that the inferred topology indicates a multiple cladogenesis within a short geological period, rather than a methodical artefact. Divergence time estimates, based on previous estimates of several node ages, range from 13.9 to 14.9 million years for the radiation event, however with very broad confidence interval. To associate the radiation with a narrower geological time we consider palaeogeographic and palaeoclimatic data, assuming that the Lacertinae probably evolved in Central Europe and W Asia after the collision of Africa and Eurasia. We suggest that this radiation may date to the late Langhian (ca. 14-13.5 million years) when geological events caused abrupt changes in regional water-land distribution and climate, offering a window of distinct conditions.

The Strongyloides (Nematoda) of sheep and the predominant Strongyloides of cattle form at least two different, genetically isolated populations.

Strongyloides sp. (Nematoda) are very wide spread small intestinal parasites of vertebrates that can form a facultative free-living generation. Most authors considered all Strongyloides of farm ruminants to belong to the same species, namely Strongyloides papillosus (Wedl, 1856). Here we show that, at least in southern Germany, the predominant Strongyloides found in cattle and the Strongyloides found in sheep belong to separate, genetically isolated populations. While we did find mixed infections in cattle, one form clearly dominated. This variety, in turn, was never found in sheep, indicating that the two forms have different host preferences. We also present molecular tools for distinguishing the two varieties, and an analysis of their phylogenetic relationship with the human parasite Strongyloides stercoralis and the major laboratory model species Strongyloides ratti. Based on our findings we propose that Strongyloides from sheep and the predominant Strongyloides from cattle should be considered separate species as it had already been proposed by [Brumpt, E., 1921. Recherches sur le determinisme des sexes et de l'evolution des Anguillules parasites (Strongyloides). Comptes rendu hebdomadaires des séances et mémoires de la Société de Biologie et de ses filiales 85, 149-152], but was largely ignored by later authors. For nomenclature, we follow [Brumpt, E., 1921. Recherches sur le determinisme des sexes et de l'evolution des Anguillules parasites (Strongyloides). Comptes rendu hebdomadaires des séances et mémoires de la Société de Biologie et de ses filiales 85, 149-152] and use the name S. papillosus for the Strongyloides of sheep and the name Strongyloides vituli for the predominant Strongyloides of cattle.

Phylogeny of the nematode genus Pristionchus and implications for biodiversity, biogeography and the evolution of hermaphroditism.

BACKGROUND: The nematode Pristionchus pacificus has originally been developed as a satellite organism for comparison to Caenorhabditis elegans. A 10X coverage of the whole genome of P. pacificus is available, making P. pacificus the first non-Caenorhabditis nematode with a fully sequenced genome. The macroevolutionary comparison between P. pacificus and C. elegans has been complemented by microevolutionary studies of closely related strains and species within the genus Pristionchus. In addition, new understanding of the biology of Pristionchus from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 Pristionchus species. Two remarkable features of the Pristionchus-beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus Pristionchus. RESULTS: Here, we provide a robust phylogeny of all 18 available Pristionchus species based on a set of 27 ribosomal protein genes encompassing a total of 10,971 bp. The phylogenetic tree provides evidence for North American and European clades, which are embedded in a deeper clade that includes Asian species. It also indicates putative invasion events. Of the 18 Pristionchus species, 13 are gonochoristic and five are hermaphroditic. The phylogeny indicates that all hermaphroditic species have arisen independently within the genus Pristionchus. CONCLUSION: Combined ribosomal protein cDNA data can provide the basis for reconstruction of a robust phylogenetic framework for microevolutionary and biogeographic analyses. An additional major implication of our studies is the use of Pristionchus for nematode biodiversity assessments. While some species are represented by more than 100 isolates, others were found less than four times. Such patterns were observed on all continents and in all phylogenetic clades indicating that species asymmetry is a widespread phenomenon, which can now be further investigated by molecular tools.

The free-living generation of the nematode Strongyloides papillosus undergoes sexual reproduction.

The nematode genus Strongyloides consists of parasites that live as parthenogenetic females in the small intestines of their hosts. They can also form a facultative free-living generation with males and females. Recently, research on Strongyloides cellular and molecular biology has concentrated on Strongyloides ratti and Strongyloides stercoralis. We propose that the related nematode Strongyloides papillosus, a common parasite of ruminants, is well suited for comparative and evolutionary studies and we show that it is phylogentically basal to S. ratti and S. stercoralis. Based on cytological observations several reports have proposed that Strongyloides males do not contribute genetically to the next generation, leaving open the question of why males still exist. In contrast, the only study employing molecular markers showed that S. ratti males do pass on genetic material. Here, we demonstrate that in S. papillosus males also contribute molecular genetic markers to the next generation. This is interesting for two reasons. First, it shows that S. papillosus is amenable to genetic analysis and second, it indicates that sexual reproduction is more common in Strongyloides than previously assumed.

The nematode Pristionchus pacificus (Nematoda: Diplogastridae) is associated with the oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae) in Japan.

Pristionchus pacificus has been developed as a nematode satellite organism in evolutionary developmental biology. Detailed studies of vulva development revealed multiple differences in genetic and molecular control in P. pacificus compared to the model organism Caenorhabditis elegans. To place evolutionary developmental biology in a comprehensive evolutionary context, such studies have to be complemented with ecology. In recent field studies in western Europe and eastern North America we found 11 Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. However, P. pacificus was not commonly found in association with scarab beetles in these studies. Here, we describe the results of a similar survey of scarab beetles in Japan. Pristionchus pacificus was the most common Pristionchus species on scarab beetles in Japan, with 40 out of 43 (93%) isolates. The other Pristionchus isolates represent three novel species, which we refer to as Pristionchus sp. 11, Pristionchus sp. 14, and Pristionchus sp. 15. Thirty-seven of the established P. pacificus strains were found on the oriental beetle Exomala orientalis. Laboratory studies with the sex pheromone (Z)-7-tetradecen-2-one of the oriental beetle revealed that P. pacificus shows strong olfactory attraction to the beetle's sex pheromone, which provides a potential mechanism for the recognition and interaction of P. pacificus and E. orientalis. Together, this study identifies P. pacificus as the most common Pristionchus nematode in field studies in Japan, identifies E. orientalis as an important host species, and provides the basis for the ecology of P. pacificus.

Sex, bugs and Haldane's rule: the nematode genus Pristionchus in the United States.

BACKGROUND: The nematode Pristionchus pacificus has been developed as a satellite organism in evolutionary developmental biology for comparison to Caenorhabditis elegans. Comparative studies have revealed major differences in the regulation of developmental processes between P. pacificus and C. elegans. To place evolutionary developmental biology and the observed developmental differences between species in a comprehensive evolutionary context, such studies have to be complemented with ecological aspects. Knowledge about the ecology of the organism in question might indicate specific environmental conditions that can result in developmental adaptations and could account for species differences in development. To this end, we have started to investigate the ecology of Pristionchus nematodes. In recent field studies in Western Europe we found six Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. This Pristionchus-beetle association provides the unique opportunity to combine research in evolutionary developmental biology with ecology. However, it remains unknown how general these findings from Europe are on a global scale. RESULTS: Here, we describe the Pristionchus species associated with scarab and Colorado potato beetles in the Eastern United States and show striking transatlantic differences and unexpected evolutionary and ecological patterns. Two hundred eighty of 285 (98%) isolates from American scarab beetles belong to five Pristionchus species, all of which are different from the European species. We describe four of them as novel Pristionchus species. The five American Pristionchus species fall into a single phylogenetic clade and have a male-female (gonochoristic) mode of reproduction, whereas the majority of European isolates are hermaphroditic. Crosses between the two most closely related species, P. aerivorus and P. pseudaerivorus n. sp., follow Haldane's rule in that heterogametic F1 males are inviable. We observed P. aerivorus and P. pseudaerivorus n. sp. coexisting on the same scarab beetle and obtained two cases of F1 hybrids from wild beetles. Finally, the Colorado potato beetle is associated with the same nematode, P. uniformis in the United States and Europe. Given the introduction of the Colorado potato beetle to Europe in 1877, our results suggest that P. uniformis was introduced together with its beetle vector. CONCLUSION: Taken together, the Pristionchus-beetle association provides a powerful tool for studying biodiversity, biogeography, speciation and species invasion on a global scale.

Nematodes of the genus Pristionchus are closely associated with scarab beetles and the Colorado potato beetle in Western Europe.

Evolutionary developmental biology examines how changes in developmental programmes give rise to developmental and, ultimately, morphological novelty. To this end, comparisons of related but distinct organisms have to be performed. The diplogastrid nematode Pristionchus pacificus has been developed as a satellite system for a detailed comparison of various developmental processes to the model organism Caenorhabditis elegans, a rhabditid nematode. In addition to developmental and genetic studies, a genomic platform has been established to analyse the biology of this organism. However, only little is known about where and how Pristionchus pacificus and its relatives live in the wild. Here we show that nematodes of the genus Pristionchus live in close association with scarabaeoid beetles and the Colorado potato beetle. In total, we generated 371 isogenic female lines from 4242 beetles collected at 25 sampling sites all over Europe. Isogenic female lines were subjected to sequence analysis and mating experiments for species determination. The 371 isolates fell into six species. Two hermaphroditic species account for about 60% of the collected nematodes. We found Pristionchus maupasi almost exclusively on cockchafers and Pristionchus entomophagus predominantly on dung beetles. Colorado potato beetles carried the gonochoristic species Pristionchus uniformis, which was only rarely observed on scarabaeoid beetles. We describe the initial evidence for the association of Pristionchus nematodes with beetles and provide a phylogeny based on sequence analysis of the small subunit ribosomal RNA gene.

Persistence of Mhc heterozygosity in homozygous clonal killifish, Rivulus marmoratus: implications for the origin of hermaphroditism.

The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced. Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose approximately 20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci, as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by mutations, each of which spread through the entire population and was fixed independently in the emerging clones.

Macrophage migration inhibitory factor (MIF) of jawed and jawless fishes: implications for its evolutionary origin.

The macrophage migration inhibitory factor (MIF) is a cytokine produced by T lymphocytes and macrophages in response to inflammatory stimuli. We sequenced MIF cDNA clones of two jawless fishes, the sea lamprey (Petromyzon marinus) and the North Atlantic hagfish (Myxine glutinosa), as well as of the jawed (cichlid) fish Paralabidochromis chilotes. The fish MIF-encoding genes have the same exon-intron organization as the mammalian MIF genes and are present in one copy per haploid genome. Secondary and tertiary structure predictions suggest that the fish MIF proteins have a topology characteristic of the entire MIF-family of proteins. Phylogenetic analysis separates the known nematode members of the family into two groups, one having a sister group relationship with the mammalian D-dopachrome tautomerase (DDT) proteins and the other being related to vertebrate MIFs. It also reveals a high degree of convergent evolution among the members of the family. Finally, it suggests that the divergence of MIF and DDT occurred before the emergence of nematodes in metazoan evolution.

Characterization of genetic diversity in the nematode Pristionchus pacificus from population-scale resequencing data.

The hermaphroditic nematode Pristionchus pacificus is an established model system for comparative studies with Caenorhabditis elegans in developmental biology, ecology, and population genetics. In this study, we present whole-genome sequencing data of 104 P. pacificus strains and the draft assembly of the obligate outcrossing sister species P. exspectatus. We characterize genetic diversity within P. pacificus and investigate the population genetic processes shaping this diversity. P. pacificus is 10 times more diverse than C. elegans and exhibits substantial population structure that allows us to probe its evolution on multiple timescales. Consistent with reduced effective recombination in this self-fertilizing species, we find haplotype blocks that span several megabases. Using the P. exspectatus genome as an outgroup, we polarized variation in P. pacificus and found a site frequency spectrum (SFS) that decays more rapidly than expected in neutral models. The SFS at putatively neutral sites is U shaped, which is a characteristic feature of pervasive linked selection. Based on the additional findings (i) that the majority of nonsynonymous variation is eliminated over timescales on the order of the separation between clades, (ii) that diversity is reduced in gene-rich regions, and (iii) that highly differentiated clades show very similar patterns of diversity, we conclude that purifying selection on many mutations with weak effects is a major force shaping genetic diversity in P. pacificus.

Quantitative assessment of the nematode fauna present on Geotrupes dung beetles reveals species-rich communities with a heterogeneous distribution.

Pristionchus spp. nematodes exhibit several traits that might serve as pre-adaptations to parasitism. Under harsh environmental conditions, these nematodes can arrest development and form dauer larvae. In addition, they have been shown to live in necromenic association with a range of beetles, including dung beetles ( Geotrupes stercorosus ) on which, for example, Pristionchus entomophagus is commonly found. It has been argued that the formation of dauer larvae and the association with invertebrates represent intermediate steps towards parasitism. To better understand necromenic associations, and to gain information on Pristionchus spp. abundance and the general species composition on dung beetles, we extracted all the nematode fauna present on 114 individuals of G. stercorosus. By direct sequencing using the 18S SSU, we provide a barcode for all nematodes isolated from the beetle samples. In total, 5,002 dauer-stage nematodes were sequenced, which included Pristionchus spp., Koerneria spp. (Diplogastridae), Pelodera spp. (Rhabditidae), and Strongyloidea as well as Spirurida. Intensities of infection varied from over 1,000 nematodes isolated from a single G. stercorosus to none, with Pelodera spp. being the most abundant group isolated. This study presents the first quantitative data on the Pristionchus spp. infection of beetles.

The phylogeny of the family Lacertidae (Reptilia) based on nuclear DNA sequences: convergent adaptations to arid habitats within the subfamily Eremiainae.

The family Lacertidae encompasses more than 250 species distributed in the Palearctis, Ethiopis and Orientalis. Lacertids have been subjected in the past to several morphological and molecular studies to establish their phylogeny. However, the problems of convergent adaptation in morphology and of excessively variable molecular markers have hampered the establishment of well supported deeper phylogenetic relationships. Particularly the adaptations to xeric environments have often been used to establish a scenario for the origin and radiation of major lineages within lacertids. Here we present a molecular phylogenetic study based on two nuclear marker genes and representatives of 37 lacertid genera and distinct species groups (as in the case of the collective genus Lacerta). Roughly 1600 bp of the nuclear rag1 and c-mos genes were sequenced and analyzed. While the results provide good support to the hitherto suggested main subfamilies of Gallotiinae (Gallotia and Psammodromus), Eremiainae and Lacertinae [Harris, D.J., Arnold, E.N., Thomas, R.H., 1998. Relationships of lacertid lizards (Reptilia: Lacertidae) estimated from mitochondrial DNA sequences and morphology. Proc. R. Soc. Lond. B 265, 1939-1948], they also suggest unexpected relationships. In particular, the oriental genus Takydromus, previously considered the sister-group to the three subfamilies, is nested within Lacertinae. Moreover, the genera within the Eremiainae are further divided into two groups, roughly corresponding to their respective geographical distributions in the Ethiopian and the Saharo-Eurasian ranges. The results support an independent origin of adaptations to xeric conditions in different subfamilies. The relationships within the subfamily Lacertinae could not be resolved with the markers used. The species groups of the collective genus Lacerta show a bush-like topology in the inferred Bayesian tree, suggesting rapid radiation. The composition of the subfamilies Eremiainae and Lacertinae as well as their phylogeography are discussed.

Unusual origin of a nuclear pseudogene in the Italian wall lizard: intergenomic and interspecific transfer of a large section of the mitochondrial genome in the genus Podarcis (Lacertidae).

Two distinct cytochrome b-like sequences were discovered in the genome of Podarcis sicula. One of them represents a nuclear copy of a mitochondrial sequence (numt-sic) differing by 14.3% from the authentic mitochondrial (mt) sequence obtained from the same individual. This numt, however, differs by only 2.7% from the mt sequence found in one population of Podarcis muralis, a related species in which no corresponding numt was detected. The numt-sic sequence extends over at least 7637 bp and is homologous to a section of the mt genome spanning from the tRNA-Lys to the tRNA-Pro gene. Premature mt stop codons were detected in two of the nine protein coding genes of numt-sic. The distribution of substitutions among the three codon positions and the transition/transversion ratio of the numt-sic sequence resemble, with few exceptions, those of functional mt genes, indicating a rather recent transfer to the nucleus. Phylogenetic analyses performed on the data set including P. sicula numt-cytb sequences as well as mt-cytb sequences from the same individuals and mt sequences of various P. muralis populations suggest that numt-sic originated in P. muralis. In a geographic survey, P. sicula populations belonging to different mt lineages, covering most of the distribution area, were screened for the presence of numt-sic and for a 15-bp duplication polymorphism in the numt-nd5 sequence. Our results suggest that numt-sic has spread rapidly through the species range via sexual transmission, thereby being transferred to populations belonging to well-separated mt lineages that diverged 1-3 Mya.

Nuclear and mtDNA-based phylogeny of southern African sand lizards, Pedioplanis (Sauria: Lacertidae).

The diversity of lacertid lizards in Africa is highest in the southern African subcontinent, where over two-thirds of the species are endemic. With eleven currently recognized species, Pedioplanis is the most diverse among the southern African genera. In this study we use 2200 nucleotide positions derived from two mitochondrial markers (ND2 and 16S rRNA) and one nuclear gene (RAG-1) to (i) assess the phylogeny of Pedioplanis and (ii) estimate divergence time among lineages using the relaxed molecular clock method. Individual analyses of each gene separately supported different nodes in the phylogeny and the combined analysis yielded more well supported relationships. We present the first, well-resolved gene tree for the genus Pedioplanis and this is largely congruent with a phylogeny derived from morphology. Contrary to previous suggestions Heliobolus/Nucras are sister to Pedioplanis. The genus Pedioplanis is monophyletic, with P. burchelli/P. laticeps forming a clade that is sister to all the remaining congeners. Two distinct geographic lineages can be identified within the widespread P. namaquensis; one occurs in Namibia, while the other occurs in South Africa. The P. undata species complex is monophyletic, but one of its constituent species, P. inornata, is paraphyletic. Relationships among the subspecies of P. lineoocellata are much more complex than previously documented. An isolated population previously assigned to P. l. pulchella is paraphyletic and sister to the three named subspecies. The phylogeny identifies two biogeographical clades that probably diverged during the mid-Miocene, after the development of the Benguella Current. This probably led to habitat changes associated with climate and, in conjunction with physical barriers (Great Escarpment), contributed towards speciation within the genus Pedioplanis.

Distinct patterns of genetic variation in Pristionchus pacificus and Caenorhabditis elegans, two partially selfing nematodes with cosmopolitan distribution.

Hermaphroditism has evolved several times independently in nematodes. The model organism Caenorhabditis elegans and Pristionchus pacificus are self-fertile hermaphrodites with rare facultative males. Both species are members of different families: C. elegans belongs to the Rhabditidae and P. pacificus to the Diplogastridae. Also, both species differ in their ecology: C. elegans is a soil-dwelling nematode that is often found in compost heaps. In contrast, field studies in Europe and North America indicate that Pristionchus nematodes are closely associated with scarab beetles. In C. elegans, several recent studies have found low genetic diversity and rare out-crossing events. Little is known about diversity levels and population structure in free-living hermaphroditic nematodes outside the genus Caenorhabditis. Taking a comparative approach, we analyse patterns of molecular diversity and linkage disequilibrium in 18 strains of P. pacificus from eight countries and four continents. Mitochondrial sequence data of P. pacificus isolates reveal a substantially higher genetic diversity on a global scale when compared to C. elegans. A mitochondrial-derived hermaphrodite phylogeny shows little geographic structuring, indicating several worldwide dispersal events. Amplified fragment length polymorphism and single strand conformation polymorphism analyses demonstrate a high degree of genome-wide linkage disequilibrium, which also extends to the mitochondrial genome. Together, these findings indicate distinct patterns of genetic variation of the two species. The low level of genetic diversity observed in C. elegans might reflect a recent human-associated dispersal, whereas the P. pacificus diversity might reflect a long-lasting and ongoing insect association. Thus, despite similar lifestyle characteristics in the laboratory, the reproductive mode of hermaphroditism with rare facultative males can result in distinct genetic variability patterns in different ecological settings.

Multiple copies of coding as well as pseudogene c-mos sequence exist in three lacertid species.

The analysis of a 581 bp section of the nuclear gene c-mos revealed multiple copies of putative functional sequences as well as pseudogenes in three closely related lacertid species Lacerta laevis, L. kulzeri and L. cyanisparsa. A phylogenetic analysis of c-mos in comparison with a molecular phylogeny based on the mitochondrial cytochrome b gene supports our findings. The study also provides new insights into the phylogenetic relationships of L. cyanisparsa and L. laevis. Pseudogenes of the three species share 11 single-nucleotide substitutions, a 1 bp deletion and a premature stop codon but differ by group-specific mutations. This result suggests that the c-mos gene has become duplicated and subsequently silenced already in the common ancestor of the three species. Sequence divergence suggests that the duplication and the loss of function occurred in the late Miocene/early Pliocene, i.e., about 5 million years ago. Indications of gene conversion are discussed. We suggest that future studies using c-mos for phylogenetic studies should provide evidence for the orthology of the sequences compared.