Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

Asymmetric allelic introgression across a hybrid zone of the coal tit (Periparus ater) in the central Himalayas.

In the Himalayas, a number of secondary contact zones have been described for vicariant vertebrate taxa. However, analyses of genetic divergence and admixture are missing for most of these examples. In this study, we provide a population genetic analysis for the coal tit (Periparus ater) hybrid zone in Nepal. Intermediate phenotypes between the distinctive western "spot-winged tit" (P. a. melanolophus) and Eastern Himalayan coal tits (P. a. aemodius) occur across a narrow range of <100 km in western Nepal. As a peculiarity, another distinctive cinnamon-bellied form is known from a single population so far. Genetic admixture of western and eastern mitochondrial lineages was restricted to the narrow zone of phenotypically intermediate populations. The cline width was estimated 46 km only with a center close to the population of the cinnamon-bellied phenotype. In contrast, allelic introgression of microsatellite loci was asymmetrical from eastern P. a. aemodius into far western populations of phenotypic P. a. melanolophus but not vice versa. Accordingly, the microsatellite cline was about 3.7 times wider than the mitochondrial one.

Convergent Losses of TLR5 Suggest Altered Extracellular Flagellin Detection in Four Mammalian Lineages.

Toll-like receptors (TLRs) play an important role for the innate immune system by detecting pathogen-associated molecular patterns. TLR5 encodes the major extracellular receptor for bacterial flagellin and frequently evolves under positive selection, consistent with coevolutionary arms races between the host and pathogens. Furthermore, TLR5 is inactivated in several vertebrates and a TLR5 stop codon polymorphism is widespread in human populations. Here, we analyzed the genomes of 120 mammals and discovered that TLR5 is convergently lost in four independent lineages, comprising guinea pigs, Yangtze river dolphin, pinnipeds, and pangolins. Validated inactivating mutations, absence of protein-coding transcript expression, and relaxed selection on the TLR5 remnants confirm these losses. PCR analysis further confirmed the loss of TLR5 in the pinniped stem lineage. Finally, we show that TLR11, encoding a second extracellular flagellin receptor, is also absent in these four lineages. Independent losses of TLR5 and TLR11 suggest that a major pathway for detecting flagellated bacteria is not essential for different mammals and predicts an impaired capacity to sense extracellular flagellin.

Genetic admixture despite ecological segregation in a North African sparrow hybrid zone (Aves, Passeriformes, Passer domesticus × Passer hispaniolensis).

Under different environmental conditions, hybridization between the same species might result in different patterns of genetic admixture. Particularly, species pairs with large distribution ranges and long evolutionary history may have experienced several independent hybridization events over time in different zones of overlap. In birds, the diverse hybrid populations of the house sparrow (Passer domesticus) and the Spanish sparrow (Passer hispaniolensis) provide a striking example. Throughout their range of sympatry, these two species do not regularly interbreed; however, a stabilized hybrid form (Passer italiae) exists on the Italian Peninsula and on several Mediterranean islands. The spatial distribution pattern on the Eurasian continent strongly contrasts the situation in North Africa, where house sparrows and Spanish sparrows occur in close vicinity of phenotypically intermediate populations across a broad mosaic hybrid zone. In this study, we investigate patterns of divergence and admixture among the two parental species, stabilized and nonstabilized hybrid populations in Italy and Algeria based on a mitochondrial marker, a sex chromosomal marker, and 12 microsatellite loci. In Algeria, despite strong spatial and temporal separation of urban early-breeding house sparrows and hybrids and rural late-breeding Spanish sparrows, we found strong genetic admixture of mitochondrial and nuclear markers across all study populations and phenotypes. That pattern of admixture in the North African hybrid zone is strikingly different from i) the Iberian area of sympatry where we observed only weak asymmetrical introgression of Spanish sparrow nuclear alleles into local house sparrow populations and ii) the very homogenous Italian sparrow population where the mitogenome of one parent (P. domesticus) and the Z-chromosomal marker of the other parent (P. hispaniolensis) are fixed. The North African sparrow hybrids provide a further example of enhanced hybridization along with recent urbanization and anthropogenic land-use changes in a mosaic landscape.

Convergent vomeronasal system reduction in mammals coincides with convergent losses of calcium signalling and odorant-degrading genes.

The vomeronasal system (VNS) serves crucial functions for detecting olfactory clues often related to social and sexual behaviour. Intriguingly, two of the main components of the VNS, the vomeronasal organ (VNO) and the accessory olfactory bulb, are regressed in aquatic mammals, several bats and primates, likely due to adaptations to different ecological niches. To detect genomic changes that are associated with the convergent reduction of the VNS, we performed the first systematic screen for convergently inactivated protein-coding genes associated with convergent VNS reduction, considering 106 mammalian genomes. Extending previous studies, our results support that Trpc2, a cation channel that is important for calcium signalling in the VNO, is a predictive molecular marker for the presence of a VNS. Our screen also detected the convergent inactivation of the calcium-binding protein S100z, the aldehyde oxidase Aox2 that is involved in odorant degradation, and the uncharacterized Mslnl gene that is expressed in the VNO and olfactory epithelium. Furthermore, we found that Trpc2 and S100z or Aox2 are also inactivated in otters and Phocid seals for which no morphological data about the VNS are available yet. This predicts a VNS reduction in these semi-aquatic mammals. By examining the genomes of 115 species in total, our study provides a detailed picture of how the convergent reduction of the VNS coincides with gene inactivation in placental mammals. These inactivated genes provide experimental targets for studying the evolution and biological significance of the olfactory system under different environmental conditions.

A strategy for the conservation of biodiversity on mid-ocean ridges from deep-sea mining.

Mineral exploitation has spread from land to shallow coastal waters and is now planned for the offshore, deep seabed. Large seafloor areas are being approved for exploration for seafloor mineral deposits, creating an urgent need for regional environmental management plans. Networks of areas where mining and mining impacts are prohibited are key elements of these plans. We adapt marine reserve design principles to the distinctive biophysical environment of mid-ocean ridges, offer a framework for design and evaluation of these networks to support conservation of benthic ecosystems on mid-ocean ridges, and introduce projected climate-induced changes in the deep sea to the evaluation of reserve design. We enumerate a suite of metrics to measure network performance against conservation targets and network design criteria promulgated by the Convention on Biological Diversity. We apply these metrics to network scenarios on the northern and equatorial Mid-Atlantic Ridge, where contractors are exploring for seafloor massive sulfide (SMS) deposits. A latitudinally distributed network of areas performs well at (i) capturing ecologically important areas and 30 to 50% of the spreading ridge areas, (ii) replicating representative areas, (iii) maintaining along-ridge population connectivity, and (iv) protecting areas potentially less affected by climate-related changes. Critically, the network design is adaptive, allowing for refinement based on new knowledge and the location of mining sites, provided that design principles and conservation targets are maintained. This framework can be applied along the global mid-ocean ridge system as a precautionary measure to protect biodiversity and ecosystem function from impacts of SMS mining.

Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Combined genotyping, microbial diversity and metabolite profiling studies on farmed Mytilus spp. from Kiel Fjord.

The blue mussel Mytilus is a popular food source with high economical value. Species of the M. edulis complex (M. edulis, M. galloprovincialis and M. trossulus) hybridise whenever their geographic ranges overlap posing difficulties to species discrimination, which is important for blue mussel aquaculture. The aim of this study was to determine the genetic structure of farmed blue mussels in Kiel Fjord. Microbial and metabolic profile patterns were studied to investigate a possible dependency on the genotype of the bivalves. Genotyping confirmed the complex genetic structure of the Baltic Sea hybrid zone and revealed an unexpected dominance of M. trossulus alleles being in contrast to the predominance of M. edulis alleles described for wild Baltic blue mussels. Culture-dependent and -independent microbial community analyses indicated the presence of a diverse Mytilus-associated microbiota, while an LC-MS/MS-based metabolome study identified 76 major compounds dominated by pigments, alkaloids and polyketides in the whole tissue extracts. Analysis of mussel microbiota and metabolome did not indicate genotypic dependence, but demonstrated high intraspecific variability of farmed mussel individuals. We hypothesise that individual differences in microbial and metabolite patterns may be caused by high individual plasticity and might be enhanced by e.g. nutritional condition, age and gender.

Combining hydrodynamic modelling with genetics: can passive larval drift shape the genetic structure of Baltic Mytilus populations?

While secondary contact between Mytilus edulis and Mytilus trossulus in North America results in mosaic hybrid zone formation, both species form a hybrid swarm in the Baltic. Despite pervasive gene flow, Baltic Mytilus species maintain substantial genetic and phenotypic differentiation. Exploring mechanisms underlying the contrasting genetic composition in Baltic Mytilus species will allow insights into processes such as speciation or adaptation to extremely low salinity. Previous studies in the Baltic indicated that only weak interspecific reproductive barriers exist and discussed the putative role of adaptation to environmental conditions. Using a combination of hydrodynamic modelling and multilocus genotyping, we investigate how oceanographic conditions influence passive larval dispersal and hybrid swarm formation in the Baltic. By combining our analyses with previous knowledge, we show a genetic transition of Baltic Mytilus species along longitude 12°-13°E, that is a virtual line between Malmö (Sweden) and Stralsund (Germany). Although larval transport only occurs over short distances (10-30 km), limited larval dispersal could not explain the position of this genetic transition zone. Instead, the genetic transition zone is located at the area of maximum salinity change (15-10 psu). Thus, we argue that selection results in weak reproductive barriers and local adaptation. This scenario could maintain genetic and phenotypic differences between Baltic Mytilus species despite pervasive introgressive hybridization.

Mitochondrial DNA Analyses Indicate High Diversity, Expansive Population Growth and High Genetic Connectivity of Vent Copepods (Dirivultidae) across Different Oceans.

Communities in spatially fragmented deep-sea hydrothermal vents rich in polymetallic sulfides could soon face major disturbance events due to deep-sea mineral mining, such that unraveling patterns of gene flow between hydrothermal vent populations will be an important step in the development of conservation policies. Indeed, the time required by deep-sea populations to recover following habitat perturbations depends both on the direction of gene flow and the number of migrants available for re-colonization after disturbance. In this study we compare nine dirivultid copepod species across various geological settings. We analyze partial nucleotide sequences of the mtCOI gene and use divergence estimates (FST) and haplotype networks to infer intraspecific population connectivity between vent sites. Furthermore, we evaluate contrasting scenarios of demographic population expansion/decline versus constant population size (using, for example, Tajima's D). Our results indicate high diversity, population expansion and high connectivity of all copepod populations in all oceans. For example, haplotype diversity values range from 0.89 to 1 and FST values range from 0.001 to 0.11 for Stygiopontius species from the Central Indian Ridge, Mid Atlantic Ridge, East Pacific Rise, and Eastern Lau Spreading Center. We suggest that great abundance and high site occupancy by these species favor high genetic diversity. Two scenarios both showed similarly high connectivity: fast spreading centers with little distance between vent fields and slow spreading centers with greater distance between fields. This unexpected result may be due to some distinct frequency of natural disturbance events, or to aspects of individual life histories that affect realized rates of dispersal. However, our statistical performance analyses showed that at least 100 genomic regions should be sequenced to ensure accurate estimates of migration rate. Our demography parameters demonstrate that dirivultid populations are generally large and continuously undergoing population growth. Benthic and pelagic species abundance data support these findings.

North African hybrid sparrows (Passer domesticus, P. hispaniolensis) back from oblivion - ecological segregation and asymmetric mitochondrial introgression between parental species.

A stabilized hybrid form of the house sparrow (Passer domesticus) and the Spanish sparrow (P. hispaniolensis) is known as Passer italiae from the Italian Peninsula and a few Mediterranean islands. The growing attention for the Italian hybrid sparrow and increasing knowledge on its biology and genetic constitution greatly contrast the complete lack of knowledge of the long-known phenotypical hybrid sparrow populations from North Africa. Our study provides new data on the breeding biology and variation of mitochondrial DNA in three Algerian populations of house sparrows, Spanish sparrows, and phenotypical hybrids. In two field seasons, the two species occupied different breeding habitats: Spanish sparrows were only found in rural areas outside the cities and bred in open-cup nests built in large jujube bushes. In contrast, house sparrows bred only in the town centers and occupied nesting holes in walls of buildings. Phenotypical hybrids were always associated with house sparrow populations. House sparrows and phenotypical hybrids started breeding mid of March, and most pairs had three successive clutches, whereas Spanish sparrows started breeding almost one month later and had only two successive clutches. Mitochondrial introgression is strongly asymmetric because about 75% of the rural Spanish sparrow population carried house sparrow haplotypes. In contrast, populations of the Italian hybrid form, P. italiae, were genetically least diverse among all study populations and showed a near-fixation of house sparrow haplotypes that elsewhere were extremely rare or that were even unique for the Italian Peninsula. Such differences between mitochondrial gene pools of Italian and North African hybrid sparrow populations provide first evidence that different demographic histories have shaped the extant genetic diversity observed on both continents.

One extinct turtle species less: Pelusios seychellensis is not extinct, it never existed.

Pelusios seychellensis is thought to be a freshwater turtle species endemic to the island of Mahé, Seychelles. There are only three museum specimens from the late 19(th) century known. The species has been never found again, despite intensive searches on Mahé. Therefore, P. seychellensis has been declared as "Extinct" by the IUCN and is the sole putatively extinct freshwater turtle species. Using DNA sequences of three mitochondrial genes of the historical type specimen and phylogenetic analyses including all other species of the genus, we provide evidence that the description of P. seychellensis was erroneously based on a widely distributed West African species, P. castaneus. Consequently, we synonymize the two species and delete P. seychellensis from the list of extinct chelonian species and from the faunal list of the Seychelles.

Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females.

Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.

Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production.

The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26-AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.

Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice.

The mammary gland undergoes a complex set of changes to establish copious milk secretion at parturition. To test the hypothesis that signaling through the Rho pathway plays a role in secretory activation, transgenic mice expressing a constitutively activated form of the Rho effector protein PKN1 in the mammary epithelium were generated. PKN1 activation had no effect in late pregnancy but inhibited milk secretion after parturition, diminishing the ability of transgenic dams to support a litter. Mammary gland morphology as well as increased apoptosis and expression of IFGBP5 and TGFbeta3 suggest precocious involution in these animals. Furthermore, tight junction sealing at parturition was impaired in transgenic mammary glands as demonstrated by intraductal injection of [14C]sucrose. Consistent with this finding, tight junction sealing in response to glucocorticoid stimulation was highly impaired in EpH4 mammary epithelial cells expressing constitutively activated PKN1, whereas expression of a dominant-negative PKN1 mutant resulted in accelerated tight junction sealing in vitro. Tight junction formation was not impaired as demonstrated by the correct localization of occludin and ZO1 at the apical cell borders. Our results provide evidence that PKN1 participates in the regulation of tight junction sealing in the mammary gland by interfering with glucocorticoid signaling.

Heteroplasmy suggests paternal co-transmission of multiple genomes and pervasive reversion of maternally into paternally transmitted genomes of mussel (Mytilus) mitochondrial DNA.

Marine mussels of the genus Mytilus have two types of mitochondrial DNA with separate paternal and maternal inheritance. Females are homoplasmic for an F genome that is transmitted to all offspring, whereas males are heteroplasmic for this F genome and for a highly diverged (> 20%) M genome that is transmitted only to sons. Here we provide phylogenetic evidence based on lrRNA sequence data that most of the paternal genomes in European M. trossulus have an introgressive female M. edulis origin and are nearly indistinguishable in sequence from F types of M. trossulus. This observation is best explained by the hypothesis that introgressed F type molecules have recently invaded the paternal route and have assumed the role of M molecules, then resetting to zero the time of sequence divergence between M and F lineages. European M. trossulus shows a high prevalence of males heteroplasmic for three different mitochondrial DNA types all having the same two paternal types and the same maternal type, consistent with paternal co-transmission of multiple genomes. Co-transmission of the same genomes must apparently operate uninterruptedly for several generations in spite of the very different evolutionary origin of the specific molecules that are transmitted paternally and maternally in European M. trossulus.