SESSION 1 - MOLECULAR EVOLUTION

Exploring the role of mutation and selection on synonymous variability in metazoan mitochondrial genomes

S. Castellana¹, S. Vicario², G. Donvito³, C. Saccone⁴

Synonymous variability in all nuclear protein coding genes of model organisms is somehow constrained in order to prevent ribosome slowdown during translation, because these could increase the probability of the incorporation of un-corrected amino acid and consequently the production of toxic misfolded protein. We investigated how synonymous variability evolves in mitochondrial genomes of Vertebrata and Insecta in order to detect events of selective pressures acting on synonymous codons.
At first, we estimated a one-dimensional index of codon usage bias (ENC) for every genes in the collected genomes (138 insect and 1173 vertebrate ‘RefSeq’ mtDNA), comparing it with different factors, such as the same index calculated under a mutational scenario. Then, we compared different codon usage and base composition (on third codon positions) likelihood models, trying to evaluate if mutation is gene or genome specific and is the only evolutionary force that determines the codon usage bias.
At last, we collect 45 groups of complete cogeneric vertebrate mtDNAs, for analyzing the relationship between synonymous and non-synonymous substitution rates (‘d_n’, ‘d_s’) within each group.
We found that codon usage bias is mainly dependent on mutation (which is gene-specific), but about 15% of its variability is associated to gene and species factors. Base composition likelihood models confirmed that mutational input is gene-specific, while the model in which codon usage is inferred by this input is rejected by the 20 and 40% of insect and vertebrate species, respectively.
There is no correlation between non-synonymous and synonymous substitution rates among the protein coding genes in each vertebrate cogeneric dataset. This is in contrast with the hypothesis of purifying selection acting on nuclear genes.
In the future, we are thinking to improve the modeling of codon usage by taking account the effect of neighboring base on the mutation bias of synonymous sites.

1 Dipartimento di Genetica e Microbiologia, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy.
2 Istituto di Tecnologie Biomediche, CNR, sede di Bari, Italy.
3 Istituto Nazionale di Fisica Nucleare, sede di Bari, Italy.
4 Dipartimento di Biochimica e Biologia Molecolare ‘E.Quagliariello’, Università degli Studi di Bari ‘Aldo Moro’, Bari, Italy.

SESSION 1 - MOLECULAR EVOLUTION

Short Interspersed Elements activity, adaptation and genomic distribution in termite genome

A. Luchetti, B. Mantovani ¹

Transposable elements (TEs) are self-replicating DNA sequences, widespread in eukaryotic genomes: jumping across the host genome, TEs interact either positively or negatively with gene and genome functionalities. Evolutionary models proposed so far to describe the dynamics of TEs within genomes rely on the so-called “selfish DNA” theory, in an equilibrium between selection and mobilization capacity.
Moreover, beside the simple TEs accumulation, also their distribution within genomes seems to be shaped by either selective pressures or local recombination rate. Here, we present the evolutionary history and genomic distribution of four SINEs (short, non-autonomous elements) discovered in termite (Insecta, Isoptera) genome through DNA libraries sequencing and Genbank EST collection scanning.
Two SINEs were found only within the Isopteran lineage, accordingly with age distribution analysis, while the other two arose before the splitting Blattoidea/Isoptera (~150 Myr ago). One of them (Taluc) shares a conserved sequence block with other SINEs isolated in Orthoptera and Diptera, as already observed for some SINEs in mammals and vertebrates. It also exhibits 3’ sequence changes across cockroaches and termite taxa, suggesting a retropositional adaptation in different genomic contexts.
Flanking region annotation showed a preferential insertion of the four SINEs in non-coding region, with significant links to microsatellite loci. In the DNA random clone library, the older element (Talud) is more frequently found near coding regions with respect to the other three elements. This genomic distribution parallels the one observed for the human SINE Alu, where a clear explanation for this distribution has not been yet determined. Selective mechanisms as well as peculiar organismal traits are here proposed to explain the observed biology of termite SINEs.

1 Dip. Biologia Evoluzionistica Sperimentale, Università di Bologna, Bologna, Italy.

SESSION 1 - MOLECULAR EVOLUTION

Molecular evolution of a gene cluster of female serine proteases involved in post-mating mechanisms of the malaria mosquito Anopheles gambiae

F. Tammaro¹, E. Mancini¹, F. Baldini², D. Rogers³, A. Via⁴, D. Raimondo⁴, P. George⁵, I. Sharakhov⁵, A. Tramontano⁴, F. Catteruccia^2,3, A. Della Torre¹

Proteins involved in reproduction evolve rapidly due to positive selection resulting from intersexual interaction. In Drosophila, rapid evolution driven by positive selection has been detected in proteases of the female lower reproductive tract (LRT) known to interact with proteins of the male ejaculate. Similarly, several proteases specifically expressed in the female LRT of Anopheles gambiae are likely to interact with male accessory gland products transferred to females upon mating that induce a series of physiological post-mating responses in females.
Here we report data on the molecular evolution in five members of the A. gambiae complex (A. gambiae s.s., A. arabiensis, A. quadriannulatus, A. melas and A. merus) of a clusters of 3 LRT specific genes potentially involved in post-mating mechanisms. The 3 LRT-specific genes encode serine proteases that are down-regulated after mating, two of which expressed in the atrium (and likely to digest the mating plug) and one in the spermatheca. Adaptive evolution was detected in several codons of the 3 genes and evidences of episodic selection were also found. The high level of replacement polymorphisms found in all 3 proteases suggests that these duplicated genes might experience relaxed evolutionary constraints that could be important to rapidly explore and eventually fix new advantageous variants. Moreover, the structural modeling of these proteins highlighted important differences in their substrate specificity and allowed to localize and better evaluate the effects of amino acid replacements in sites evolving under selective pressures.
Novel approaches will be developed to characterize the proteolytic activity of these proteases and, thus, to better interpret the observed evolutionary patterns. Elucidating the functional role of this gene family would in fact allow to understand its importance in the reproductive success of A. gambiae species, and to eventually improve malaria vector control strategies.

1 ‘Sapienza’ Università di Roma, Dip. Scienze di Sanità Pubblica, Rome, Italy.
2 Universita’ di Perugia, Dip. Medicina Sperimentale e Scienze biochimiche, Perugia, Italy.
3 Imperial College, London, UK.
4 ‘Sapienza’ Università di Roma, Dip. Scienze Biochimiche, Rome, Italy.
5 Dept. of Entomology, Virgina Tech, Blacksburg, USA.

SESSION 1 - MOLECULAR EVOLUTION

The evolutionary dynamics of plasmids and chromosomes inhabiting the same bacterial cytoplasm

I. Maida, M. Fondi, M.C. Papaleo, E. Perrin, R. Fani ¹

Despite the key-role of plasmids in the horizontal spreading of genetic information within the prokaryotic community, their evolutionary dynamics has been poorly explored. Particularly interesting from an evolutionary viewpoint is the cross-talk existing between the different DNA molecules (plasmids and chromosomes) inhabiting the same cytoplasm. In order to shed some light on this issue, we performed a detailed analysis of the genome of Burkholderia vietnamiensis G4, a β-proteobacterium belonging to the Burkholderia genus. We focused the attention on this micro-organism since its genome consisted of five plasmids and three chromosomes of different size; hence, strain G4 can be considered as a model microorganism to analyze the molecular mechanisms responsible of the gene rearrangements occurring between different DNA molecules of the same cell. To this purpose, we adopted the pipeline Blast2Network (B2N). This software allows the visualization of evolutionary relationships among a datasets of sequences through the building up of identity-based similarity networks.
The aims of our work were: i) the identification and the analysis of the possible evolutionary relationships existing within and among the five B. vietnamiensis G4 plasmids at both intra- and intermolecular level, ii) the analysis of the gene flow (if any) existing between the chromosomes and plasmids of different size inhabiting the same cytoplasm.
Data obtained revealed: i) the existence of intra-molecular rearrangements mainly due to duplication events that occurred in different stages of plasmids evolution; ii) an extensive gene flow between both different plasmids and plasmids and chromosomes; iii) these genetic rearrangements involved single genes, multiple genes, operons and/or gene clusters; iv) as might be expected, mobile genetic elements have played and are still playing a key-role in the recombination events occurring between different molecules; v) complex evolutionary pathways leading to the structure of the G4 plasmids.

1 Laboratory of Microbial and Molecular Evolution, Department of Evolutionary Biology, University of Florence, Firenze, Italy.

SESSION 1 - MOLECULAR EVOLUTION

A survey of mitochondrial evolution in Metazoa: large variability in little genomes

R. Lupi, C. Gissi ¹

The animal mitochondrial genome (mtDNA) has been traditionally used as molecular marker for phylogenetic reconstructions but the study of the evolutionary history of this small genome can also help to unravel the hidden link between structural and functional genomic features. In this respect, the animal mtDNA can be regarded as a model in comparative genomics, whose use is also enhanced by the availability of complete sequences for about 2000 species.
In spite of the biased taxon sampling, the analysis of the present mtDNA dataset is doubtless promising to reconstruct the evolutionary history of this entire genome in a wide phylogenetic range and to identify possible differences in its evolutionary trend among different lineages.
In order to carefully analyze the plethora of available mt sequences of Metazoa, we have developed a specialized mtDNA database, MitoZoa, collecting (nearly) complete mtDNA entries whose annotations have been significantly corrected/improved using a semi-automatic reannotation pipeline. MitoZoa has been designed both to address comparative analyses of genomic features, such as gene order, non-coding regions and gene content, and to help comparisons at short evolutionary distances, such as in congeneric species. Here, we will briefly present the MitoZoa database and the preliminary results of our investigation on the mtDNA evolutionary dynamics in Metazoa: we have investigated the variability of basic mitogenomic features, such as gene content, gene compactness, and base composition, and the trend of gene order rearrangements in the main metazoan lineages.
In fast-evolving lineages, the same features have also been investigated in congeneric species, i.e. in closely-related species where saturation of evolutionary changes is unlikely to occur.
Our data show that the mtDNA plasticity of Metazoa is higher than previously thought and that the mt evolutionary trend has changed several times, and sometimes dramatically, in the metazoan phylogenetic tree.

1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.

SESSION 1 - MOLECULAR EVOLUTION

Mithocondrial DNA (mtDNA) temporal variation in wild and domestic pigs from a North-Eastern Italian site

M. Lari¹, S. Vai1, M. Romandini², E. Rizzi³, G. Corto³, P. Visentini⁴, G. De Bellis³, D. Caramelli¹, G. Bertorelle⁵

Ancient DNA analysis of faunal remains is a useful tool to reconstruct past migration events and different aspects of a domestication process. In the last five years, particular attention has been addressed using both modern and ancient DNA data to clarify the complex domestication processes responsible of the transition between the wild board and the domestic pig (Sus scrofa).
In this study we focused our attention on a single site in Northern Italy, Biarzo Shelter (Udine). This choice is justified by at least three characteristics of this site. First of all, Sus scrofa is the most represented species at all stratigraphic levels, with large number of specimens available. Second, Biarzo Shelter represents the only Northern Italy site with continuous stratigraphic records of the Pleistocene-Olocene transition.
This continuity provides an opportunity to directly monitor modifications in both archaeozoological parameters and genetic traits in a ~6000 years time frame from the Upper Palaeolithic to the Neolithic. Third, the geographic area of the site represents a likely connection region between the Balkans, Italy, and Central Europe, both for animal migrations and for cultural exchanges. We selected 28 remains recovered from five different stratigraphic units assigned to three different archeological contexts (Epigravettian, Mesolithic and Neolithic). Following standard ancient DNA procedures, we analyzed a small fragment of the mitochondrial DNA (~80bp) that, despite the short length, has been showed to be highly informative. Our preliminary results suggest that a variation of the frequency of different haplotypes occurred through time, possibly related to the early stages of the pig domestication process.
In addition, we recovered in two Mesolithic samples a sequence motif observed today only in the Near East and which was previously associated to the Neolithic diffusion of herding and farming lifestyles.

1 Dept. of Evolutionary Biology, University of Firenze, Firenze, Italy.
2 Dept. of Biology and Evolution, Paleobiology Prehistory and Anthropology Section, University of Ferrara, Ferrara, Italy.
3 Institute for Biomedical Technologies, National Research Council (ITB – CNR), Segrate (Milano), Italy.
4 Museo Friulano di Storia Naturale, Udine, Italy.
5 Dept. of Biology and Evolution, University of Ferrara, Ferrara, Italy.

SESSION 1 - MOLECULAR EVOLUTION

The sexually antagonistic genes of Drosophila melanogaster

P. Innocenti¹

When selective pressures differ between males and females, the genes experiencing these conflicting evolutionary forces are said to be sexually antagonistic. Although the phenotypic effect of these genes has been documented in both wild and laboratory populations, their identity, number, and location remains unknown. We used a combination of hemiclonal analysis and genomics technology (microarrays) to ascertain the existence of intralocus sexual conflict and to investigate its genetic basis. One hundred hemiclonal lines were established and assayed for both male and female relative fitness in a replicated design under competitive conditions. We found significant genetic variation for both male and female relative fitness, as well as a significant negative genetic correlation between the sexes – thereby confirming the existence of intralocus sexual conflict.
In order to identify which loci contribute to fitness variation and the sexually antagonistic effect, we combined data on sex-specific fitness and genome-wide transcript abundance in a quantitative genetic framework, and identified a group of candidate genes experiencing sexually antagonistic selection in the adult, which correspond to 8% of Drosophila melanogaster genes. As predicted, the X chromosome is enriched for these genes, but surprisingly they represent only a small proportion of the total number of sex-biased transcripts, indicating that the latter is a poor predictor of sexual antagonism. Furthermore, the majority of genes whose expression profiles showed a significant relationship with either male or female adult fitness are also sexually antagonistic. These results provide a first insight into the genetic basis of intralocus sexual conflict and indicate that genetic variation for fitness is dominated and maintained by sexual antagonism, potentially neutralizing any indirect genetic benefits of sexual selection.

1 Uppsala University, Uppsala, Sweden.

SESSION 1 - MOLECULAR EVOLUTION

The Midichloria mitochondrii genome project

D. Sassera, S. Epis, M. Montagna, F. Commandatore, C. Bandi ¹

The hard tick Ixodes ricinus is an arthropod of great importance under a medical and veterinary point of view, as it can be vector of a number of bacterial, viral and protozoan diseases (e.g. Lyme disease caused by Borrelia spp.). Furthermore this tick species harbours a recently described intracellular symbiont: Midichloria mitochondrii. This alphaproteobacterium is a member of the Rickettsiales, a bacterial order that encompasses pathogens (Rickettsia, Ehrlichia, Anaplasma), manipulators of the host reproduction (Wolbachia of arthropods) and also mutualistic symbionts (Wolbachia of nematodes). The Rickettsiales are considered to be the closest relatives of mitochondria, bearing a resemblance of the alphaproteobacterial mitochondrial ancestor that entered the protoeukariotic cell two billion years ago. But M. mitochondrii is not only phylogenetically close to the mitochondrion, it also presents a unique type of interaction with it.
M. mitochondrii is in fact the only bacterium ever described able to invade the mitochondria of any metazoan. Transmission electron microscopy (TEM) photographs clearly show that M. mitochondrii not only resides in the cytoplasm of oocytes and of accessory ovarian cells of its host, but it can also colonize mitochondria. In particular bacteria localize in the intramembrane space, between the inner and outer membranes of the organelles. TEM images suggest that M. mitochondrii consumes the mitochondrial matrix and multiplies within the remnants of the organelle, thus indicating a parasitic relationship with the host. Molecular data, however, indicate a 100% prevalence of M. mitochondrii in wild-collected females of I. ricinus, which is usually regarded as a sign of beneficial symbiosis.
The sequencing of the genome is a feasible strategy in order to obtain a better understating of the biology of this symbiont, of the interaction with the host and with the mitochondria. The unique localization of M. mitochondrii and the current lack of cell cultures make it challenging to obtain sufficient quantities of pure bacterial DNA. In order to overcome this challenge, we developed a specific method, which we hereby describe. A semi-engorged I. ricinus female was collected and the ovary was extracted. Single oocytes were isolated by micromanipulation, then grouped in pools of 10-20 and incubated in a slightly hypotonic medium that allowed the formation of small holes in the cell membrane. The cytoplasms leaking from the holes were collected and multiple displacement amplification (MDA) was performed in order to obtain micrograms of M. mitochondrii DNA. MDA products were qPCR tested in order to assess uniform amplification of the genome. Furthermore the possible presence of contaminating I. ricinus nuclear DNA was also investigated with qPCR. It was thus possible to select uniformly amplified MDA products that contained minimal I. ricinus DNA contamination. Two MDA products were subjected to pyro sequencing by 454 (half titanium run and a quarter GS-FLX paired-ends run) and Sanger sequencing (500 clones, both ends).
The genome was assembled using Mira assembler, Bambus scafolder, Gap4 for contig viewing and ad-hoc bioinformatics tools for finishing. Inverse-PCRs were performed to close gaps and disambiguate repetitive regions. Phylogenomics analyses were performed (MrBayes, PhyML) in order to assess the exact phylogenetic placement of M. mitochondrii, in particular in respect to bacteria of the order Rickettsiales and to mitochondria. Results of these analyses as well as other genome study results (currently in progress) will be presented.

1 Sezione di Patologia Generale e Parassitologia, DIPAV, Facoltà di Veterinaria, Università degli Studi di Milano, Milano, Italy.

SESSION 2 - BIODIVERSITY AND EVOLUTION

Origin ad evolution of the modern coral reef fish fauna

F. Santini, G. Carnevale, M.E. Alfaro ¹

Coral reefs occupy less than 2% of marine surface, yet about 40% of the approximately 170000 marine fish species live predominantly or exclusively on coral reefs. Earlier studies of tetraodontiform fishes (puffers, box- and triggerfish) showed that reef-associated fish clades are significantly more diverse than non reef clades, suggesting that coral reefs have increased fish diversification rates.
Here we test whether reef-association has driven diversification in other fish clades as well using time-calibrated phylogenies from 28 reef-associated clades. Analysis of diversification rates for 42 groups based upon method of moments estimates (Magallon and Sanderson, 2001), indicates that reef clades have higher diversification rates than teleost fish as a whole. However we find that most named reef clades are not significantly more diverse than percomorphs.
We also apply recently developed comparative methods to test for exceptionally rapid or slow diversification events within reef families and across time periods.

1 Department of Ecology and Evolutionary Biology University of California at Los Angeles, Los Angeles, USA.

SESSION 2 - BIODIVERSITY AND EVOLUTION

Ecogeographical variation in amphibian body size: from patterns to processes

G.F. Ficetola, A. Romano, S. Scali, E. Padoa-Schioppa ¹

Ecogeographical variation is the co-variation between biological features and geographical or ecological features (e.g., latitude, temperature, precipitation). Body size displays intraspecific ecogeographical variation in many ectotherms, with populations with large body size associated to particular climates. However, the mechanisms determining such variation in ectothermic vertebrates are poorly understood. Here we analysed body size variation in two taxa of urodelans: the Italian crested newt Triturus carnifex and the spectacled salamanders Salamandrina terdigitata and S. perspicillata.
We used an information-theoretic approach to assess the support of multiple evolutionary and ecological processes proposed to explain body size variation: heat conservation; endurance to thermal range, seasonality, starvation resistance, water availability, environmental productivity, parental investment and evolutionary history. We obtained body size for 2639 newts and 3850 salamanders covering the whole range of the species, and ecogeographical information on the populations.
Our models included spatial autocorrelation and genetic information, to tease apart spatial and evolutionary processes. For newts, populations with large body size were associated to cold climates and areas with high primary productivity; sexual dimorphism increased in cold climates. Evolutionary history of populations had a significant role but explained little variation.
This suggests that local adaptations (particularly to improve heat balance and optimize parental investment) and phenotypic plasticity are the most likely drivers of body size variation. Conversely, for Salamandrina most variation was explained by mortality and deep genetic structuring, suggesting that body size was mostly determined by ancient evolutionary processes, while recent or ecological processes had a minor role. Our analysis shows that multiple processes drive body size variation in amphibians.
The difficulty to find generalities is due to the complexity of ongoing processes; the comparison of multiple mechanism can allow to understand ecogeographical variation.

1 Università degli Studi di Milano Bicocca, Dipartimento di Scienze dell’Ambiente e del Territorio, Milano, Italy.

SESSION 2 - BIODIVERSITY AND EVOLUTION

Barcoding meiofauna: the MoDNA (Morphology and DNA) project on DNA barcoding and phylogeny of Tardigrada

M. Cesari¹, T. Marchioro¹, R. Guidetti¹, L. Rebecchi¹, T. Altiero¹, F. Vicente², Y. Kiosya³, N. Guil⁴, O. Lisi⁵, R. Bertolani¹

The MoDNA Project has been undertaken to study the taxonomy and phylogeny of Tardigrada by joining molecular and morphological approaches. Tardigrades are important elements of meiofauna from marine, limnic and terrestrial environments, including hostile-life habitats.
Tardigrade taxonomy is almost exclusively based on morphological descriptions of over 1000 species. Therefore, the DNA barcoding method has been performed, with the aim to add and link molecular information to the morphological appearance of each individual specimen. The whole tardigrade must be used for molecular analysis, with consequent difficulties in correlating molecular sequences and morphology. We have perfected methods which allow to retain individual morphological information either from the animal before it was used for DNA extraction, or using an egg shell (when ornamented) as a voucher specimen and extracting DNA from the corresponding newborn, or using the animal as a voucher specimen and its eggs for DNA extraction. Combining such morphological information (LM and SEM) with sex ratio and karyological data, it was possible to obtain a good understanding of different species groups. First results regard the Paramacrobiotus richtersi group, the Macrobiotus hufelandi group, several species of the genus Ramazzottius, and the supposedly widespread Milnesium tardigradum.
For this study a fragment of the mtDNA COI gene was analyzed in more than 200 specimens sampled in Europe and America, including many type localities. This new approach has allowed us to barcode 31 species. Some of them can also be distinguished by fine morphological characters, others by karyological and sex ratio information, others only by very different DNA sequences (cryptic species).
A phylogenetic analysis using 18S nuclear gene and morphology was also performed on 69 specimens, considering several eutardigrade families.
The presence of four new different superfamilies and of new relationships inside them have been highlighted. Other collaborations related to this project are in progress.

1 Dipartimento di Biologia, Università di Modena e Reggio Emilia, Italy.
2 Centre for Environmental Biology and Department of Animal Biology, University of Lisbon, Portugal.
3 Department of Genetics and Cytology, Kharkov National University, Ukraine.
4 Department of Biodiversity and Evolutionary Biology, National Museum of Natural History (CSIC), Spain.
5 Dipartimento di Biologia Animale “Marcello La Greca”, Università di Catania, Italy.

SESSION 2 - BIODIVERSITY AND EVOLUTION

A multigenic molecular phylogeny and biogeography of the “Haenydra” lineage (Coleoptera, Hydraenidae, genus Hydraena)

M. Trizzino, P.A. Audisio, G. Antonini, E. Mancini, I. Ribera

Hydraena Kugelann represents the largest genus within the water beetle family Hydraenidae, with about 650 species widely distributed all over the world and several hundreds not yet described.
In a recent cladistic analysis, based on morphological characters, Hydraena s.l. was splitted in two subgenera: Hydraenopsis and Hydraena s.str. Moreover, within Hydraena s.str, some derived and well-supported monophyletic clades were recognised, and defined as “lineages”. Among them, the “Haenydra” lineage, previously considered by many authors as a valid genus/subgenus, includes 88 species distributed exclusively in western Palaearctic, from Portugal to Iran, but absent in North Africa. According mainly to male genitalia features, “Haenydra” species could be divided into several species groups and complexes.
The aim of the present work was to investigate the molecular phylogeny of the whole “Haenydra” lineage using both mitochondrial (COI, 16s rDNA, NAD1, tRNALeu) and nuclear (18s rDNA, 28s rDNA) markers, in order to clarify the evolutionary relationships among the different species groups and complexes, and to confirm the phylogenetic position of the “Haenydra” lineage within the large genus Hydraena.
In fact, although the monophyly of “Haenydra” is generally accepted, hydraenid specialists have been often discordant about the phylogenetic position of the lineage within Hydraena, sometimes considering it a basal taxon, sometimes a derived lineage.
Furthermore, molecular clocks were used to investigate the natural history of Hydraena s.str., and particularly of “Haenydra”.
Preliminarly results well supported the monophyly and the derived phylogenetic position of the “Haenydra” lineage, that was splitted in two major monophyletic clades: the H. gracilis and the H. dentipes clades, each one including several sub-clades, often corresponding to previously defined species groups/complexes. According to our calibration, the origin of “Haenydra” was estimated to be at ca. 8.3 MY, whereas relevant geological events such as Messinian salinity crisis and Pleistocene Glacial Cycles have been pivotal for the great morphologic diversification of the lineage.

SESSION 2 - BIODIVERSITY AND EVOLUTION

Conservation of Galápagos land iguanas: genetic monitoring and predictions of a long-term program on the island of Santa Cruz

A. Fabiani, S. Rosa, E. Trucchi, C. Marquez, H.L. Snell, H. M. Snell, W.T. Aguilera, G. Gentile ¹

The distribution of the Galápagos land iguanas (Conolophus subcristatus) has been strongly affected by human activities in the last century. Previously widespread throughout the whole archipelago, today they inhabit only few islands, with populations often small and isolated. In this study, we analysed the population genetic structure of land iguanas from Santa Cruz Island to investigate the genetic implications of a semi-captive conservation program that started in middle 1970s and is still ongoing.
Nine microsatellites were used to measure the level of genetic variability and to detect potential evidence of inbreeding and genetic sub-structure.
Furthermore, we used Approximate Bayesian Computation (ABC), together with software packages for coalescent-based simulations, to test a priori hypotheses in different demographic scenarios. Despite the abrupt reduction in size of the original population, no evidence of inbreeding was found and the levels of genetic variability found place between those of undisturbed populations of the archipelago.
Nevertheless, the source and the repatriated populations started differentiating (FST = 0.016) and genetic sub-structure was found. Following our results and the simulation of possible future scenarios, we suggest the genetic measures that should be adopted to avoid further genetic variability depletion and preserve this vulnerable endemic species.

1 Dipartimento di Biologia, Università degli Studi di Roma Tor Vergata, Rome, Italy.

SESSION 2 - BIODIVERSITY AND EVOLUTION

Molecular tools help resolving a taxonomic enigma: the case of Paradoxornis webbianus and P. alphonsianus (Aves, Paradoxornithidae)

A. Galimberti¹, A. Crottini^1,2,3, A. Boto⁴, L. Serra⁵, M. Barbuto¹, A. Bellati⁵, S.G. Baccei¹, M. Casiraghi¹

Abstract omesso per volontà del primo A.

Breve descrizione dello studio:
Il lavoro indaga se due gruppi di passeracei viventi in Italia dal 1995, nella riserva naturale della Palude Brabbia (VA), ma di certa provenienza cinese, costituiscano due specie distinte o due diverse forme della stessa specie.
L'analisi molecolare basata su markers mitocondriali mostra la quasi completa identità fra i due gruppi. Ciò, unitamente ad altri dati derivanti dallo studio di individui dei luoghi cinesi di origine della specie, porta alla conclusione che i due gruppi già nei luoghi di origine erano varianti simpatriche della stessa specie. (G.L.)

1 Università degli Studi di Milano Bicocca, ZooPlantLab, Dipartimento di Biotecnologie e Bioscienze, Milano, Italy (tgalimba@gmail.com).
2 Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany.
3 Universita’ degli Studi di Milano, Dipartimento di Biologia, Sezione di Zoologia e Citologia, Milano, Italy.
4 Stazione Ornitologica Palude Brabbia, Sesto Calende (VA), Italy.
5 ISPRA., Ozzano Emilia (BO), Italy.
6 Università degli studi di Pavia, Dipartimento di Biologia Animale, Pavia, Italy.

SESSION 3 - PHYLOGENESIS

Disentangling the evolutionary history of the Tubifex species complex (Annelida, Clitellata): a comparison between molecular phylogeny and cytogenetics

R. Marotta^1,2§, A. Crottini^2,3§, C. Fondello², E. Raimondi⁴, M. Ferraguti²

Tubifex Tubifex Muller 1774 is a common species in the Lambro River (Milano) where it forms complex tubificid-dominated communities. Recently, an integrative taxonomy approach, using molecular and morphological analyses, brought to the species status validation of T. blanchardi and revealed the co-existence of several sympatric cryptic evolutionary lineages.
A cytogenetic approach was used to investigate the evolutionary pattern of species differentiation in this species complex. Indeed, the analyses of chromosome pattern and variability can help disentangling the evolutionary processes (e.g.: polyploidy speciation) that shaped the complex relationships characterizing these taxa. Only few scattered cytogenetic data were so far available for T. Tubifex, and no data exist for T. blanchardi. We performed a cytogenetic analysis in parallel with a molecular analysis that allowed for lineage attribution. About 80 cocoons of T. Tubifex and T. blanchardi were collected and dissected.
For each cocoon half of the embryos were used for genetic analyses (by sequencing a fragment of the mitochondrial 16S rRNA gene) and the other half was processed for the cytogenetic assays. Our preliminary results show that four different euploid chromosome sets are present across the Tubifex’ 16S rRNA gene phylogeny, as confirmed also by a FISH assay (with an autologous 18S rDNA molecular probe), and that there are three genetically differentiated tetraploid taxa.
All analysed T. blanchardi have 50 chromosomes (n=25), whereas the chromosome number of the analyzed T. Tubifex specimens varies from 75 (triploids), to 100 (tetraploid) and 150 (esaploid), with typically large metacentrics and small acrocentric chromosome pairs. These results are compatible with the hypothesis that consecutive polyploidyzation events may have played a creative role in shaping the evolution of the Tubifex community of the Lambro River.

1 Italian Institute of Technology (IIT), Genova, Italy.
2 Università degli Studi di Milano, Dipartimento di Biologia, Sezione di Zoologia e Citologia, Milano, Italy.
3 Division of Evolutionary Biology, Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany.
4 Università degli Studi di Pavia, Dipartimento di Genetica e Microbiologia “A. Buzzati Traverso”, Pavia, Italy.
§ Both authors equally contributed to this study.

SESSION 3 - PHYLOGENESIS

Re-framing model choice in phylogeographic Approximate Bayesian Computation

S. Vicario, E. Trucchi, V. Sbordoni ¹

Likelihood estimate when comparing complex evolutionary hypotheses, as in phylogeography, is often an unaffordable mathematical task. Methods that rely on approximate Bayesian computation (ABC) may constitute a valid alternative in such a situation though user need to be extremely careful in applying the method and in choosing and comparing the models. We will focus i) on the possibility of adopting a full-data approach in model-choice without a tolerance threshold (Full Data Resample) rather than a Local Weighted Regression of a simulated subset closest to the observed data and ii) on the estimate of the goodness-of-fit of simulated on the observed data by using a posterior predictive statistics to assess overall models adequacy. The latter can be used to both evaluate the representativeness of the models in respect of the observed data and to test the efficiency of the chosen summary statistics.
Efficacy of the two methods targeted to estimate relative posterior of competing hypotheses was analyzed in different test conditions: the entertained model set include or not the correct model. Using the same simulated data we show the utility of adequacy to spot when no correct model is entertained. The Full Data Resample method can be considered a conservative and extremely accurate approach. The swapping for this high accuracy is a high level of indecision when the right answer is available. Conversely, the Local Weighted Regression method enhances the contrast in posterior estimates thus quite always resulting in a decision among competing scenarios. Being this approach structured in “anyway” finding an answer, it was fenced in a very high level of errors in our false attribution test. Since in phylogeography the probability to include the true hypothesis among those tested is very rare, we strongly suggest to use both methods when testing alternative evolutionary hypotheses.

1 University of Roma Tor Vergata, Roma, Italy.

SESSION 3 - PHYLOGENESIS

Dating arthropod relationships using soft constrains and phylogenomic datasets: methodological and paleoecological implications

O. Rota-Stabelli¹, A. Daley², D. Pisani¹

With a documented fossil record of 520 my and more than a million living species, arthropods are the most abundant group of animals on earth, accounting for 80% of the metazoan biodiversity.
Still, knowledge of their relationships is source of vigorous debate and their exemplar radiation is mainly unexplored by molecules, in particular by long phylogenomic datasets. Here we use three distinct large phylogenomic and mitogenomic datasets of up to 198 genes and 80 species to reconstruct and date the relationships within the arthropod and among their ecdysozoan outgroup.
We show that these three datasets agree in supporting morphologically recognised groups such as Pancrustacea, Mandibulata and Chelicerata, while relationships among (pan)crustacean classes are disputable and in clear discordance with those proposed by Regier and colleagues (2010, Nature). We calibrated our molecules with 30 soft constrains based on fossil records spanning from the Precambrian to the Cretaceous, and described evolution using the best fitting replacement and clock models. Results suggest that while the arthropods and their immediate Onycophora, Tardigrada, Nematoda and Scalidophora outgroups originated in the Ediacaran or even earlier, radiation of crustaceans major groups occurred in the Cambrian and that of chelicerates, insects and myriapods in the Orodovician.
From a methodological point of view, most of the estimates varied depending on the type and length of dataset, suggesting that particular care should be given for the selection of molecular marker and that the ideal approach should be to cross-test clock studies with more than one marker. From a paleontological perspective, our results imply that extant crustacean classes appeared at the end of the Cambrian explosion. Crustaceans radiation overlaps with the beginning of trilobites decay, suggesting an ecological displacement of the latter by crustaceans.

1 Department of Biology, The National University of Ireland, Maynooth, Ireland.
2 Department of Earth Sciences, Palaeobiology, Uppsala University, Uppsala, Sweden.

SESSION 3 - PHYLOGENESIS

Phylogenetic representativeness: a new method for evaluating taxon sampling in evolutionary studies

F. Plazzi, R.R. Ferrucci, M. Passamonti ¹

Taxon sampling is a key point in phylogenetic studies. Incomplete, biased, or improper taxon sampling can lead to misleading results in reconstructing phylogenies. Several methods are available to optimize taxon choice, involving genetic distance evaluations, interruption of long branches, etc.
However, all are conceived as a posteriori tests, i. e. they can only be carried out after data collection, when most of the experimental work is already done. We think it would be very useful to have an a priori test, allowing to compute and predict goodness of taxon sampling before the phylogenetic work starts. The established taxonomy of the group under study (although not necessary the “true” one), which is usually available from literature, may help in this: given a master list of organisms, it is possible to measure to which degree different subsamples do represent the whole assemblage.
For this reason, we proposed a new method to assess the representativeness of a given taxon sampling, by developing the Clarke and Warwick statistics on taxonomic distinctness. Our method aims to measure the phylogenetic representativeness of a given sample or set of samples, and it is based entirely on the pre-existing available taxonomy of the ingroup. Moreover, our method also accounts for instability and discordance in systematics: we conceived a randomization algorithm on master lists, mimicking taxonomic revisions, therefore addressing test reliability. A Python-based script suite, called PhyRe, was developed to implement all analyses, and it is available for free download at www.mozoolab.net.
We showed that this method is sensitive and allows direct discrimination between representative and unrepresentative samples. It is also informative about the addition of taxa to improve taxonomic coverage of the ingroup. Provided that the investigators’ expertise is mandatory in this field, phylogenetic representativeness makes up an objective touchstone in planning phylogenetic studies.

1 Università di Bologna, Bologna, Italy.

SESSION 3 - PHYLOGENESIS

One tree with many dates: assessing the variation of time estimates across different datasets

F. Nardi¹

Inference of divergence times, or dating, is becoming a major topic in phylogenetics, and methodologies and software implementations have been made available that can handle multi-gene datasets. Nevertheless, while a number of procedures have been devised in phylogenetic tree construction to test for congruence across datasets prior to concatenation and to test for the opportunity to combine or partition data, such procedures are not as developed and widely available in dating analyses.
Here we suggest the use of internal congruence, the rather obvious observation that, if multiple datasets are applied to estimate dates on the same topology, some will produce similar date estimates and some will produce more deviant date estimates. We suggest that under the assumption that single gene datasets are independent estimators of dates along the tree, internal congruence could provide a workable framework to assess the performance in dating of a given dataset and to detect outliers.
We introduce a metric to compare how much two trees with identical topology differ in terms of date estimates and a stepwise exclusion algorithm, derived from an unrelated application in Real-Time PCR analysis, to rank the datasets from the most to the least internally congruent.
The procedure is applied to full mitochondrial genome data, with four groups (insect orders Lepidoptera, Diptera, Hymenoptera and Coleoptera) of 14 datasets each (mitochondrial rRNA genes and PCGs except ATP8). We show, using a randomization test, that there is a rather unique set of genes that consistently provide more similar, or internally congruent, dates.
A possible use of this rank to select datasets to be used in a multi-dataset analyses aimed at estimating divergence times is further discussed, as well as its effect over the wideness of confidence intervals.

1 Dip. Biologia Evolutiva, Università di Siena, Siena, Italy.

SESSION 3 - PHYLOGENESIS

Assessing molecular divergence time estimates and the placement of alternative calibration points in the primate tree

L. Pozzi, J.A. Hodgson, T.R. Disotell ¹

The origins and time of divergence of primates have been difficult to resolve, due to incomplete sampling of early fossil taxa and to the relatively short window of time in which major lineages diverged from each other. The main source of contention is related to the discordance between molecular and fossil estimates: while no putative crown primate fossils date older than 55Ma, most molecule-based estimates extended the origins of crown primates well before the Cretaceous–Tertiary (K-T) boundary, with estimates up to 90Ma. While a clear understanding of the morphological radiation of clade is affected by the completeness of the fossil record and the identification of clear synapomorphies, molecular estimates can be biased by several sources of error, including heterogeneity in molecular rates and the use of potentially erroneous fossil calibrations.
We present molecular dating analyses for primates using complete mitochondrial genomes for 60 primate species and 40 species within mammals. We apply an uncorrelated relaxed-clock analysis that accommodates for heterogeneity in rates of evolution across different branches and takes into account uncertainties in phylogenetic relationships and the fossil record. We explore the effects of 18 calibration points chosen across mammals on the divergence-time estimates for major clades within the primate tree by using a cross-validation method that identifies inconsistent fossils when multiple fossil calibrations are available for a clade.
In contrast with previous mitogenomic studies, our results support primate monophyly and the position of tarsiers as sister group of Anthropoidea, with the exclusion of flying lemurs. Our dating results support a long fuse model of evolution, with an origin in the Late Cretaceous, and the radiation of all major lineages in the early Tertiary. Our study shows the utility of fossil concordance analyses to critically assess calibration points to be used in estimating divergence times within primates.

1 New York University, New York, USA.

SESSION 3 - PHYLOGENESIS

Population structure and gene flow of European landraces of the common bean (Phaseolus vulgaris L. )

S.A. Angioi¹, D. Rau¹, G. Attene¹, L. Nanni², E. Bellucci², G. Logozzo³, V. Negri⁴, P.L. Spagnoletti Zeuli³, R. Papa²

The pathways of dissemination of common bean into and across Europe were very complex, with a number of introductions from Americas that were combined with direct exchanges between European and other Mediterranean countries. We analyzed a large collection of European landraces of P. vulgaris with six chloroplast microsatellite (cpSSR) loci and two unlinked nuclear loci (for phaseolin types and Pv-Shatterproof1). We compared the genetic structure and the level of diversity of this collection with a group of American individuals representative of the Andean and Mesoamerican gene pools.
The results show that the majority of the European common bean landraces are of Andean origin. Moreover, bottleneck due to the introduction of P. vulgaris into the Old World, was very weak for chloroplast analysis but of greater intensity for nuclear analysis. Finally, a relatively high proportion of the European bean germplasm has derived from hybridization between the Andean and Mesoamerican gene pools. Based on the analysis of the distribution of genetic diversity and hybrid individuals across European countries, we suggest that the entire European continent can be regarded as a secondary diversification centre for P. vulgaris. Lastly, we outline the relevance of these inter-gene-pool hybrids for plant breeding.

1 Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi di Sassari, Sassari, Italy.
2 Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Ancona, Italy.
3 Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della Basilicata, Potenza, Italy.
4 Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Perugia, Italy.

SESSION 3 - PHYLOGENESIS

The origin of retinal binding domain. A phylogenomics investigation of the origin of vision in Metazoa

R. Feuda, S. Hamilton, S.J. Longhorn, J.O. McInerney, D. Pisani ¹

Vision in animals is mediated by opsins, transmembrane G-protein coupled receptors (GPCR) which are able to react in the presence of light and promote a signaling cascade in the cell. Animals possess two main classes of visual opsin: protosomes (most of the invertebrates) use R-opsin, while vertebrates use C-opsins; additionally, most animals also posses RGR opsins which increase the efficiency of the visual process. Opsins are also known in Cnidara (jellyfishes and corals) but it is not clear whether cnidarian opsins represent C-, R-, or a third, unrelated opsin class. More precisely, cnidarian Opsins have been suggested as the sister group of a clade composed by the R and the RGR opsins, to include both C- and R- opsins, or as the sister group of all the other animal Opsins (R-, C-, and RGR).
Reconstructing a correct opsin phylogeny is key to understand the origin of animal vision, as uncertainty about the topology has significantly hampered our comprehension of the molecular and system processes underlying the origin of light sensitivity and vision in animals.
We used three non-bilaterian animals (two Cnidarians, and one Placozoan) and all the available bilaterian genomes (both vertebrates and invertebrates) to investigate opsin evolution, and evaluate whether we could clarify the relationships among the opsin classes, with special reference to the cnidarian opsins. In addition, we investigated the presence of opsin-like sequences in Placozoa (the sister group of Cnidaria plus Bilateria).
We show that the cnidarian opsins are either R-, or C-opsins, and that early opsin evolution most likely involved two gene duplications and one loss. Furthermore we found that the closest outgroup of the opsins is an ophan GPCR family found only in Nematostella and Tricloplax.

1 Bioinformatics Unit, Biology Department National Universtiy of Ireland Maynooth, Ireland.

SESSION 4 - ORGANISM EVOLUTION

Comparative neuroanatomy of ascidian and amphioxus larvae: inferences on the nervous system of the chordate ancestor

R. Pennati¹, S. Candiani², G. Zega¹, F. De Bernardi¹, M. Pestarino²

Tunicates and Cephalochordates are invertebrate chordates and occupy key phylogenetic positions to understand the evolution of chordate characters. They are exclusively marine, the adults are filter-feeding animals and develop through a lecitotrofic swimming larva. Their larvae display typical chordate central nervous systems, consisting of a dorsal neural tube, which develops from a neural plate.
Although the gross neuroanatomy of protochordate larvae have been extensively studied by conventional analysis, we used molecular tools to dissect the fine details of nervous system components in the larvae of the ascidian Ciona intestinalis and of the lancelet Branchiostoma floridae.
During differentiation, neurons acquire an important phenotypic character given by the neurotransmitter that is released to excite or inhibit the target cells.
We analyzed the differentiation of serotonergic, GABAergic, cholinergic, dopaminergic, glutamatergic and glycinergic neurons in the central nervous systems of our animal models by studying the expression pattern of neurotransmitter synthesis genes. Moreover, we localized some metabotropic receptors of serotonin, GABA and acetylcholine to get a more complete framework of the functional organization of neural networks. We obtained two maps of the distribution of the main neurotransmitters that allowed us to compare the basic functional organization of the nervous system among chordates.
We noted that some functional regions are conserved among the CNS of ascidian larva and that of cephalochordates and vertebrates, thus revealing features shared by all chordates that possibly could be already present in their last common ancestor.

1 Department of Biology. University of Milano, Milano, Italy.
2 Department of Biology. University of Genova, Genova, Italy.

SESSION 4 - ORGANISM EVOLUTION

Dormancy strategies in tardigrades: evolutionary aspects and ecological meanings

R. Guidetti, T. Altiero, R. Bertolani, L. Rebecchi ¹

Dormancy includes any form of resting stage, regardless of cues required for induction or termination. It involves a temporary suspension of active life, a reduced or suspended metabolism and a developmental standstill. One of the few phyla including species able to suspend the metabolism in any stage of the life cycle performing the extreme form of dormancy (cryptobiosis) is represented by tardigrades.
Tardigrades are aquatic animals distributed all over the world in many water and terrestrial environments, frequently inhabiting unpredictable (e.g. mosses, lichens, etc.) or hostile-life habitats (e.g. deserts, polar regions, etc.). Dormancy includes any form of resting stage, regardless of cues required for their induction or termination. It involves a temporary suspension of active life, a reduced or suspended metabolism and a developmental standstill. Dormancy in tardigrades is characterized by a wide variety of adaptive strategies . This unequalled diversity of strategies is represented by anhydrobiosis, cryobiosis, anoxybiosis, osmobiosis, encystment, cyclomorphosis and resting eggs.
These dormancy forms evolved to withstand a wide range of unfavorable environmental conditions and can be grouped into quiescence and diapause according to their inducing factors, performing process, duration, and of course, adaptive meanings. The evolution of tardigrade dormancy strategies received little attention though it had a strong impact on their life cycle, ecology and evolution.
A comparative analysis of the tardigrade resting stages will be performed focusing our attention to their evolution and ecological meaning.

1 Department of Biology, University of Modena and Reggio Emilia, Italy.

SESSION 4 - ORGANISM EVOLUTION

The mitochondrial bottleneck: strict sex-specific mtDNA segregation in the germline of the DUI species Venerupis philippinarum (Bivalvia Veneridae)

F. Ghiselli, L. Milani, M. Passamonti ¹

Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of Strict Maternal Inheritance (SMI) of metazoan mitochondria and has been found in several bivalve species. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-Type), the other through sperm (M-Type). Moreover, M- and F-Types experience peculiar tissue distributions, with males being heteroplasmic in their soma, while females usually not.
We performed a Real-Time Multiplex qPCR analysis on the Manila clam Venerupis philippinarum to quantify M- and F-Types in somatic tissues, gonads and gametes, as well as mtDNA duplication rates during early embryo development. In most male somatic tissues, the MType is largely predominant; something similar is found for a few females too, actually showing MmtDNAs in their soma.
On the contrary, in the germline we evidenced a strict sex-specific mtDNA segregation, since both sperm and eggs do carry exclusively M- and F-Type respectively. Because of this, we propose that the sex-specific mtDNA transmission is achieved through a 3-checkpoint process. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the early embryo development (that we named checkpoints #1 and #2) are already known for DUI species; we propose for the first time, a third checkpoint that would act when Primordial Germ Cells (PGCs) are first formed and would work for both mtDNAs. DUI provides a privileged point of view to unveil the processes by which mitochondria enter the germline (i.e. the “mitochondrial bottleneck”) and the evidences we observed on a strict selection of sex-specific mtDNAs in the germline could be a first step in that direction. Actually, a sound working hypothesis is that these processes might be based on similar molecular mechanisms in SMI species.

1 Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Bologna, Italy.

SESSION 4 - ORGANISM EVOLUTION

The early emergence of a central nervous system in basal metazoans

A. Dell’Anna¹, G. Zega¹, S. Piraino², C. Di Benedetto³, A. Leone⁴, X. Bailly⁵, P. Pagliara², R. Pennati¹

A shared feature of cnidarian and bilaterian animals is the presence of a nervous system composed of interconnected neural cells. The number of neural cells, the complexity of their connectivity, and their organization into regionalized neural assemblies show a great deal of variation in all animal groups. The evolutionary origin of this neuroanatomical complexity and diversity in eumetazoan nervous systems represents a fundamental problem in neurobiology. Recently, a number of comparative developmental and genetic analyses suggested that the diversity of nervous systems of protostomes and deuterostomes arose from a common evolutionary origin (Lichtneckert and Reichert, 2005; Denes et al., 2007; Arendt et al., 2008; Lichtneckert and Reichert 2008) and that the urbilaterian nervous system was already a remarkably complex centralized structure (De Robertis, 2008). To shed light on this question, we analyzed and compared the nervous system of the larva of the cnidarian Clava multicornis and of the acoel Symsagittifera roscofenses since cnidarians are now regarded as early bilateral animals whereas acoels as the basalmost triploblastic metazoans (Baguña et al 2008). Immunohistological analysis showed that the nervous system of the cnidarian planula larva possesses a surprising high level of histological and functional organization with a strikingly large and ordered array of sensory cells located at the anterior pole. Conversely the nervous system of the acoel exhibits a stereotypical organization and is formed by six longitudinal neurite bundles and an anterior concentration of sensory cells. Our observations suggest that cephalization and the potential to differentiate a centralized nervous system early occurred at the dawn of Bilateria radiation in the last common ancestor of cnidarians and acoels. Further steps of nervous system evolution could have been the acquisition of a stereotypical organization of the nervous elements linked to evolution of unidirectional synapses.

1 Università di Milano, Dip. Biologia, Sezione Biologia Riproduttiva e Funzionale, Milano, Italy.
2 Università del Salento, DISTEBA, Lecce, Italy.
3 Università di Milano, Dip. Biologia, Sezione Zoologia e Citologia, Milano, Italy.
4 CNR-ISPA, Lecce, Italy.
5 Station Biologique de Roscof, France.

SESSION 4 - ORGANISM EVOLUTION

Evolutionary relationships between Zootoca vivipara subspecies as inferred from patterns of genetic variation in Alpine populations

L. Cornetti¹

Transition from oviparous to viviparous reproductive modality, which is likely to have a pronounced effect on the reproductive success and the fitness of the organisms concerned, is a relevant evolutionary event. Squamate reptiles are of particular interest for studying this shift as it has been shown that viviparity has evolved from oviparity far more often in squamate reptiles than in all other lineages of vertebrates.
The lacertid lizard Zootoca vivipara is one of the few squamate species with both oviparous and viviparous populations. In most of the range, from the British Isles and central France into Scandinavia and eastern Russia, populations are viviparous, whereas two distinct, allopatric oviparous populations are restricted to the southern margin of the range.
The ‘western oviparous group’, recently ascribed to subspecies Z. vivipara louislantzi, is found in southern France and northern Spain, while the ‘eastern oviparous group’, corresponding to the subspecies Z. vivipara carniolica is located in northern Italy, southern Austria, Slovenia and Croatia. The presence of Z. vivipara vivipara and Z. vivipara carniolica in Central and Eastern Alps makes this region an interesting area to investigate about taxonomical and ecological distribution of these two morphologically undistinguishable subspecies.
We collected a small amount of tissue tail of 193 samples of Zootoca vivipara sp.in Central and Eastern Alps. We analysed cytb gene (mtDNA), a nuclear gene (c-Mos, oocyte maturation factor, maybe related to reproductive modality) and nine nuclear microsatellites. This study allowed us to describe taxonomical and ecological distribution of the two subspecies in Central and Eastern Alps along with their evolutionary relationships. We analysed genetic variation between subspecies, within each subspecies and population structure within subspecies.
All these data concur to gain insights about the evolutionary shift from oviparity to viviparity.

1 Fondazione Edmund Mach.

SESSION 4 - ORGANISM EVOLUTION

Origin and domestication of the common bean (Phaseolus vulgaris L.)

E. Bitocchi¹, E. Bellucci ¹, D. Goretti¹, S. Angioi², F. Desiderio¹, M. Rossi¹, G. Logozzo ³, L. Nanni¹, D. Rau², V. Negri⁴, P. Spagnoletti Zeuli³, G. Attene², R. Papa¹

Phaseolus vulgaris is characterised by two geographic gene pools, one located in Mesoamerica and the second in the southern Andes, with parallel geographic structures in their wild and domesticated beans. In the 1980s, a wild P. vulgaris population was discovered in Ecuador and northern Peru, which was described as a new distinct wild gene pool. This population was suggested to be the ancestral populations of the common bean on the basis of sequence studies of the genes coding for phaseolin, the main seed-storage protein. The occurrence of independent domestication events in Mesoamerica and the Andes is well established; however, the number of domestication events that occurred in the two different regions remains a topic of discussion.
We have carried out various studies using multilocus molecular markers with the wild and domesticated genotypes of the common bean, including an analysis of the structure of the European landraces. Recently, we analysed a representative panel containing both wild and domesticated P. vulgaris (215 accessions overall) at the nucleotide diversity level, for five independent genes. Moreover, we analysed the effects of domestication in Mesoamerica on the nucleotide diversity of 48 loci.
Here, we present a summary of the results obtained from these studies, with particular focus on the origin of the species and the effects of domestication on the common bean genome. Our data indicate a new scenario for the structure and evolution of wild P. vulgaris, especially questioning the Peru-Ecuador origin, and arguing in favour of a Mesoamerican origin of the species. Moreover, the data support the occurrence of a single domestication event in Mesoamerica, and they also tend to support the same scenario in the Andes.

1 Dipartimento di Scienze Ambientali e delle Produzioni Vegetali (SAPROV), Università Politecnica delle Marche, Ancona, Italy.
2 Dipartimento di Scienze Agronomiche e Genetica Vegetale Agraria, Università degli Studi di Sassari, Sassari, Italy.
3 Dipartimento di Biologia Difesa e Biotecnologie Agro-forestali, Università degli Studi della Basilicata, Potenza, Italy.
4 Dipartimento di Biologia Vegetale e Biotecnologie Agro-ambientali e Zootecniche, Università degli Studi di Perugia, Perugia, Italy.

SESSION 4 - ORGANISM EVOLUTION

Pristionchus uniformis, should I stay or should I go. Host switching in a nematode

I. D'Anna¹

Pristionchus pacificus is an established model system in evolutionary and developmental biology. Studies of other Pristionchus species, such as the closely related gonochoristic Pristionchus uniformis, can help us gain a comprehensive understanding of the hermaphroditic P. pacificus. Nematodes of the genus Pristionchus have a species-specific necromenic association predominantly with scarab beetle.
One interesting exception is Pristionchus uniformis, one of a few Pristionchus species that shows host switching. In particular, P. uniformis is observed in both the chrysomelid Leptinotarsa decemlineata (Colorado potato beetle), and scarab beetles. In this study we provide a first insight into the ecology and genome organization of the species P. uniformis. Specifically, we present the reconstruction of P. uniformis chromosomes by generating a genetic linkage map based on 48 nuclear markers, tested on a meiotic mapping panel of 42 inbred lines. We can show evidence for macrosynteny and are currently investigating if colinearity is also conserved between P. pacificus and P. uniformis.
In parallel, we plan to integrate this newly acquired knowledge of the P. uniformis genome to identify the genetic basis of the unique association between the nematode P. uniformis and the toxic Colorado potato beetle. Indeed P. uniformis is the only nematode species that we could isolate from both North America and Western Europe and from both the long living scarab beetles and the leaf beetle Leptinotarsa decemlineata, which is characterized by a much shorter life cycle.
We are currently investigating if the absence of other nematodes in the Colorado potato beetle could be justified by the toxicity in the beetle hemolymph and how P. uniformis has evolved a resistance to it.

1 Max Planck Institute for Developmental Biology, Department for Evolutionary Biology.

SESSION 4 - ORGANISM EVOLUTION

DNA barcoding a tool to detect cryptic species? Amblyomma cajennense, a case of study

M. Montagna¹, E. Ferri², L. Beati³, D. Sassera¹, M. Casiraghi², S. Epis¹, C. Bandi¹

The purpose of the Consortium for the Barcode of Life (CBOL) is to obtain a complete molecular “catalogue of life”. The main goal is to simplify identification of any kind of biological sample, but also to create a world DNA-sequence bank, purpose that is getting more and more important every day, considering the rising rates in species extinction. Barcoding means identification of taxa using a simple molecular approach, consisting of a PCR and subsequent sequencing of the obtained product. DNA barcoding is also useful to help the detection of cryptic species. The barcoding approach can be very useful for ticks, as identification of tick samples based on morphology requires experience and a high level of knowledge of these arthropods, furthermore most discriminating morphological features are sometimes visible only in adult specimens, making species identification of immature stages simply impossible in a number of cases.
The main problem with barcoding is being able to correctly evaluate the capability of molecular markers to discriminate between different species. A molecular marker with high interspecific variability but high intraspecific identity needs to be used, in order to obtain a “barcoding gap” necessary to discriminate between different species. The genus Amblyomma comprises 126 species of hard ticks, 57 restricted to Neotropical region, among them there are species that are thought to mask “cryptic species”. Correct identification of all the species belonging to this genus of the family Ixodidae is often difficult, in fact through the years there have been a number of changes in the taxonomic keys for the identification of certain species as well as in some species names. In order to aid the identification of species of the genus Amblyomma, here we present the testing of a barcoding approach on 560 samples from 69 species of the genus Amblyomma with specific attention on comprehensive sampling strategy of Amblyomma cajennense (106 specimens) from different population (11 localities) of the area in which the species is distributed.
As suggested by CBOL protocols, our PCR target was a fragment of 610 bp of the mitochondrial gene cytochrome c oxidase subunit 1 (COI). Amplification of this target in PCR was obtained with universal primers for diverse metazoan invertebrates LCO1490 and HCO2198. PCR was successful on all samples, allowing us to perform DNA sequencing and to obtain the desired 610 bp DNA fragment for all 560 samples. The obtained sequence database was tested with a specific leave-one-out test, estimating the genetic distance, in order to assess the presence of the “identity percentage gap” between intra- and inter- specific variation. The barcoding approach was coherent with the morphological identification of the samples in most cases, but a few situations of disagreement arose. In details we examined the variability of COI fragment (610 bp) in Amblyomma cajennense samples and we found that the intra specific variation of specimens belonging to different populations reach value typical for inter- specific variation.

1 DIPAV, Sezione di Patologia Generale e Parassitologia, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy.
2 Dipartimento di Biotecnologie e Bioscienze, ZooPlantLab, Università degli Studi di Milano Bicocca, Milano, Italy.
3 U.S. National Tick Collection, IAP-Georgia Southern University, Statesboro, USA.

SESSION 5 - HUMAN EVOLUTION

Pre-Neolithic signals of population expansion in Bantu speakers detected by high resolution analysis of Y-chromosome lineages

V. Montano¹, V. Marcari¹, C. Batini⁴, G. Berniell-Lee², O. Anayale³, D. Comas², G. Destro-Bisol¹

Bantu speaking populations represent a subject of particular interest for linguistics, archaeologists and molecular anthropologists. While linguistic data suggests that a population expansion driven by Neolithic revolution (~ 3-5 kya ago) could have been responsible for the spreading of Bantu languages, from the genetic point of view the effective role of population expansion in the whole process has not been clarified.
We studied 17 populations from Nigeria, Cameroon, Gabon and Congo, which are thought to be in continuity with the groups who took part to the “Western stream” of the Bantu expansion. A total of 505 male individuals were typed for 20 SNPs and 17 STRs of the Y-chromosome.
We detected signals of structure which seem to not follow a global pattern of geographic distribution according to the analysis of spatial PCs (Jombart et al., 2008). Using Batwing software (Wilson et al., 2003) under a model of constant population size followed by a demographic expansion, we estimated a time since expansion of 8 kya from an ancestral population size of approximately 2800 individuals.
Dividing the dataset with a geographic criterion, more recent estimates of time since expansion were obtained for Gabonese populations relative to Nigeria and Cameroon (~5.6, ~11.0 and ~9.0 kya, respectively). This was paralleled by a larger ancestral effective population size (~3600 for Gabon vs ~1600 for Nigeria and Cameroon), apparently mirroring the higher genetic variability in Gabon both at the population and haplogroup level. Accordingly, we suggest that population expansion events are likely to have occurred in sub Saharan Africa before the Neolithic age. However, only Gabonese region seems to have undergone strong recent migration processes.

1 Dipartimento di Biologia Ambientale, Università di Roma “La Sapienza”, Rome, Italy; Istituto Italiano di Antropologia, Rome, Italy.
2 Unitat de Biologia Evolutiva, Department de Ciencies Experimentals i de la Salut, Universitat “Pompeu Fabra”, Barcelona, Spain.
3 Department of Zoology, Ibadan University, Ibadan, Nigeria.
4 Department of Genetics, University of Leicester, UK.

SESSION 5 - HUMAN EVOLUTION

Sensory drive of colour signals in humans

F.T. Wehrle¹

Sensory drive is a broad concept which assesses the evolutionary dependences of similar animal behaviours in different contexts. Sensory and behavioural adaptations in an ancestral context (preexisting bias) are thought to impose selection pressures on new traits emerging in more recent contexts and thus to “drive” their evolution. These selection pressures may either favour traits that maximally stimulate the sensory system of the recipient (sensory exploitation) or traits that optimally match the original stimulus (sensory trap). In Catarrhine primates (old world monkeys, apes and humans), colour stimuli in the food context are thought to have coevolved with trichromatic colour vision in a way that signals of good food quality trigger attraction behaviour.
This attraction is thought to impose selection pressure on pelage and skin colouration as signals for mate choice in the more recent sexual context. This context is particularly important in humans, who live in large, interdependent social groups and who are known to signal sexually relevant content like hormonal state or health via changes in skin colouration. With innovative computerised methods we experimentally assess the selection pressures imposed on colour signals in the food and the sexual context in humans. Based on our results we discuss the applicability of the sensory drive models to the evolution of human skin colour signals as well as their limitations.

1 Albert-Ludwigs University Freiburg, Germany.

SESSION 5 - HUMAN EVOLUTION

No evidence of Neandertal admixture in Cro-Magnoid and modern European mitochondrial genomes

S. Ghirotto, F. Tassi, A. Benazzo, G. Barbujani ¹

Neandertals, the archaic human form documented in Eurasia until 29,000 years ago, share no mitochondrial haplotype with modern Europeans. Whether this means that the two groups were reproductively isolated is controversial, and indeed nuclear data have been interpreted as suggesting that they admixed. We explored the range of demographic parameters that may have generated the observed mitochondrial diversity, simulating 2.5 million genealogies under five evolutionary models differing as for the relationships among Neandertals, modern Europeans and Cro-Magnoids, the anatomically modern humans who coexisted with Neandertals for millennia. We compared by Approximate Bayesian Computation the simulation results with mitochondrial diversity in 7 Neandertal, 3 Cro-Magnoid, and 150 opportunely chosen modern Europeans. A model of genealogical continuity between Cro-Magnoid and Europeans, with no Neandertal contribution, received overwhelming support from the analyses.
The maximum degree of Neandertal admixture was estimated at 0.3%, one order of magnitude less than suggested by studies of the Neandertal nuclear genome. However, results based on nuclear and mitochondrial data might be reconciled if smaller population sizes led to faster lineage sorting for mitochondrial DNA, and Neandertals shared a longer period of common ancestry with the non-African’s than with the African’s ancestors.

1 Dipartimento di Biologia ed Evoluzione, Università di Ferrara, Ferrara, Italy.

SESSION 5 - HUMAN EVOLUTION

Humans and their environment: the evolution of loci involved in metabolism of exogenous molecules in globally diverse populations

S. Fuselli, P. Maisano Delser ¹

Genes involved in biotransformation of exogenous substances are particularly interesting from the evolutionary point of view because of their role as mediators between the organism and the environment. The chemical environment varies significantly with diet, climate, lifestyle, etc., and thus in humans a great deal of inter-ethnic differentiation is expected for these genes.
Here we analyzed the degree of human population differentiation at 614 single nucleotide polymorphisms (SNPs) in 121 genes coding for biotransformation proteins of exogenous substances in 52 globally diverse populations from the Human Genome Diversity Panel (HGDP). To identify the type of genes and genetic variants preferentially targeted by selection, we applied a statistical approach that considers the degree of population differentiation (Fst) looking for signals of adaptation enriched in our candidate genes. Under an assumption of neutrality, the geographic structure of genetic diversity is determined by demographic history which affect all loci similarly. By contrast, natural selection acts in a locus- specific manner and local positive selection in particular tends to increase Fst.
The same approach was used to test the hypothesis that the selective regime acting on genes involved in biotransformation of exogenous molecules changed when human populations shifted from an economy based hunting and gathering to one in which food was produced by farming and animal breeding, in the Neolithic period. To this aim we compared present-day HGDP hunting-gathering populations with geographically close food producers from East Asia and Sub-Saharan Africa.
Our analyses show that, in general, genes involved in biotransformation of exogenous compounds are not enriched of SNPs showing signals of adaptive evolution, both globally and comparing hunter-gatherers and food producers. However, specific categories of genes and sites show patterns indicating that population history alone is not enough to explain the observed genetic differences between different subsistence economies.

1 Università di Ferrara, Ferrara, Italy.

SESSION 5 - HUMAN EVOLUTION

Identifying genetic bases of human adaptation to high altitude

C. Chianella, A. Panziera, P. Maisano Delser, S. Fuselli ¹

High-altitude is the only environment colonized by modern humans where no behavioral buffering is available to face the peculiar and unavoidable stress: hypoxia. Life at low oxygen pressure needs specific biological adaptations to deliver enough oxygen to maintain aerobic metabolism. In the last decades many studies investigated the relationship between altitude and adaptive phenotypic traits, but to date no association between genetic variants and adaptive traits to hypoxia-related stress have been found in native American highlands.
Here we aim at identifying the genetic bases of human adaptation to high altitude by studying the evolution of candidate genes.
To this goal we (i) identified candidate genes by means of a bioinformatic approach, (ii) studied their molecular evolution comparing different species and (iii) began to investigate their genetic variation in Andean native populations.
The transcription factor HIF 1a, a master regulator of O₂ homeostasis, and additional 8 loci encoding for proteins that either regulate HIF1 activity (PHD1,2,3 and VHL) or represent its targets (NOS3, EDN1, EPO, VEGFA), were selected as candidate genes. Applying phylogenetic methods for comparative analysis of DNA we found that the candidate genes mostly evolved under purifying selection, as expected when important metabolic activity is conserved. Few codons were identified showing signature of adaptive evolution, and may represent good targets for the high altitude human population study.
We collected DNA and phenotypic measures from individuals of three Andean populations living at 3000-4000 m. Our re-sequencing analysis of coding and regulatory regions of two candidate genes, EPO and VHL, showed that these loci are extremely conserved also within humans, the few polymorphic sites being located in regulatory regions. Further candidate genes re-sequencing and comparison with low altitude populations from the same geographic regions will allow us to define a possible adaptive role of the genetic variation identified in our study.

1 Dipartimento di Biologia ed Evoluzione, Università di Ferrara, Ferrara, Italy.

SESSION 6 - EVOLUTIONARY THEORY

Fitness landscapes and surfaces of selective value

E. Serrelli¹

The notion of “fitness landscapes” was presented by Sewall Wright in 1932. Its influence in evolutionary biology was extensive in several directions up to the present day. One direction consists in studies that built “fitness landscapes” although, according to my analysis, they employed only a part of Wright’s ideas - i.e. the one concerning “surfaces of selective value” (cf. Wright 1988) - focusing on one or few genetic or phenotypic traits of the studied systems. The model Wright fostered in 1932 was about the entire genotypic space of a Mendelian population, characterized by huge dimensionality.
The lack of formal tools and computational power have prevented its actual construction, but understanding the original idea and how it differs from the realized models seems useful, all the most after the recent proposal by Sergey Gavrilets (e.g. Gavrilets 1997; 2004) of revising the overall structure of the genotype space. Understanding crucial differences is necessary here as well: for example, the newly proposed diagrams - namely, nearly flat, holey surfaces - do not represent the whole genotypic space, but the existence and properties of “nearly neutral networks” within it. The latter are fundamental for building particular speciation models called “spontaneous clusterization” (Gavrilets 2010).
I will present, on the one hand, Wright’s primal proposal and the revision advanced by Gavrilets, on the other hand, the fruitful “surface of selective value” method, that consists in (1) representing genetic or phenotypic variants as points that are distributed on a bi-dimensional surface, so that the distance between points be proportional to the “reachability” between variants; (2) extruding such a surface along a third, orthogonal dimension that represents the considered variants’ fitness. The method aids the study of the role of fitness and other factors in evolutionary dynamics.

1 Università degli Studi di Milano Bicocca, Dipartimento di Scienze Umane per la Formazione “Riccardo Massa”, Milano, Italy.

SESSION 6 - EVOLUTIONARY THEORY

Development of a new evolutionary theory

G. Damiani¹, E, Capelli²

Many experimental evidences, as for example the distribution of retroelements and DNA mutations in complex genomes, lead to the discovery of mechanisms based on epigenetic signals, editing of nucleic acids, reverse transcription, and micro-recombination, that promote DNA changes and horizontal transfer of genetic information between cells and organisms. The natural genetic engineering systems are triggered into action by a metabolic shift from an anabolic-syntropic phase to a catabolic-entropic one. The Lotka-Volterra oscillation between these two alternate and complementary phases at the levels of molecules, cells, organisms, and populations might explain many evolutionary dynamics and the emergence of collective behaviours and fractal structures near the critical points in many physical and biological systems.
In the catabolic phase, the genetic apparatus and the Darwinian processes allow the reproduction and conservation of biological systems. In the anabolic phase, the epigenetic apparatus and the Lamarckian processes produce innovative adaptations in response to environmental variations. In complex animals, the acquired immunity regulates the selective proliferation of the cells with adaptive mutations during the physiological responses to stresses. The same immunotrophic process allows the maternal selection of embryos with adaptive mutations and maintains the metabolic biodiversity of animal populations. A better understanding of how the genetic apparatus interacts with the environment by means of the epigenetic systems will allow the design of interventions to reduce the population load of complex diseases.

1 Istituto di Genetica Molecolare, CNR, Pavia, Italy.
2 Dipartimento di Biologia Animale, Università di Pavia, Pavia, Italy

References
The Yin and Yang of anti-Darwinian epigenetics and Darwinian genetics. Damiani G., Riv Biol. 2007. 100(3):361-402.
Corrections to chance fluctuations: quantum mind in biological evolution? Damiani G., Riv Biol. 2009. 102(3): 421-448.

SESSION 6 - EVOLUTIONARY THEORY

Genetic consequence of habitat fragmentation during a range expansion

S. Mona, L. Excofier ¹

Habitat fragmentation is often defined as a process where the habitat of a species is broken into smaller and isolated patches, accompanied (or not) by a loss of the total area available.
Here we investigated the genetic consequences of habitat fragmentation during a range expansion. Range expansions are known to leave specific footprint on the genetic diversity of a meta-population both in homogeneous and in some form of heterogeneous environment. Habitat fragmentation can be considered as the simplest form of environmental heterogeneity, i.e. a mixture of habitat and non-habitat.
To model habitat fragmentation during a range expansion, we simulated a forward expansion in a two-dimensional square array of 50x50 demes. The array was partitioned into group of demes (patches) by adding barrier to dispersal defining 100 patches of 5x5 demes. Demes were allowed to exchange migrants with the surroundings demes at a rate m1 if belonging to the same patch and at rate m2 if a barrier is present (with m2 < m1). Demographic and migration histories were then used to generate genetic diversity in a sample of genes under a coalescent approach. Sampling was performed at different scales: demes, patches, region (group of four neighbouring patches) and at the landscape level.
By varying the carrying capacity (k), m and time of the expansion, we found they all exert a strong influence on the genetic diversity of the meta-population, particularly when sampling within demes and patches (but not at the region and landscape level). Moreover, we found that genealogies at all sampling levels depend on k and m separately, rather than on their product. Finally, we performed additional simulations removing the barriers sometimes after the expansion, to determine how fast genetic diversity can be restored.
We found that, under many conditions, the process may take a large number of generations.

1 University of Bern, Berna, Svizzera.

SESSION 6 - EVOLUTIONARY THEORY

"Extended synthesis": a progressive shift of research program

T. Pievani¹

The theory of evolution shows a 150 years history of theoretical and empirical extensions and revisions, without any apparent radical change of “paradigm”. We would like to present here a tentative extension of the neo-Darwinian structure of evolutionary theory. The ongoing transition from the Modern Synthesis to a so-called “Extended Synthesis” is interpreted through the Methodology of Scientific Research Programs, proposed by the epistemologist Imre Lakatos and updated. The current situation in evolutionary biology is represented by a “progressive” shift of the neo-Darwinian research program, moving from the quite rigid framework of the Modern Synthesis to the more inclusive and pluralistic “core” and “protective belt” of the Extended Synthesis. So, we argument against the idea that we would have now more “theories of evolution” or a new “theory” with post-Darwinian features.
What we actually see at the front of the researches in evolutionary biology is quite different: an extended and strongly corroborated neo-Darwinian core (with its four main factors: variation and inheritance, natural selection, genetic drift, migration and macro-evolutionary effects), surrounded by a protective belt of auxiliary assumptions in progress, concerning i.e. multiple patterns about modes and rhythms of speciation, levels of selection, the effective agency of natural selection in the genome, the relationships between evolution and development, epigenetics, phenotypic plasticity, endosymbiosis.
This analysis of the rational and continuous dynamics of growth of biological knowledge seems much needed also for a critical examination of some popular controversies about evolution.

1 University of Milan - Bicocca, Milano, Italy.