Showing papers in "BMC Evolutionary Biology in 2008"

Bayesian inference of population size history from multiple loci

Abstract: Effective population size (N e ) is related to genetic variability and is a basic parameter in many models of population genetics. A number of methods for inferring current and past population sizes from genetic data have been developed since JFC Kingman introduced the n-coalescent in 1982. Here we present the Extended Bayesian Skyline Plot, a non-parametric Bayesian Markov chain Monte Carlo algorithm that extends a previous coalescent-based method in several ways, including the ability to analyze multiple loci. Through extensive simulations we show the accuracy and limitations of inferring population size as a function of the amount of data, including recovering information about evolutionary bottlenecks. We also analyzed two real data sets to demonstrate the behavior of the new method; a single gene Hepatitis C virus data set sampled from Egypt and a 10 locus Drosophila ananassae data set representing 16 different populations. The results demonstrate the essential role of multiple loci in recovering population size dynamics. Multi-locus data from a small number of individuals can precisely recover past bottlenecks in population size which can not be characterized by analysis of a single locus. We also demonstrate that sequence data quality is important because even moderate levels of sequencing errors result in a considerable decrease in estimation accuracy for realistic levels of population genetic variability.

Dynamics and evolution of the inverted repeat-large single copy junctions in the chloroplast genomes of monocots.

Abstract: Various expansions or contractions of inverted repeats (IRs) in chloroplast genomes led to fluxes in the IR-LSC (large single copy) junctions. Previous studies revealed that some monocot IRs contain a trnH-rps19 gene cluster, and it has been speculated that this may be an evidence of a duplication event prior to the divergence of monocot lineages. Therefore, we compared the organizations of genes flanking two IR-LSC junctions in 123 angiosperm representatives to uncover the evolutionary dynamics of IR-LSC junctions in basal angiosperms and monocots. The organizations of genes flanking IR-LSC junctions in angiosperms can be classified into three types. Generally each IR of monocots contains a trnH-rps19 gene cluster near the IR-LSC junctions, which differs from those in non-monocot angiosperms. Moreover, IRs expanded more progressively in monocots than in non-monocot angiosperms. IR-LSC junctions commonly occurred at polyA tract or A-rich regions in angiosperms. Our RT-PCR assays indicate that in monocot IRA the trnH-rps19 gene cluster is regulated by two opposing promoters, S10 A and psbA. Two hypotheses are proposed to account for the evolution of IR expansions in monocots. Based on our observations, the inclusion of a trnH-rps19 cluster in majority of monocot IRs could be reasonably explained by the hypothesis that a DSB event first occurred at IRB and led to the expansion of IRs to trnH, followed by a successive DSB event within IRA and lead to the expansion of IRs to rps19 or to rpl22 so far. This implies that the duplication of trnH-rps19 gene cluster was prior to the diversification of extant monocot lineages. The duplicated trnH genes in the IRB of most monocots and non-monocot angiosperms have distinct fates, which are likely regulated by different expression levels of S10 A and S10 B promoters. Further study is needed to unravel the evolutionary significance of IR expansion in more recently diverged monocots.

Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera.

Abstract: Moths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, Lampronia capitella, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (Z,Z)-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis. The reconstructed evolutionary tree of desaturases evidenced two ditrysian-specific lineages (the Δ11 and Δ9 (18C>16C)) to have orthologs in the primitive moth L. capitella despite being absent in Diptera and other insect genomes. Four acyl-CoA desaturase cDNAs were isolated from the pheromone gland, three of which are related to Δ9-desaturases whereas the fourth cDNA clusters with Δ11-desaturases. We demonstrated that this transcript (Lca-KPVQ) exclusively accounts for both steps of desaturation involved in pheromone biosynthesis. This enzyme possesses a Z11-desaturase activity that allows transforming the palmitate precursor (C16:0) into (Z)-11-hexadecenoic acid and the (Z)-9-tetradecenoic acid into the conjugated intermediate (Z,Z)-9,11-tetradecadienoic acid. The involvement of a single Z11-desaturase in pheromone biosynthesis of a non-ditrysian moth species, supports that the duplication event leading to the origin of the Lepidoptera-specific Δ11-desaturase gene subfamily took place before radiation of ditrysian moths and their divergence from other heteroneuran lineages. Our findings uncover that this novel class of enzymes affords complex combinations of unique unsaturated fatty acyl-moieties of variable chain-lengths, regio- and stereo-specificities since early in moth history and contributes a notable innovation in the early evolution of moth-pheromones.

The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands

Abstract: The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database. Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences. The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.

Mitochondrial genomes reveal an explosive radiation of extinct and extant bears near the Miocene-Pliocene boundary

Abstract: Despite being one of the most studied families within the Carnivora, the phylogenetic relationships among the members of the bear family (Ursidae) have long remained unclear. Widely divergent topologies have been suggested based on various data sets and methods. We present a fully resolved phylogeny for ursids based on ten complete mitochondrial genome sequences from all eight living and two recently extinct bear species, the European cave bear (Ursus spelaeus) and the American giant short-faced bear (Arctodus simus). The mitogenomic data yield a well-resolved topology for ursids, with the sloth bear at the basal position within the genus Ursus. The sun bear is the sister taxon to both the American and Asian black bears, and this clade is the sister clade of cave bear, brown bear and polar bear confirming a recent study on bear mitochondrial genomes. Sequences from extinct bears represent the third and fourth Pleistocene species for which complete mitochondrial genomes have been sequenced. Moreover, the cave bear specimen demonstrates that mitogenomic studies can be applied to Pleistocene fossils that have not been preserved in permafrost, and therefore have a broad application within ancient DNA research. Molecular dating of the mtDNA divergence times suggests a rapid radiation of bears in both the Old and New Worlds around 5 million years ago, at the Miocene-Pliocene boundary. This coincides with major global changes, such as the Messinian crisis and the first opening of the Bering Strait, and suggests a global influence of such events on species radiations.

hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions

Abstract: Hsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family. We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65–80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages. The human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.

Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties

Abstract: The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection. Here we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures. We found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.

Phylogeny and biogeography of African Murinae based on mitochondrial and nuclear gene sequences, with a new tribal classification of the subfamily

Abstract: Within the subfamily Murinae, African murines represent 25% of species biodiversity, making this group ideal for detailed studies of the patterns and timing of diversification of the African endemic fauna and its relationships with Asia. Here we report the results of phylogenetic analyses of the endemic African murines through a broad sampling of murine diversity from all their distribution area, based on the mitochondrial cytochrome b gene and the two nuclear gene fragments (IRBP exon 1 and GHR). A combined analysis of one mitochondrial and two nuclear gene sequences consistently identified and robustly supported ten primary lineages within Murinae. We propose to formalize a new tribal arrangement within the Murinae that reflects this phylogeny. The diverse African murine assemblage includes members of five of the ten tribes and clearly derives from multiple faunal exchanges between Africa and Eurasia. Molecular dating analyses using a relaxed Bayesian molecular clock put the first colonization of Africa around 11 Mya, which is consistent with the fossil record. The main period of African murine diversification occurred later following disruption of the migration route between Africa and Asia about 7–9 Mya. A second period of interchange, dating to around 5–6.5 Mya, saw the arrival in Africa of Mus (leading to the speciose endemic Nannomys), and explains the appearance of several distinctive African lineages in the late Miocene and Pliocene fossil record of Eurasia. Our molecular survey of Murinae, which includes the most complete sampling so far of African taxa, indicates that there were at least four separate radiations within the African region, as well as several phases of dispersal between Asia and Africa during the last 12 My. We also reconstruct the phylogenetic structure of the Murinae, and propose a new classification at tribal level for this traditionally problematic group.

Prezygotic reproductive isolation between Saccharomyces cerevisiae and Saccharomyces paradoxus

Abstract: Matings between different Saccharomyces sensu stricto yeast species produce sexually sterile hybrids, so individuals should avoid mating with other species. Any mechanism that reduces the frequency of interspecific matings will confer a selective advantage. Here we test the ability of two closely-related Saccharomyces sensu stricto species to select their own species as mates and avoid hybridisation. We set up mate choice tests, using five independently isolated pairs of species, in which individual germinating spores were presented with the opportunity to mate either with a germinating spore of their own species or with a germinating spore of the other species. For all five strain pairs, whether a S. cerevisiae or S. paradoxus occupies the role of "chooser" strain, the level of hybridisation that is observed between the two species is significantly lower than would be expected if mates were selected at random. We also show that, overall, S. cerevisiae exhibited a stronger own-species preference than S. paradoxus. Prezygotic reproductive isolation is well known in higher organisms but has been largely overlooked in yeast, an important model microbe. Here we present the first report of prezygotic reproductive isolation in Saccharomyces. Prezygotic reproductive isolation may be important in yeast speciation or yeast species cohesion, and may have evolved to prevent wasted matings between different species. Whilst yeast has long been used as a genetic model system, little is known about yeast in the wild. Our work sheds light on an interesting aspect of yeast natural behaviour: their ability to avoid costly interspecific matings.

Giant viruses, giant chimeras: The multiple evolutionary histories of Mimivirus genes

Abstract: Although capable to evolve, viruses are generally considered non-living entities because they are acellular and devoid of metabolism. However, the recent publication of the genome sequence of the Mimivirus, a giant virus that parasitises amoebas, strengthened the idea that viruses should be included in the tree of life. In fact, the first phylogenetic analyses of a few Mimivirus genes that are also present in cellular lineages suggested that it could define an independent branch in the tree of life in addition to the three domains, Bacteria, Archaea and Eucarya. We tested this hypothesis by carrying out detailed phylogenetic analyses for all the conserved Mimivirus genes that have homologues in cellular organisms. We found no evidence supporting Mimivirus as a new branch in the tree of life. On the contrary, our phylogenetic trees strongly suggest that Mimivirus acquired most of these genes by horizontal gene transfer (HGT) either from its amoebal hosts or from bacteria that parasitise the same hosts. The detection of HGT events involving different eukaryotic donors suggests that the spectrum of hosts of Mimivirus may be larger than currently known. The large number of genes acquired by Mimivirus from eukaryotic and bacterial sources suggests that HGT has been an important process in the evolution of its genome and the adaptation to parasitism.

Evolutionary history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii, Characidae) in Mesoamerica reveals multiple morphological homoplasies

Abstract: Mesoamerica is one of the world's most complex biogeographical regions, mostly due to its complex geological history. This complexity has led to interesting biogeographical processes that have resulted in the current diversity and distribution of fauna in the region. The fish genus Astyanax represents a useful model to assess biogeographical hypotheses due to it being one of the most diverse and widely distributed freshwater fish species in the New World. We used mitochondrial and nuclear DNA to evaluate phylogenetic relationships within the genus in Mesoamerica, and to develop historical biogeographical hypotheses to explain its current distribution. Analysis of the entire mitochondrial cytochrome b (Cytb) gene in 208 individuals from 147 localities and of a subset of individuals for three mitochondrial genes (Cytb, 16 S, and COI) and a single nuclear gene (RAG1) yielded similar topologies, recovering six major groups with significant phylogeographic structure. Populations from North America and Upper Central America formed a monophyletic group, while Middle Central America showed evidence of rapid radiation with incompletely resolved relationships. Lower Central America lineages showed a fragmented structure, with geographically restricted taxa showing high levels of molecular divergence. All Bramocharax samples grouped with their sympatric Astyanax lineages (in some cases even with allopatric Astyanax populations), with less than 1% divergence between them. These results suggest a homoplasic nature to the trophic specializations associated with Bramocharax ecomorphs, which seem to have arisen independently in different Astyanax lineages. We observed higher taxonomic diversity compared to previous phylogenetic studies of the Astyanax genus. Colonization of Mesoamerica by Astyanax before the final closure of the Isthmus of Panama (3.3 Mya) explains the deep level of divergence detected in Lower Central America. The colonization of Upper Mesoamerica apparently occurred by two independent routes, with lineage turnover over a large part of the region. Our results support multiple, independent origins of morphological traits in Astyanax, whereby the morphotype associated with Bramocharax represents a recurrent trophic adaptation. Molecular clock estimates indicate that Astyanax was present in Mesoamerica during the Miocene (~8 Mya), which implies the existence of an incipient land-bridge connecting South America and Central America before the final closure of the Isthmus of Panama (~3.3 Mya).

The evolutionary costs of immunological maintenance and deployment

Abstract: The evolution of disease resistance and immune function may be limited if increased immunocompetence comes at the expense of other fitness-determining traits. Both the maintenance of an immune system and the deployment of an immune response can be costly, and the observed costs may be evaluated as either physiological or evolutionary in origin. Evolutionary costs of immunological maintenance are revealed as negative genetic correlations between immunocompetence and fitness in the absence of infection. Costs of deployment are most often studied as physiological costs associated with immune system induction, however, evolutionary costs of deployment may also be present if genotypes vary in the extent of the physiological cost experienced. In this study we analyzed evolutionary and physiological costs of immunity in two environments representing food-limited and food-unlimited conditions. Patterns of genetic variation were estimated in females from 40 'hemiclone families' isolated from a population of D. melanogaster. Phenotypes evaluated included fecundity, weight measures at different time periods and resistance to Providencia rettgeri, a naturally occurring Gram-negative pathogen of D. melanogaster. In the food-limited environment we found a negative genetic correlation between fecundity in the absence of infection and resistance, indicative of an evolutionary cost of maintenance. No such correlation was observed in the food-unlimited environment, and the slopes of these correlations significantly differed, demonstrating a genotype-by-environment interaction for the cost of maintenance. Physiological costs of deployment were also observed, but costs were primarily due to wounding. Deployment costs were slightly exaggerated in the food-limited environment. Evolutionary costs of immunological deployment on fecundity were not observed, and there was only marginally significant genetic variation in the cost expressed by changes in dry weight. Our results suggest that the costs of immunity may be an important factor limiting the evolution of resistance in food-limited environments. However, the significant genotype-by-environment interaction for maintenance costs, combined with the observation that deployment costs were partially mitigated in the food-unlimited environment, emphasizes the importance of considering environmental variation when estimating patterns of genetic variance and covariance, and the dubious nature of predicting evolutionary responses to selection from quantitative genetic estimates carried out in a single environment.

A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish.

Abstract: A large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression. Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues. Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish.

On the origins of arrestin and rhodopsin

Abstract: G protein coupled receptors (GPCRs) are the most numerous proteins in mammalian genomes, and the most common targets of clinical drugs. However, their evolution remains enigmatic. GPCRs are intimately associated with trimeric G proteins, G protein receptor kinases, and arrestins. We conducted phylogenetic studies to reconstruct the history of arrestins. Those findings, in turn, led us to investigate the origin of the photosensory GPCR rhodopsin. We found that the arrestin clan is comprised of the Spo0M protein family in archaea and bacteria, and the arrestin and Vps26 families in eukaryotes. The previously known animal arrestins are members of the visual/beta subfamily, which branched from the founding "alpha" arrestins relatively recently. Curiously, we identified both the oldest visual/beta arrestin and opsin genes in Cnidaria (but not in sponges). The arrestin clan has 14 human members: 6 alphas, 4 visual/betas, and 4 Vps26 genes. Others recently showed that the 3D structure of mammalian Vps26 and the biochemical function of the yeast alpha arrestin PalF are similar to those of beta arrestins. We note that only alpha arrestins have PY motifs (known to bind WW domains) in their C-terminal tails, and only visual/betas have helix I in the Arrestin N domain. We identified ciliary opsins in Cnidaria and propose this subfamily is ancestral to all previously known animal opsins. That finding is consistent with Darwin's theory that eyes evolved once, and lends some support to Parker's hypothesis that vision triggered the Cambrian explosion of life forms. Our arrestin findings have implications on the evolution of GPCR signaling, and on the biological roles of human alpha arrestins.

Sperm competition: linking form to function

Abstract: Background Using information from physics, biomechanics and evolutionary biology, we explore the implications of physical constraints on sperm performance, and review empirical evidence for links between sperm length and sperm competition (where two or more males compete to fertilise a female's eggs). A common theme in the literature on sperm competition is that selection for increased sperm performance in polyandrous species will favour the evolution of longer, and therefore faster swimming, sperm. This argument is based on the common assumption that sperm swimming velocity is directly related to sperm length, due to the increased thrust produced by longer flagella.

Single mitochondrial gene barcodes reliably identify sister-species in diverse clades of birds

Abstract: DNA barcoding of life using a standardized COI sequence was proposed as a species identification system, and as a method for detecting putative new species. Previous tests in birds showed that individuals can be correctly assigned to species in ~94% of the cases and suggested a threshold of 10× mean intraspecific difference to detect potential new species. However, these tests were criticized because they were based on a single maternally inherited gene rather than multiple nuclear genes, did not compare phylogenetically identified sister species, and thus likely overestimated the efficacy of DNA barcodes in identifying species. To test the efficacy of DNA barcodes we compared ~650 bp of COI in 60 sister-species pairs identified in multigene phylogenies from 10 orders of birds. In all pairs, individuals of each species were monophyletic in a neighbor-joining (NJ) tree, and each species possessed fixed mutational differences distinguishing them from their sister species. Consequently, individuals were correctly assigned to species using a statistical coalescent framework. A coalescent test of taxonomic distinctiveness based on chance occurrence of reciprocal monophyly in two lineages was verified in known sister species, and used to identify recently separated lineages that represent putative species. This approach avoids the use of a universal distance cutoff which is invalidated by variation in times to common ancestry of sister species and in rates of evolution. Closely related sister species of birds can be identified reliably by barcodes of fixed diagnostic substitutions in COI sequences, verifying coalescent-based statistical tests of reciprocal monophyly for taxonomic distinctiveness. Contrary to recent criticisms, a single DNA barcode is a rapid way to discover monophyletic lineages within a metapopulation that might represent undiscovered cryptic species, as envisaged in the unified species concept. This identifies a smaller set of lineages that can also be tested independently for species status with multiple nuclear gene approaches and other phenotypic characters.

The evolution of body size under environmental gradients in ectotherms: why should Bergmann's rule apply to lizards?

Abstract: The impact of environmental gradients on the evolution of life history traits is a central issue in macroecology and evolutionary biology. A number of hypotheses have been formulated to explain factors shaping patterns of variation in animal mass. One such example is Bergmann's rule, which predicts that body size will be positively correlated with latitude and elevation, and hence, with decreasing environmental temperatures. A generally accepted explanation for this phenotypic response is that as body mass increases, body surface area gets proportionally smaller, which contributes to reduced rates of heat-loss. Phylogenetic and non-phylogenetic evidence reveals that endotherms follow Bergmann's rule. In contrast, while previous non-phylogenetic studies supported this prediction in up to 75% of ectotherms, recent phylogenetic comparative analyses suggest that its validity for these organisms is controversial and less understood. Moreover, little attention has been paid to why some ectotherms conform to this rule, while others do not. Here, we investigate Bergmann's rule in the six main clades forming the Liolaemus genus, one of the largest and most environmentally diverse genera of terrestrial vertebrates. A recent study conducted on some species belonging to four of these six clades concluded that Liolaemus species follow Bergmann's rule, representing the only known phylogenetic support for this model in lizards. However, a later reassessment of this evidence, performed on one of the four analysed clades, produced contrasting conclusions. Our results fail to support Bergmann's rule in Liolaemus lizards. Non-phylogenetic and phylogenetic analyses showed that none of the studied clades experience increasing body size with increasing latitude and elevation. Most physiological and behavioural processes in ectotherms depend directly upon their body temperature. In cold environments, adaptations to gain heat rapidly are under strong positive selection to allow optimal feeding, mating and predator avoidance. Therefore, evolution of larger body size in colder environments appears to be a disadvantageous thermoregulatory strategy. The repeated lack of support for Bergmann's rule in ectotherms suggests that this model should be recognized as a valid rule exclusively for endotherms.

Evolution of gastropod mitochondrial genome arrangements

Abstract: Gastropod mitochondrial genomes exhibit an unusually great variety of gene orders compared to other metazoan mitochondrial genome such as e.g those of vertebrates. Hence, gastropod mitochondrial genomes constitute a good model system to study patterns, rates, and mechanisms of mitochondrial genome rearrangement. However, this kind of evolutionary comparative analysis requires a robust phylogenetic framework of the group under study, which has been elusive so far for gastropods in spite of the efforts carried out during the last two decades. Here, we report the complete nucleotide sequence of five mitochondrial genomes of gastropods (Pyramidella dolabrata, Ascobulla fragilis, Siphonaria pectinata, Onchidella celtica, and Myosotella myosotis), and we analyze them together with another ten complete mitochondrial genomes of gastropods currently available in molecular databases in order to reconstruct the phylogenetic relationships among the main lineages of gastropods. Comparative analyses with other mollusk mitochondrial genomes allowed us to describe molecular features and general trends in the evolution of mitochondrial genome organization in gastropods. Phylogenetic reconstruction with commonly used methods of phylogenetic inference (ME, MP, ML, BI) arrived at a single topology, which was used to reconstruct the evolution of mitochondrial gene rearrangements in the group. Four main lineages were identified within gastropods: Caenogastropoda, Vetigastropoda, Patellogastropoda, and Heterobranchia. Caenogastropoda and Vetigastropoda are sister taxa, as well as, Patellogastropoda and Heterobranchia. This result rejects the validity of the derived clade Apogastropoda (Caenogastropoda + Heterobranchia). The position of Patellogastropoda remains unclear likely due to long-branch attraction biases. Within Heterobranchia, the most heterogeneous group of gastropods, neither Euthyneura (because of the inclusion of P. dolabrata) nor Pulmonata (polyphyletic) nor Opisthobranchia (because of the inclusion S. pectinata) were recovered as monophyletic groups. The gene order of the Vetigastropoda might represent the ancestral mitochondrial gene order for Gastropoda and we propose that at least three major rearrangements have taken place in the evolution of gastropods: one in the ancestor of Caenogastropoda, another in the ancestor of Patellogastropoda, and one more in the ancestor of Heterobranchia.

Broadly sampled multigene trees of eukaryotes.

Abstract: Our understanding of the eukaryotic tree of life and the tremendous diversity of microbial eukaryotes is in flux as additional genes and diverse taxa are sampled for molecular analyses. Despite instability in many analyses, there is an increasing trend to classify eukaryotic diversity into six major supergroups: the 'Amoebozoa', 'Chromalveolata', 'Excavata', 'Opisthokonta', 'Plantae', and 'Rhizaria'. Previous molecular analyses have often suffered from either a broad taxon sampling using only single-gene data or have used multigene data with a limited sample of taxa. This study has two major aims: (1) to place taxa represented by 72 sequences, 61 of which have not been characterized previously, onto a well-sampled multigene genealogy, and (2) to evaluate the support for the six putative supergroups using two taxon-rich data sets and a variety of phylogenetic approaches. The inferred trees reveal strong support for many clades that also have defining ultrastructural or molecular characters. In contrast, we find limited to no support for most of the putative supergroups as only the 'Opisthokonta' receive strong support in our analyses. The supergroup 'Amoebozoa' has only moderate support, whereas the 'Chromalveolata', 'Excavata', 'Plantae', and 'Rhizaria' receive very limited or no support. Our analytical approach substantiates the power of increased taxon sampling in placing diverse eukaryotic lineages within well-supported clades. At the same time, this study indicates that the six supergroup hypothesis of higher-level eukaryotic classification is likely premature. The use of a taxon-rich data set with 105 lineages, which still includes only a small fraction of the diversity of microbial eukaryotes, fails to resolve deeper phylogenetic relationships and reveals no support for four of the six proposed supergroups. Our analyses provide a point of departure for future taxon- and gene-rich analyses of the eukaryotic tree of life, which will be critical for resolving their phylogenetic interrelationships.

Mitogenomic evaluation of the historical biogeography of cichlids toward reliable dating of teleostean divergences

Abstract: Recent advances in DNA sequencing and computation offer the opportunity for reliable estimates of divergence times between organisms based on molecular data. Bayesian estimations of divergence times that do not assume the molecular clock use time constraints at multiple nodes, usually based on the fossil records, as major boundary conditions. However, the fossil records of bony fishes may not adequately provide effective time constraints at multiple nodes. We explored an alternative source of time constraints in teleostean phylogeny by evaluating a biogeographic hypothesis concerning freshwater fishes from the family Cichlidae (Perciformes: Labroidei). We added new mitogenomic sequence data from six cichlid species and conducted phylogenetic analyses using a large mitogenomic data set. We found a reciprocal monophyly of African and Neotropical cichlids and their sister group relationship to some Malagasy taxa (Ptychochrominae sensu Sparks and Smith). All of these taxa clustered with a Malagasy + Indo/Sri Lankan clade (Etroplinae sensu Sparks and Smith). The results of the phylogenetic analyses and divergence time estimations between continental cichlid clades were much more congruent with Gondwanaland origin and Cretaceous vicariant divergences than with Cenozoic transmarine dispersal between major continents. We propose to add the biogeographic assumption of cichlid divergences by continental fragmentation as effective time constraints in dating teleostean divergence times. We conducted divergence time estimations among teleosts by incorporating these additional time constraints and achieved a considerable reduction in credibility intervals in the estimated divergence times.

Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken

Abstract: Domestication of chicken is believed to have occurred in Southeast Asia, especially in Indus valley. However, non-inclusion of Indian red jungle fowl (RJF), Gallus gallus murghi in previous studies has left a big gap in understanding the relationship of this major group of birds. In the present study, we addressed this issue by analyzing 76 Indian birds that included 56 G. g. murghi (RJF), 16 G. g. domesticus (domestic chicken) and 4 G. sonneratii (Grey JF) using both microsatellite markers and mitochondrial D-loop sequences. We also compared the D-loop sequences of Indian birds with those of 779 birds obtained from GenBank. Microsatellite marker analyses of Indian birds indicated an average FST of 0.126 within G. g. murghi, and 0.154 within G. g. domesticus while it was more than 0.2 between the two groups. The microsatellite-based phylogenetic trees showed a clear separation of G. g. domesticus from G. g. murghi, and G. sonneratii. Mitochondrial DNA based mismatch distribution analyses showed a lower Harpending's raggedness index in both G. g. murghi (0.001515) and in Indian G. g. domesticus (0.0149) birds indicating population expansion. When meta analysis of global populations of 855 birds was carried out using median joining haplotype network, 43 Indian birds of G. g. domesticus (19 haplotypes) were distributed throughout the network sharing haplotypes with the RJFs of different origins. Our results suggest that the domestication of chicken has occurred independently in different locations of Asia including India. We found evidence for domestication of Indian birds from G. g. spadiceus and G. g. gallus as well as from G. g. murghi, corroborating multiple domestication of Indian and other domestic chicken. In contrast to the commonly held view that RJF and domestic birds hybridize in nature, the present study shows that G. g. murghi is relatively pure. Further, the study also suggested that the chicken populations have undergone population expansion, especially in the Indus valley.

Correlates of substitution rate variation in mammalian protein-coding sequences

Abstract: Rates of molecular evolution in different lineages can vary widely, and some of this variation might be predictable from aspects of species' biology. Investigating such predictable rate variation can help us to understand the causes of molecular evolution, and could also help to improve molecular dating methods. Here we present a comprehensive study of the life history correlates of substitution rate variation across the mammals, comparing results for mitochondrial and nuclear loci, and for synonymous and non-synonymous sites. We use phylogenetic comparative methods, refined to take into account the special nature of substitution rate data. Particular attention is paid to the widespread correlations between the components of mammalian life history, which can complicate the interpretation of results. We find that mitochondrial synonymous substitution rates, estimated from the 9 longest mitochondrial genes, show strong negative correlations with body mass and with maximum recorded lifespan. But lifespan is the sole variable to remain after multiple regression and model simplification. Nuclear synonymous substitution rates, estimated from 6 genes, show strong negative correlations with body mass and generation time, and a strong positive correlation with fecundity. In contrast to the mitochondrial results, the same trends are evident in rates of nonsynonymous substitution. A substantial proportion of variation in mammalian substitution rates can be explained by aspects of their life history, implying that molecular and life history evolution are closely interlinked in this group. The strength and consistency of the nuclear body mass effect suggests that molecular dating studies may have been systematically misled, but also that methods could be improved by incorporating the finding as a priori information. Mitochondrial synonymous rates also show the body mass effect, but for apparently quite different reasons, and the strength of the relationship with maximum lifespan provides support for the hypothesis that mtDNA damage is causally linked to aging.

Dopamine receptor genetic polymorphisms and body composition in undernourished pastoralists: An exploration of nutrition indices among nomadic and recently settled Ariaal men of northern Kenya

Abstract: Minor alleles of the human dopamine receptor polymorphisms, DRD2/TaqI A and DRD4/48 bp, are related to decreased functioning and/or numbers of their respective receptors and have been shown to be correlated with body mass, height and food craving. In addition, the 7R minor allele of the DRD4 gene is at a higher frequency in nomadic compared to sedentary populations. Here we examine polymorphisms in the DRD2 and DRD4 genes with respect to body mass index (BMI) and height among men in two populations of Ariaal pastoralists, one recently settled (n = 87) and the other still nomadic (n = 65). The Ariaal live in northern Kenya, are chronically undernourished and are divided socially among age-sets. Frequencies of the DRD4/7R and DRD2/A1 alleles were 19.4% and 28.2%, respectively and did not differ between the nomadic and settled populations. BMI was higher in those with one or two DRD4/7R alleles in the nomadic population, but lower among the settled. Post-hoc analysis suggests that the DRD4 differences in BMI were due primarily to differences in fat free body mass. Height was unrelated to either DRD2/TaqI A or DRD4/48 bp genotypes. Our results indicate that the DRD4/7R allele may be more advantageous among nomadic than settled Ariaal men. This result suggests that a selective advantage mediated through behaviour may be responsible for the higher frequency of the 7R alleles in nomadic relative to sedentary populations around the world. In contrast to previous work, we did not find an association between DRD2 genotypes and height. Our results support the idea that human phenotypic expression of genotypes should be rigorously evaluated in diverse environments and genetic backgrounds.

Non-homogeneous models of sequence evolution in the Bio++ suite of libraries and programs

Abstract: Accurately modeling the sequence substitution process is required for the correct estimation of evolutionary parameters, be they phylogenetic relationships, substitution rates or ancestral states; it is also crucial to simulate realistic data sets. Such simulation procedures are needed to estimate the null-distribution of complex statistics, an approach referred to as parametric bootstrapping, and are also used to test the quality of phylogenetic reconstruction programs. It has often been observed that homologous sequences can vary widely in their nucleotide or amino-acid compositions, revealing that sequence evolution has changed importantly among lineages, and may therefore be most appropriately approached through non-homogeneous models. Several programs implementing such models have been developed, but they are limited in their possibilities: only a few particular models are available for likelihood optimization, and data sets cannot be easily generated using the resulting estimated parameters. We hereby present a general implementation of non-homogeneous models of substitutions. It is available as dedicated classes in the Bio++ libraries and can hence be used in any C++ program. Two programs that use these classes are also presented. The first one, Bio++ Maximum Likelihood (BppML), estimates parameters of any non-homogeneous model and the second one, Bio++ Sequence Generator (BppSeqGen), simulates the evolution of sequences from these models. These programs allow the user to describe non-homogeneous models through a property file with a simple yet powerful syntax, without any programming required. We show that the general implementation introduced here can accommodate virtually any type of non-homogeneous models of sequence evolution, including heterotachous ones, while being computer efficient. We furthermore illustrate the use of such general models for parametric bootstrapping, using tests of non-homogeneity applied to an already published ribosomal RNA data set.

Mining metadata from unidentified ITS sequences in GenBank: a case study in Inocybe (Basidiomycota)

Abstract: The lack of reference sequences from well-identified mycorrhizal fungi often poses a challenge to the inference of taxonomic affiliation of sequences from environmental samples, and many environmental sequences are thus left unidentified. Such unidentified sequences belonging to the widely distributed ectomycorrhizal fungal genus Inocybe (Basidiomycota) were retrieved from GenBank and divided into species that were identified in a phylogenetic context using a reference dataset from an ongoing study of the genus. The sequence metadata of the unidentified Inocybe sequences stored in GenBank, as well as data from the corresponding original papers, were compiled and used to explore the ecology and distribution of the genus. In addition, the relative occurrence of Inocybe was contrasted to that of other mycorrhizal genera. Most species of Inocybe were found to have less than 3% intraspecific variability in the ITS2 region of the nuclear ribosomal DNA. This cut-off value was used jointly with phylogenetic analysis to delimit and identify unidentified Inocybe sequences to species level. A total of 177 unidentified Inocybe ITS sequences corresponding to 98 species were recovered, 32% of which were successfully identified to species level in this study. These sequences account for an unexpectedly large proportion of the publicly available unidentified fungal ITS sequences when compared with other mycorrhizal genera. Eight Inocybe species were reported from multiple hosts and some even from hosts forming arbutoid or orchid mycorrhizae. Furthermore, Inocybe sequences have been reported from four continents and in climate zones ranging from cold temperate to equatorial climate. Out of the 19 species found in more than one study, six were found in both Europe and North America and one was found in both Europe and Japan, indicating that at least many north temperate species have a wide distribution. Although DNA-based species identification and circumscription are associated with practical and conceptual difficulties, they also offer new possibilities and avenues for research. Metadata assembly holds great potential to synthesize valuable information from community studies for use in a species and taxonomy-oriented framework.

Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis

Abstract: The Wuschel related homeobox (WOX) family proteins are key regulators implicated in the determination of cell fate in plants by preventing cell differentiation. A recent WOX phylogeny, based on WOX homeodomains, showed that all of the Physcomitrella patens and Selaginella moellendorffii WOX proteins clustered into a single orthologous group. We hypothesized that members of this group might preferentially share a significant part of their function in phylogenetically distant organisms. Hence, we first validated the limits of the WOX13 orthologous group (WOX13 OG) using the occurrence of other clade specific signatures and conserved intron insertion sites. Secondly, a functional analysis using expression data and mutants was undertaken. The WOX13 OG contained the most conserved plant WOX proteins including the only WOX detected in the highly proliferating basal unicellular and photosynthetic organism Ostreococcus tauri. A large expansion of the WOX family was observed after the separation of mosses from other land plants and before monocots and dicots have arisen. In Arabidopsis thaliana, AtWOX13 was dynamically expressed during primary and lateral root initiation and development, in gynoecium and during embryo development. AtWOX13 appeared to affect the floral transition. An intriguing clade, represented by the functional AtWOX14 gene inside the WOX13 OG, was only found in the Brassicaceae. Compared to AtWOX13, the gene expression profile of AtWOX14 was restricted to the early stages of lateral root formation and specific to developing anthers. A mutational insertion upstream of the AtWOX14 homeodomain sequence led to abnormal root development, a delay in the floral transition and premature anther differentiation. Our data provide evidence in favor of the WOX13 OG as the clade containing the most conserved WOX genes and established a functional link to organ initiation and development in Arabidopsis, most likely by preventing premature differentiation. The future use of Ostreococcus tauri and Physcomitrella patens as biological models should allow us to obtain a better insight into the functional importance of WOX13 OG genes.

Costs and benefits of multiple resistance to insecticides for Culex quinquefasciatus mosquitoes

Abstract: The evolutionary dynamics of xenobiotic resistance depends on how resistance mutations influence the fitness of their bearers, both in the presence and absence of xenobiotic selection pressure. In cases of multiple resistance, these dynamics will also depend on how individual resistance mutations interact with one another, and on the xenobiotics applied against them. We compared Culex quinquefasciatus mosquitoes harbouring two resistance alleles ace-1 R and Kdr R (conferring resistance to carbamate and pyrethroid insecticides, respectively) to mosquitoes bearing only one of the alleles, or neither allele. Comparisons were made in environments where both, only one, or neither type of insecticide was present. Each resistance allele was associated with fitness costs (survival to adulthood) in an insecticide-free environment, with the costs of ace-1 R being greater than for Kdr R . However, there was a notable interaction in that the costs of harbouring both alleles were significantly less than for harbouring ace-1 R alone. The two insecticides combined in an additive, synergistic and antagonistic manner depending on a mosquito's resistance status, but were not predictable based on the presence/absence of either, or both mutations. Insecticide resistance mutations interacted to positively or negatively influence a mosquito's fitness, both in the presence or absence of insecticides. In particular, the presence of the Kdr R mutation compensated for the costs of the ace-1 R mutation in an insecticide-free environment, suggesting the strength of selection in untreated areas would be less against mosquitoes resistant to both insecticides than for those resistant to carbamates alone. Additional interactions suggest the dynamics of resistance will be difficult to predict in populations where multiple resistance mutations are present or that are subject to treatment by different xenobiotics.

Post-Messinian Evolutionary Relationships Across the Sicilian Channel: Mitochondrial and Nuclear Markers Link a New Green Toad From Sicily to African Relatives

Abstract: Little attention has been paid to the consequences of the last landbridge between Africa and Sicily on Mediterranean biogeography. Previous paleontological and scarce molecular data suggest possible faunal exchange later than the well-documented landbridge in the Messinian (5.3 My); however, a possible African origin of recent terrestrial Sicilian fauna has not been thoroughly tested with molecular methods. To gain insight into the phylogeography of the region, we examine two mitochondrial and two nuclear markers (one is a newly adapted intron marker) in green toads (Bufo viridis subgroup) across that sea barrier, the Strait of Sicily. Extensive sampling throughout the western Mediterranean and North Africa revealed a deep sister relationship between Sicilian (Bufo siculus n.sp.) and African green toads (B. boulengeri) on the mitochondrial and nuclear level. Divergence times estimated under a Bayesian-coalescence framework (mtDNA control region and 16S rRNA) range from the Middle Pliocene (3.6 My) to Pleistocene (0.16 My) with an average (1.83 to 2.0 My) around the Pliocene/Pleistocene boundary, suggesting possible land connections younger than the Messinian (5.3 My). We describe green toads from Sicily and some surrounding islands as a new endemic species (Bufo siculus). Bufo balearicus occurs on some western Mediterranean islands (Corsica, Sardinia, Mallorca, and Menorca) and the Apennine Peninsula, and is well differentiated on the mitochondrial and nuclear level from B. siculus as well as from B. viridis (Laurenti), whose haplotype group reaches northeastern Italy, north of the Po River. Detection of Calabrian B. balearicus haplotypes in northeastern Sicily suggests recent invasion. Our data agree with paleogeographic and fossil data, which suggest long Plio-Pleistocene isolation of Sicily and episodic Pleistocene faunal exchange across the Strait of Messina. It remains unknown whether both species (B. balearicus, B. siculus) occur in sympatry in northern Sicily. Our findings on green toads give the first combined mitochondrial and nuclear sequence evidence for a phylogeographic connection across the Strait of Sicily in terrestrial vertebrates. These relationships may have implications for comparative phylogeographic research on other terrestrial animals co-occurring in North Africa and Sicily.

The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship

Abstract: Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins. We have performed an evolutionary comparative analysis of cysteine proteinases C1A and C13 and their putative inhibitors in representative species of different taxonomic groups that appeared during the evolution of the Viridiplantae. The results indicate that whereas C1A cysteine proteinases are present in all taxonomic groups, cystatins and C13 cysteine proteinases are absent in some basal groups. Moreover, gene duplication events have been associated to the increasing structural and functional complexities acquired in land plants. Comparative genomic analyses have provided us valuable insights into the conservation and evolution of the cystatin inhibitory family and their putative targets, the cysteine proteinases from families C1A and C13. Functionality of both families of proteins in plants must be the result of a coevolutionary process that might have occurred during the evolution of basal and land plants leading to a complex functional relationship among them.

Cophylogeny of the anther smut fungi and their caryophyllaceous hosts: prevalence of host shifts and importance of delimiting parasite species for inferring cospeciation.

Abstract: Using phylogenetic approaches, the expectation that parallel cladogenesis should occur between parasites and hosts has been validated in some studies, but most others provided evidence for frequent host shifts. Here we examine the evolutionary history of the association between Microbotryum fungi that cause anther smut disease and their Caryophyllaceous hosts. We investigated the congruence between host and parasite phylogenies, inferred cospeciation events and host shifts, and assessed whether geography or plant ecology could have facilitated the putative host shifts identified. For cophylogeny analyses on microorganisms, parasite strains isolated from different host species are generally considered to represent independent evolutionary lineages, often without checking whether some strains actually belong to the same generalist species. Such an approach may mistake intraspecific nodes for speciation events and thus bias the results of cophylogeny analyses if generalist species are found on closely related hosts. A second aim of this study was therefore to evaluate the impact of species delimitation on the inferences of cospeciation. We inferred a multiple gene phylogeny of anther smut strains from 21 host plants from several geographic origins, complementing a previous study on the delimitation of fungal species and their host specificities. We also inferred a multi-gene phylogeny of their host plants, and the two phylogenies were compared. A significant level of cospeciation was found when each host species was considered to harbour a specific parasite strain, i.e. when generalist parasite species were not recognized as such. This approach overestimated the frequency of cocladogenesis because individual parasite species capable of infecting multiple host species (i.e. generalists) were found on closely related hosts. When generalist parasite species were appropriately delimited and only a single representative of each species was retained, cospeciation events were not more frequent than expected under a random distribution, and many host shifts were inferred. Current geographic distributions of host species seemed to be of little relevance for understanding the putative historical host shifts, because most fungal species had overlapping geographic ranges. We did detect some ecological similarities, including shared pollinators and habitat types, between host species that were diseased by closely related anther smut species. Overall, genetic similarity underlying the host-parasite interactions appeared to have the most important influence on specialization and host-shifts: generalist multi-host parasite species were found on closely related plant species, and related species in the Microbotryum phylogeny were associated with members of the same host clade. We showed here that Microbotryum species have evolved through frequent host shifts to moderately distant hosts, and we show further that accurate delimitation of parasite species is essential for interpreting cophylogeny studies.