Dissertations / Theses on the topic 'Phylogenesis'

In order to support the conservation of the Mediterranean octocorals improvements on information regarding their taxonomic units and phylogenetic relationships are strongly needed. In the present thesis work, phylogenetic analyses based on the mitochondrial mtMSH and 16S genes were performed including 15 Mediterranean octocorals species on the 56 recognized to date. Moreover, an extended datasets with Atlanto/Pacific congeners Octocorallia species was implemented to clarify their phylogenetic relationships and estimate the divergence times of the Mediterranean species. Results indicated that: 1) there are similarity and differences among molecular and morphological traits depending on the taxonomical level considered; 2) the molecular phylogeny of the Mediterranean octocorals retrace the previous relationships based on wide octocorals analyses; and 3) the divergence time among Mediterranean and Atlanto/Pacific species varies depending on analysed taxa. At higher taxonomic level, the Mediterranean trees supported the division of the Mediterranean Octocorallia into one major clade (Alcyoniina-Holaxonia) plus two unresolved branch including the single species available of Scleraxonia and Stolonifera respectively. This topology was better supported including the Atlanto/Pacific congeners species. The molecular evidence suggested that Alcyonium palmatum and Corallium rubrum species are the youngest with a divergence time estimated around 4 MYA. Particularly, C. rubrum results were in agreement with the hypothesis that recent orogenesis process of the Mediterranean Sea promoted the allopatric speciation of this specie. Increasing the sample design and implementing the emerging next-generation genomic-sequencing technologies, further studies would be able to improve the understanding of the Mediterranean octocorals phylogenetic relationships and evolution.

This research evaluates the unorthodox proposition that organisational development proceeds through the Darwinian processes of variation, selection and inheritance acting upon a non-genetic replicating code. This new replicator represents the fundamental unit of cultural transmission and was termed by evolutionist, Richard Dawkins, as the meme. The memetic position re-introduces many often neglected, sometimes shunned, evolutionary arguments into social and organisational debate by providing a naturalistic and plausible hereditary element upon which socio-cultural adaptation operates. The popularity of the neologism 'meme' initially grew through rather ad-hoc non-scientific usage on the Internet. For some time, this geekish tendency has tarnished the idea of memetics and impeded serious academic investigation into the subject. A more rigorous philosophical treatment has been provided by Daniel Dennett who has argued that, while a science of memetic cladistics may be both desirable and feasible, it remains unlikely. On the other hand one of Dawkins' most famous critics, Mary Midgley, heralds dark forebodings that one-day memes may be given actual credence. The present study necessitated the adaptation of conventional genealogical and taxonomic methods, for novel application in confirming congruence between actual organisational phylogeny and hereditary traits. One specific requirement was to develop a means of identifying, capturing and codifying such traits as meme strips for phenetic analysis. In order to handle the computational complexity inherent in the phenetic reconstruction algorithms, proprietary software had to be produced. This was extensively tested upon meme strips generated through simulated evolution. Western Christian denominational families provided a source of empirical evidence and demonstrated that the methods could be successfully applied to real organisational forms. A theological phylogeny was reliably reconstructed thereby upholding the hypothesis of cultural descent with modification based on a memetic replication. Further support for the claim was made in conjunction with the rendering of a facilities management market landscape. More importantly however, the results coming from this research suggest that the potential for formulating a science of memetics may be significantly greater than in Dennett original consideration.

Glutathione peroxidase (GPX, EC 1.11.1.9 and EC 1.11.1.12) and Carbonic anhydrase (CA, EC 4.2.1.1) are two enzymes important for a variety of adaptation/tolerance processes of organisms. CA plays a key role in osmoregulation when GPX is one of the most important enzymes involved in protection of the organism against oxidative damage. In my thesis I studied GPX and CAs from Antarctic teleosts species of Notothenidae and Bathydraconidae (Paper I-II) and CAs from intertidal teleosts (Periophtalmus sobrinus, Gobidae, and Opsanus beta, Batrachoididae (Paper II-III). Firstly, I compared the obtained sequences with those available in database for teleosts and other vertebrates (human, mouse and bird) to reconstruct the molecular phylogeny of two enzyme in teleosts (Paper I-II). Finally, I have investigated the role of CA for the osmoregulation in seawater toadfish (Opsanus beta) exposed to hypersalinity (Paper III). The GenBank database, few sequences of GPX and CAs of temperate teleosts are available and no data are available for Antarctic or intertidal fish were not present. In particular, I choose to study those species because they live in environments with peculiar chemical/physical parameters that influenced, during the evolution, their physiological, morphological and behavioural characteristics. The Antarctic fish (Papers I-II) live in a well delimited geographic zone. The Southern Ocean is comprised from the Antarctic continent to the Polar Front, a curved current continuously encircling Antarctica where cold, northward-flowing Antarctic waters meet and mix with the relatively warmer sub-Antarctic waters. In this limited zone, macro evolutionary events happened, such as radiation of some fauna components. Most of the fish species living in this zone (like nototenioidae) are presents only there. Temperature (-1.9 °C) and salinity (34.8 ppt) are lower than in temperate Oceans (Legg et al. 2009). As a consequence, the oxygen concentration is higher (9 mg l-1 (Meiner et al. 2009)) in these waters because of the higher solubility of the gas. Furthermore, these parameters are constant during the year. In this perspective, is high the interest to study the evolution of some proteins such as of GPX (Paper I), linked to hyperoxia, and of CAs (Paper II), linked to osmoregulation, to investigate if two enzymes have been an independent evolution and if them aminoacidic sequences are modified compared to sequences of temperate teleosts. The intertidal species (mudskipper Periophtalmus sobrinus (Paper II) and toadfish Opsanus beta (Paper III) both live in mangrove zones. Temperature (the average 28.9°C in Kenya (Schmitz et al. 2007) and in 25.8°C Florida (Kelble et al. 2007)) and salinity (the average 32.4 ppt in Kenya (Schmitz et al. 2007) and 33.0 ppt in Florida (Kelble et al. 2007)) are higher with respect to temperate oceans and these parameters, in contrast to Antarctic habitat, are very fluctuating. Gazy Bay (Kenya), where mudskipper lives, is subject to seasonal fluctuation due to tides. Florida Bay (Florida), where toadfish lives, is subject to seasonal fluctuation due to wet and dry season. The mudskipper was sampled in Gazy Bay (during summer of 2007), and toadfish was sampled in Florida Bay (during summer of 2009). My data show that both GPXs and CAs aminoacidic sequences are conserved in all teleosts (Paper I-II). Just a few aminoacidic changes were found in Antarctic teleosts aminoacidic sequences. Considering the active site and he aminoacidic zone toward the catalytic centre of GPX (Paper I), only the polar Arg182, conserved in all teleosts GPX-1, changes in a hydrophobic Lys in Antarctic fish enzyme (Aumann et al. 1997). In CA sequences the analysis has been focused on residues that are included within 10 Å of the zinc atom (Paper II). They are almost all conserved, with only a few aminoacidic substitutions but maintaining the same properties. Consequently we assume that the features of active site and the aminoacidic sequence toward the catalytic centre of both enzymes are well conserved in teleosts. The phylogenetic tree has different topology for GPX and CA sequences (Paper I-II). In the GPX the Antarctic teleosts group together, while in the CA tree they are divided in two different groups. The topology of GPX of Antarctic teleosts is the same as that of Antarctic teleosts obtained by molecular data (16S rRNA. (Near et al. 2004). This suggests that the CAs evolved in a separate way. In the last paper (Paper III), I measured the expression and activity of CA linked to osmoregulation. It was not possible to perform the same analysis for GPXs and CAs of other fish sequenced because the samples used were frozen while to measure activity I needed fresh samples. I found a correlation between hypersalinity and increase of CA activity and expression in gills and intestine. This suggests an involvement of gill and intestine CA in regulation of seawater fish exposed to hypersalinity
I pesci vivono negli habitat più diversi: acque dolci (fino a 0,01 ppt), salate (fino a 100 ppt), stagnanti; in zone al di sopra dei 5.200 metri (Kottelat and Chu 1988) o nelle profondità abissali di 7000 metri (Nelson 1994). Alcuni vivono in zone soggette a forti maree e tollerano brevi escursioni sulla terraferma (Sayer 2005), altri resistono a temperature di circa 44°C (es. Tilapia in Africa) o di circa -2°C (es. Trematomus nelle acque antartiche) (Nelson 1994). Per vivere e riprodursi in ambienti tanto diversi hanno sviluppato una serie di adattamenti fisiologici, morfologici e comportamentali. Il mio progetto di dottorato si è concentrato su due linee di ricerca: la filogenesi molecolare di due enzimi, la Carbonico anidrasi (CA, EC 4.2.1.1) e la Glutatione perossidasi (GPX, EC 1.11.1.9 e 1.11.1.12) di specie antartiche e intertidali; il coinvolgimento della CA nell’adattamento a condizioni di ipersalinità in una specie di teleosteo della zona intertidale. I pesci antartici vivono in acque molto fredde, vicine al punto di congelamento (-1,8 °C), e con una concentrazione salina molto bassa (34,8 ppt) (Nelson 1994). Questi parametri si collocano all’estremo inferiore della scala di temperature e salinità delle acque dove vivono pesci di mare aperto la temperatura nelle acque temperate è di circa 20°C e la salinità è intorno a 40 ppt (Nelson 1994)- ma sono molto stabili anche nel periodo annuale. La zona di mare dove vivono questi organismi rappresenta una sorta di microambiente con minime variazioni (Eastman 2005). Una conseguenza diretta dei bassi valori di temperatura e salinità è una concentrazione di ossigeno molto più elevata nel caso delle acque antartiche rispetto a quelle temperate (valori nelle acque antartiche i valori vanno da un minimo di 159 a un massimo di 413 μmol/Kg (Meiner et al. 2009) mentre nelle acque temperate il minimo registrato e <20 μmol/Kg (Fuenzalida et al. 2009)). La zona di acqua costiera dove vivono questi organismi, è circondata dalla corrente circumpolare antartica, formatasi circa 22-25 milioni di anni fa in seguito a una serie di movimenti tettonici e oceanografici, che crea una naturale barriera attraverso la quale i pesci non possono passare.(Eastman 2005) Si è formato cosi un unico sito dove si sono create delle nuove nicchie ecologiche occupate da gruppi di pesci (i Nototenoidei) che si sono sviluppati in situ.(Anderson 1999) I pesci studiati sono Trematomus bernacchii, T. eulepidotus, T. lepidorinus e Cygnodraco mawsoni, appartenenti alla famiglia dei notetioneidei, la fauna maggiormente rappresentata in termini di diversità, abbondanza e biomassa nell’Oceano Antartico (Eastman 1993). I membri di questa famiglia sono caratterizzati da diversi adattamenti fisiologici (ad es. le proteine antigelo) (Chen et al., 1997; Cheng C-HC, 2002)) e da un alto grado di diversità morfologica (Eastman 2000; Kock 1992) che li rendono adatti a sopravvivere in queste acque. I Nototenioidei sono endemici in Antartide ed è stato ipotizzato che rappresentino una radiazione adattativa avvenuta nell’Oceano Antartico (Briggs 1974; Briggs 1996) Esistono 8 famiglie di Nototenioidei per un totale di 44 generi e 129 specie. Di queste, 101 sono prettamente antartiche e solo 28 sono non antartiche (Bargelloni et al., 2000; Stankovic et al., 2002; Near et al., 2004). La zona di mangrovia è situata tra mare e terra nella zona intertidale. La collocazione geografica di questo biotopo implica elevate temperature e di conseguenza la concentrazione di ossigeno disciolto e la salinità fluttuano ampiamente durante il corso della giornata per effetto combinato delle maree e della evaporazione dell’acqua (Blaber 1997; Lowe-McConell 1987; Morton 1989). Elevata salinità, elevate temperature e l’istaurarsi di condizioni di anaerobiosi nell’acqua sono le caratteristiche peculiari di questa zona (Kathiresan 2001). La maggior parte di pesci intertidali di questi areali, mostrano diversi adattamenti di tipo fisiologico, morfologico e comportamentale (Bridges 1993; Gibson 1996; Lewis 1970). I mudskippers sono un gruppo di teleostei (Perciformes, Gobiidae, Oxudercinae) che vive tra le mangrovie; sono eurialini e adattati a cambiamenti estremi di salinità (Chew and Ip 1990), esposizione all’aria (Kok et al. 1998) ipossia (Chew SF 1990), e ad alte concentrazioni di ammonio (Ip et al. 2004). I mudskippers appartengono al gruppo monofiletico dei gobioidei (Thacker 2009; Winterbottom 1993) che ha subito una radiazione nel passaggio all’habitat marino. L’Opsanus beta (Gulf toadfish) è un teleosteo marino distribuito lungo le coste tra il Golfo del Messico e il Sud America. Vive in acque stagnanti dove vi è un continuo mescolamento di acqua salata e acqua dolce. La continua evaporazione crea un ambiente con salinità fluttuante (Lirman and Cropper 2003); ad esempio le medie di salinità calcolate nella zona di Florida Bay vanno da un minimo di 24.2 ppt in novembre a un massimo di 41.8 ppt in luglio (Kelble et al. 2007). Il toadfish tollera salinità che vanno da 5 a 60 ppt in condizioni di laboratorio (McDonald and Grosell 2006). All’aumentare della salinità si osserva un aumento della richiesta di assorbimento di sale dall’intestino; questo ha un significativo impatto sul bilancio acido-base dovuto al cambiamento nell’escrezione di CO2 nell’intestino (Genz et al. 2008). La Glutatione Perossidasi La Glutatione perossidasi GPX) è una famiglia di isozimi che catalizzano la riduzione degli idroperrosidi organici ad acqua, o alla corrispondente sostanza alcolica, usando il glutatione ridotto (GSH) come donatore di elettroni. Di questo enzima, che può essere seleno-dipendente o indipendente, ne sono state caratterizzate 4 diverse isoforme: GPX1 localizzata in fegato, polmoni e reni, GPX2 gastrointestinale, GPX3 trovata in reni, polmoni, epididimo, vasi deferenti, placenta, vescicole seminali,cuore e muscolo e GPX4 (fosfolipidica) distribuita largamente nei tessuti (Hochachka and Lutz, 2001; Margis et al., 2008). L’importanza della GPX è di preservare le cellule dai possibili danni dovuti alla produzione di H2O2 prodotto nei mitocondri come conseguenza della fosforilazione ossidativa. In diversi siti lungo la catena respiratoria mitocondriale, l’ossigeno subisce una riduzione parziale, generando l’anione superossido che è il primo di una serie di radicali (O2-), il radicale idrossilico (OH∙), l’ossigeno singoletto (1O2) e il perossido di idrogeno (H2O2)) (Miller 1993b). I radicali prodotti (ROS, reactive oxigen species) interagendo con diversi target intracellulari (lipidi, proteine e acidi nucleici) attivano meccanismi di morte cellulare e inducono varie disfunzioni cellulari ritenute responsabili di molteplici patologie. In condizioni di ipossia o iperossia la produzione di ROS è maggiore (Fink and Scandalios 2002). La Carbonico Anidrasi La carbonico anidrasi riveste un ruolo fondamentale nella reazione reversibile di idratazione, deidratazione della CO2 con produzione di H+ e HCO3– (CO2+H2O→H++HCO3-). La funzione principale della CA è la regolazione acido-base e l’osmoregolazione a livello intestinale e branchiale (Geers and Gros, 2000; Esbaugh and Tufts, 2006). La CA dei pesci studiati, probabilmente, ha sviluppato un particolare adattamento, legato all’osmoregolazione, dovuto agli ambienti ipo (oceano Antartico) e ipersalino (zona intertidale) in cui vivono. Nei mammiferi si conoscono 16 differenti isozimi che differiscono per proprietà cinetiche, distribuzione tissutale e localizzazione subcellulare. Le diverse isoforme della famiglia delle α-CA sono la I, II, III, V, VII and XIII citoplasmatiche, la IV, IX, XII,XIV, XV legate in membrana (Esbaugh and Tufts 2006). La CAII sembra essere in uno stato ancestrale nei pesci, mostrando alta attività, fino ai teleostei dove apparentemente si è duplicata in due diverse isoforme, la CAb (eritrocitaria) e la CAc (citoplasmatica) (Esbaugh et al., 2004; Esbaugh et al., 2005). Il lavoro svolto durante il mio dottorato può essere suddiviso in tre parti principali: 1. Il clonaggio e il sequenziamento di GPX di pesci antartici per individuare mutazioni aminoacidiche nella sequenza della proteina caratteristiche di queste specie e fare una ricostruzione filogenetica dell’enzima. Anche in questo caso si è partiti con un’estrazione di RNA da branchie. La ricostruzione filogenetica ci ha permesso di indagare la storia evolutiva di tali proteine confrontandole con sequenze di GPX di altri teleostei e di altri vertebrati e prendendo in considerazione la storia evolutiva degli organismi. I pesci sono stati campionati durante la XIV (1998-1999), la XVII (2001-2002) e la XXI (2005-2006) campagna italiana in Antartide. 2. Il clonaggio e il sequenziamento di CA di pesci antartici e di zone intertidali (mangrovieti e acque ipersaline). Questo è stato ottenuto partendo dall’estrazione di RNA da tessuto branchiale o intestinale di tali pesci. Ottenute le sequenze delle CA, queste sono state utilizzate per un’analisi filogenetica. Le sequenze sono state confrontate tra loro e con le sequenze di CA disponibili in rete per evidenziare la presenza di zone conservate o meno ed è stata fatta una ricostruzione tridimensionale per vedere e confrontare la distribuzione di potenziale elettrico delle stesse. La ricostruzione filogenetica è servita per delineare un’ipotetica evoluzione di tali enzimi in confronto con gli enzimi di altri teleostei e di altri vertebrati tenendo conto della storia evolutiva di tali organismi. (I pesci sono stati campionati durante la XIV (1998-1999), la XVII (2001-2002)e la XXI (2005-2006) campagna italiana in Antartide.) 3. Il clonaggio e il sequenziamento della CA del Gulf Toadfish (Opsanus beta), un teleosteo che vive in zona intertidale con tendenza all’ipersalinità. Una volta ottenuta la sequenza (partendo anche in questo caso da estrazione di RNA da tessuti) è stata fatta una ricostruzione filogenetica e delle analisi di attività ed espressione dell’enzima. I tessuti analizzati sono intestino (anteriore, medio e posteriore), retto e branchie. L’analisi è stata fatta su campioni controllo (stabulati alla stessa salinità dell’acqua in cui vivono (40ppt) e su pesci stabulati in condizioni d’ipersalinità (60ppt) per 6-12-24-48-96 ore (per quanto riguarda le misure di espressione nei vari segmenti di intestino e retto) o due settimane (per quanto riguarda misure di espressione e attività su branchie e di attività sui segmenti di intestino e retto). Questo studio è stato svolto per indagare il coinvolgimento della CA a livello intestinale e branchiale nell’osmoregolazione di pesci d’acqua salata se sottoposti a un ambiente ipersalino. Per questa parte del lavoro mi sono recata da aprile a ottobre 2009 presso il laboratorio del professor Martin Grosell (RSMAS, University of Miami, Florida, USA). Risultati e Discussione Glutatione Perossidasi La sequenza della GPX1 selenio-dipendente è stata ottenuta per i pesci antartici Trematomus bernacchii (946 bp), T. lepidorhinus (946 bp), T. eulepidotus (945 bp) e Cygnodraco mawsoni (950 bp). Tutte le sequenze sono caratterizzate da un open reading frame (ORF) di 191 amminoacidi. In posizione nucleotidica 174-176 è presente la tripletta TGA che codifica per la selenocisteina, un amminoacido facente parte della triade catalitica. La proteina sequenziata corrisponde all’isoforma 1 (citoplasmatica) perché dalla ricostruzione filogenetica ottenuta con il metodo del NJ le sequenze si pongono tutte in un cluster vicino all’isoforma 1 e separato dal gruppo dalle GPX2. Si può ipotizzare un’origine monofiletica all’interno dei teleostei antartici, che sono un clade completamente separato. Questo enzima sembra essersi evoluto nei pesci antartici indipendentemente dalle altre specie di teleostei. La ricostruzione filogenetica ottenuta conferma le odierne analisi filogenetiche sull’evoluzione delle GPX (Margis et al., 2008; Toppo et al., 2008). Le varie isoforme risultano separate da valori di bootstrap che si approssimano al 100%, dimostrando che le isoforme 1, 2 e 3 sono più vicine filogeneticamente fra loro, mentre le isoforme 7 e 8 sono più affini alla GPX4. Il cluster dei teleostei antartici risulta indipendente e ben separato dagli altri con valori di bootstrap del 100%, avvalorando l’idea di un’origine monofiletica della proteina in questo gruppo. All’interno del ramo dei teleostei antartici, le distanze evolutive appaiono molto brevi, indicando che la diversificazione della GPX nelle diverse specie è avvenuta in tempi piuttosto recenti. In questo gruppo le relazioni evolutive non sono completamente risolte, nonostante alcuni nodi siano sostenuti da alti valori di bootstrap. La GPX di C. mawsoni risulta essere il sister group delle GPX delle specie appartenenti al genere Trematomus. Analisi filogenetiche basate sullo studio delle subunità 16S e 12S dell’rRNA mitocondriale, indicano Cygnodraco mawsoni come sister group della famiglia dei Nototheniidae, alla quale appartiene anche il genere Trematomus (Bargelloni et al. 2000), confermando la ricostruzione filogenetica. Il lavoro di (Epp et al. 1983) ha determinato per la prima volta la struttura cristallografica della GPX1 bovina e ha evidenziato la triade catalitica rappresentata da selenocisteina (40), glutammina (75) e triptofano (153). E’ stato proposto che nel sito attivo la glutammina e il triptofano siano legati mediante ponti idrogeno alla selenocisteina e che attivino l’elemento redox (il selenolo) proprio grazie a questo legame che dovrebbe facilitare l’attacco nucleofilico dell’idroperossido. Questi aminoacidi sono, infatti, altamente conservati anche nei teleostei. I residui coinvolti nella formazione dei foglietti β e delle α-eliche rimangono, tranne alcune eccezioni, invariati. La regione che sembra avere un ruolo fondamentale nella capacità di dimerizzazione della proteina è rappresentata dai residui 89-95, che mancano nella GPX4, che per questo motivo presenta una struttura monomerica (Scheerer et al. 2007). Questi residui sono conservati anche nei teleostei, anche se con alcune eccezioni. La Val90 è stata sostituita nei soli teleostei antartici, da un altro amminoacido alifatico, la leucina. Mentre negli anfibi e nei mammiferi in posizione 94 è presente una glicina, nei teleostei troviamo quattro amminoacidi diversi, tutti residui polari: nei teleostei antartici troviamo una lisina (carica positiva), in H. molitrix un aspartato (carica negativa), in D. rerio un glutammato (carica negativa) e negli altri teleostei una asparagina (senza carica). Possiamo ipotizzare che tali cambiamenti aminoacidici abbiano un effetto sulla velocità e sull’efficienza della reazione catalizzata dalla GPX. Carbonico anidrasi La carbonico anidrasi di tre specie antartiche e di due specie di zona intertidale è stata sequenziata completamente (Trematomus bernacchii (1062 bp), T. lepidorinhus (1656 bp), T. eulepidotus (1563 bp) per le specie antartiche e Periophtalmus sobrinus (1217 bp) e Opsanus beta (1827 bp) per le specie intertidali). Una quarta sequenza, mancante di una parte in 3’, è stata ottenuta per Cygnodraco mawsoni (713 bp), (specie Antartica) tutte con un ORF di 260 aminoacidi (GenBank accession number: GQ443602 (T. bernacchii); GQ443601 (T. lepidorinhus); GQ443600 (T. eulepidotus); GQ443603 (Periophtalmus sobrinus). Queste sequenze saranno disponibili a partire da settembre 2010 mentre per Opsanus beta (GQ443599) la sequenza è già disponibile in rete) Le sequenze mostrano un’identità piuttosto alta con le sequenze di CAII disponibili in database (dall’80% al 72%). Per questo e perché nell’albero ottenuto con il Neighbor Joining (NJ) raggruppano nel cluster delle CAII, possiamo ritenere che si tratti dell’isoforma II. Nell’albero ottenuto sono ben visibili le separazioni tra il gruppo delle CA di membrana e quelle citoplasmatiche. Tra quest’ultime vi è un’altra separazione, tra le CAII citoplasmatiche (CAIIc) e le CAII citoplasmatiche eritrocitarie (CAIIb). Possiamo ipotizzare che la CA di Opsanus beta sia l’isoforma CAIIc, ovvero che si tratti dell’isoforma citoplasmatica e non di quella eritrocitaria (CAIIb), perché nella ricostruzione filogenetica è raggruppata insieme alla CAc di trota. La CA del mudskipper, invece, raggruppa con le isoforme CAb di trota, carpa e zebrafish. Un’altra divisione ben delineata e mostrata anche nell’albero ottenuto da Esbaugh et al., 2006, è quella esistente tra mammiferi e non mammiferi. Le CA di pesci antartici raggruppano insieme in un cluster separato dagli altri e ben supportato da alti valori di bootstrap, sottolineandone la stretta relazione filogenetica. Anche l’icefish (Chionodraco hamatus) raggruppa in questo cluster. Gli aminoacidi che compongo la triade catalitica (His 94, 96, 119) e quelli che mantengono la struttura funzionale della proteina (Thr 199 e Glu 106) sono conservati, mentre possiamo notare dei cambiamenti a livello aminoacidico propri delle sequenze di carbonico anidrasi dei pesci Antartici. In posizione 10-11 si trovano una Ala e una Asn conservate solo nelle sequenze di CA di pesci antartici, come in posizione 157 dove vi è una Ser e in posizione 190-191 con una Gly e una Cys. Carbonico anidrasi in Opsanus beta La carbonico anidrasi sequenziata (GenBank accession number GQ443599) è di 1827 paia di basi con un ORF di 260 aminoacidi. La sequenza mostra un’identità del 78% con Oncorhynchus mykiss e Pseudopleuronectes americanus, del 77-76% con le altre sequenze di CA di teleostei, e del 63-59% con anfibi e mammiferi. Le analisi filogenetiche effettuate con il metodo del NJ raggruppano la CA con le isoforme citoplasmatiche degli altri teleostei e la separano dalle isoforme di membrana (CA IV, IX, XII, XIV, XV) e dalle sequenze di mammiferi e di altri tetrapodi, in accordo con quanto riportato da altri studi. La CAII può essere suddivisa in due isoforme, la CAIIc (citoplasmatica) e la CAIIb (citoplasmatica eritrocitaria). La ricostruzione filogenetica ottenuta suggerisce che l’isoforma sequenziata sia di tipo CAIIc poiché raggruppa con le CAIIc di altri pesci. Su branchie, intestino (anteriore, medio e posteriore) e retto sono state condotte misure di espressione dell’mRNA tramite qPCR. Intestino e retto mostrano simile espressione in pesci acclimatati all’acqua salata mentre le branchie mostrano un’espressione più elevata. Nei pesci sottoposti a ipersalinità (60 ppt), si osservano alti livelli di espressione rispetto al controllo (40 ppt) nell’intestino medio e posteriore e nel retto. In particolare l’intestino medio risponde per ultimo all’ipersalinità, dopo 96 ore di esposizione, mentre il retto mostra una maggiore attività già dopo 12 ore di esposizione mantenendosi stabile per tutto il tempo. L’intestino posteriore mostra un rapido incremento di espressione dopo 6 ore di esposizione con un decremento dopo 12 e 24 ore seguito da un altro incremento dopo 96 ore. Per quanto riguarda le branchie, è stato possibile fare misure di espressione su branchie perfuse solo dopo due settimane di stabulazione all’ipersalinità. I dati ottenuti non mostrano un incremento nell’espressione in pesci a 40 ppt o a 60 ppt. L’attività, misurata su tessuti di pesci controllo (40 ppt) e su tessuti di pesci esposti per due settimane a 60 ppt mostra un significativo incremento nella frazione citosolica nei tessuti dopo l’esposizione. L’attività totale, confrontata con la citosolica, mostra un minor incremento. Diversi studi mostrano che la CA è coinvolta nell’osmoregolazione in teleostei marini. Usando inibitori (Grosell and Genz 2006), è stato dimostrato che la CA ha un ruolo chiave per la secrezione intestinale di HCO3- in teleostei marini. In questo studio è stato dimostrato il coinvolgimento di questo enzima in tessuti intestinali e branchie in risposta all’ipersalinità. Il confronto tra l’espressione e l’attività registrata rivela una diversa risposta. L’espressione di mRNA incrementa nell’intestino posteriore e nel retto con effetto non evidente a livello branchiale, intestino anteriore e medio. Invece, l’attività dell’enzima aumenta visibilmente in tutti i tessuti. Le nostre osservazioni rivelano, quindi, una maggiore espressione nei tessuti distali dell’intestino. Invece, la parte anteriore dell’intestino che sotto normali condizioni di salinità è responsabile della maggior parte dell’escrezione di HCO3- non mostra un visibile cambiamento di espressione nei tessuti di pesci acclimatati a ipersalinità. Questo è in contrasto con quanto trovato nella trota (Grosell et al. 2007), dove l’attività era maggiore nella regione anteriore dell’intestino piuttosto che nella posteriore. Nelle branchie, l’attività della CA è confrontabile con quella registrata nelle diverse regioni dell’intestino. Le nostre osservazioni possono suggerire un coinvolgimento della CA branchiale nell’osmoregolazione ad ambienti ipersalini. L’incremento della capacità della CA branchiale di idratare la CO2 in pesci sottoposti a ambienti ipersalini, può aumentare la disponibilità di HCO3- e protoni e quindi conferire una maggiore abilità di trattenere HCO3- trasportata attraverso la membrana basolaterale e/o la secrezione di protoni attraverso la membrana apicale.

The aim of the thesis is the development of algorithms to study the evolution of genomic information starting from data produced by Next Generation Sequencing (NGS) technologies. We address the problem of reconstructing evolutionary histories following two research directions which both explore algorithms for the generation (or reconstruction) of ancestral recombination graphs (or phylogenetic trees) modeling the evolution in presence of evolutionary events, such as recombination and Single Nucleotide Polymorphisms (SNPs). The first research direction regards the development of efficient algorithms for simulating complex scenarios of multiple population evolution with admixture. The aim of simulations is to obtain the resulting extant population samples and their common relevant evolutionary history captured by an ARG. We propose a backward simulation algorithm, named SimRA, for modeling complex evolutionary scenarios, which improves time and space requirements of the classical algorithm of single populations. Through extensive simulation experiments, we show that SimRA produces ARGs in compact form without compromising any accuracy. Moreover, we present the first combinatorial approach, based on persistency in topology, which detects admixture in populations. We show, based on efficient and controlled simulations computed by SimRA, that the topological framework has the potential for detecting admixture in related populations. The second research direction regards the development of efficient algorithms to reconstruct phylogenesis of contemporary species described by genomic binary characters. Established maximum parsimony models are Dollo and Camin-Sokal, both leading to NP-hard reconstruction problems. On the other hand, the perfect phylogeny, which has very efficient polynomial time algorithmic solutions, is often too restrictive for explaining the evolution of real biological data where homoplasy is present. We address the problem of reconstructing a variant of the perfect phylogeny model, the persistent phylogeny, that is more widely applicable, with the aim of retaining the computational efficiency. For this purpose, we introduce the Constrained Persistent Perfect Phylogeny problem (CPPP) which generalizes the Persistent Perfect Phylogeny (PPP) problem, by adding constraints for some observed characters. We provide a polynomial time algorithm for a particular class of instances and a parameterized algorithm for solving the general problem. We conclude the thesis with results concerning the scaffold filling computational problem which derives from the necessity of filling incomplete genomic sequences in order to maximize their similarity with a known reference genome. We consider two scaffold filling problems (One-sided and Two-sided) that are NP-hard under the maximum number of common adjacencies similarity. We design two Fixed Parameterized Tractable (FPT)-algorithms respectively for the One-side and Two-side scaffold filling problem, with only one parameter representing the number of common adjacencies between the two filled genomes.

An unknown fraction of what can be known is inaccessible to the verbalising which has otherwise made rational science so dramatically successful. The lack of a verbal currency has not extinguished these domains but their relatively diminished access has pushed them into obscurity. Despite its relative success, rational science has limits and obscuring the non-rational has foreclosed on searching it for contributions to the epistemology of human and natural phenomena. Following Vanelli (2001)*, my thesis pushes against the narrow perspective that rational science is the only legitimate mechanism for constructing new knowledge. I do this by using frameworks of comparative anthropology and psychoanalysis to interpret ‘unexplained early infant crying’ and ‘infant directed speech’, two prosaic and universal human behaviours. These two phenomena have been abundantly studied through the prism of rational science but neither has been adequately explained. I first survey the dimensions of unexplained early infant crying such as its susceptibility to cultural variation and resistance to rational explanation. That it is a human universal suggests it maybe a physical manifestation of a more deeply seated phenomenon and so I broaden the field of inquiry from that of observation of extant behaviour to human phylogeny. In so doing, I use the psychoanalytic frameworks of Winnicott and Grotstein to situate the so called “primitive agonies” as a consequence of the hominin obstetric dilemma purported to result from the habitual bipedalism by Ardipithecus ramidus ~4.4 mya. Through this synthesizing of the evidence from evolutionary anthropology, primatology and psychoanalysis, I show how the temporal, diurnal and synchronic profiles of early infant crying, including cultural variations, all become explicable as an outcome of the infant’s experience of their primitive agonies. These have no contemporary causation but emerged with the emergence of the hominin mind. A similar survey of infant directed speech, shows how unlikely it is to be a derived behaviour and that its universality again hints at deeper evolutionary roots. And so again through synthesizing phylogeny with the psychoanalytic models of Bion and Melanie Klein, I argue that premature birth of hominins exposes them to negative consequences of using that which Bion labelled in humans, the alpha-function, but which Castoriadis exposes to us as an existentially vital mental function of all sentient life. Infant directed speech can then be argued as the primary sensory stimulus to facilitate the self-organisation of the specific neural architecture from which this property of mind emerges. By engaging with the vast store of that which can be known but which is inaccessible to rational science, these seemingly trivial instances of human mental neoteny become significant knowledge pathways into the foundation of the human mind. Keywords: Hominin evolution, early infant crying, infant directed speech, primitive agonies, psychoanalytic anthropology, Winnicott, Bion, Grotstein, Castoriadis

O óxido nítrico (NO) é um radical livre gasoso que participa de uma série de processos biológicos fisiológicos sendo produzido por enzimas denominadas óxido nítrico sintases (NOS). Diversos estudos vem demonstrando sua participação na resposta inflamatória crônica, na qual as células inflamatórias, com destaque para os macrófagos, são estimuladas a sintetizarem NOS, que por esse motivo são denominadas induzíveis (iNOS) e passam a produzir o óxido nítrico que vai atuar na modulação do processo. A fim de comprovar sua participação filogenética na resposta granulomatosa, utilizou-se como modelo experimental a inoculação de onco-BCG na musculatura de tilápias-do-Nilo (Oreochromys niloticus) e de girinos de rã touro-gigante (Rana catesbeiana), na região plantar de tartarugas ?red ear? (Trachemys scripta elegans) e no coxim plantar de hamsters sírios (Mesocricetus auratus). Fragmentos da lesão foram colhidos aos 14, 28 e 42 dias pós-inoculação, fixados em Carnoy por quatro horas, sendo em seguida transferidos para álcool 70o GL. Procedeu-se a confecção de preparados histopatológicos segundo métodos de rotina que foram corados pelo método da hematoxilina e eosina. Imunoistoquímica foi realizada para a verificação da produção de óxido nítrico indiretamente através da marcação da iNOS com anticorpos anti-iNOS humana biotinilados produzidos em coelhos. Observou-se em todos os animais desenvolvimento de granulomas que mostraram tendência a maior organização aos 42 dias; as características celulares foram semelhantes, com algumas variações específicas. Constatou-se marcação imunoistoquímica em macrófagos presentes nas lesões produzidas pela inoculação de BCG em todos os grupos experimentais, exceto nos girinos aos 14 dias, cuja marcação foi irrelevante. Os resultados permitiram concluir que o óxido nítrico participa da resposta inflamatória granulomatosa, bem como a utilização de imunoistoquímica mostrou-se método eficiente para evidenciar sua produção em estudos filogenéticos. Pesquisas futuras deverão qualificar e quantificar mediadores químicos envolvidos na regulação da participação do óxido nítrico para melhor compreender sua fisiopatologia na modulação do granuloma inflamatório.
Nitric oxide (NO) is a gaseous free radical that takes part in a series of biological physiological processes. It is produced by enzymes called nitric oxide synthetases (NOS). Several studies have demonstrated its role in chronic inflammatory response, in which inflammatory cells, mainly macrophages, are stimulated to synthesize NOS, being called then inducible nitric oxide synthetases (iNOS). Nitric oxide is then produced and acts in the modulation of the process. In order to corroborate its phylogenetic role in granulomatous response, the inoculation of onco-BCG experimental model was used in the muscle of Nile tilapias (Oreochromys niloticus) and bullfrog tadpoles (Rana catesbeiana), in the plantar region of red eared sliders (Trachemys scripta elegans) and in the plantar pad of hamsters (Mesocricetus auratus). Fragments of the lesions were collected at 14, 28 and 42 days after inoculation, fixed in Carnoy for four hours, and then transferred to alcohol 70o GL. After that, histopathological slides were prepared following routine methods, and stained by hematoxylin-eosin. Immunohistochemical tests were performed in order to assess the production of nitric oxide indirectly by means of marking iNOS with biotinylated human anti-iNOS antibodies produced by rabbits. It was observed in all animals that the development of granulomas showed greater tendency of organization at 42 days; cell characteristics were similar, with some specific variations. Immunohistochemical marking was observed in macrophages present in lesions produced by BCG inoculation in all experimental groups, except in tadpoles at 14 days, which showed irrelevant marking. Results enabled the conclusion that nitric oxide takes part in granulomatous inflammatory response. Besides, the use of immunohistochemistry showed to be an efficient method for evidencing the production of nitric oxide in phylogenetic studies. Future research studies should qualify and quantify chemical mediators involved in the regulation nitric oxide role in order to better understand its physiopathology in the modulation of inflammatory granuloma.

Wolbachia is a bacterium observed in relationship with a wide array of arthropod and nematode species. This is an obligate intracellular symbiont, maternally transferred through the host oocytes. In arthropods Wolbachia is able to manipulate reproduction, using multiple strategies to increase the fitness of infected females. In nematodes the bacterium has a fundamental, and not completely understood, role in larvae development. Wolbachia infects ~50% of all the arthropod species worldwide, and in some of them it can be considered the most important sex determination factor. In contrast, Wolbachia presence is much more limited in nematodes, being present in a limited number of filarial species. The taxonomic status within the Wolbachia genus is highly debated, with the current classification dividing all strains in 14 'supergroups'. During my Ph.D. I studied the evolution of the symbiotic relationship between Wolbachia and its arthropod and nematode hosts, using genomic approaches. Indeed, during the evolution of the Wolbachia-host relationship, genetic signs have been left in the Wolbachia genomes. I worked to identify these genomic signs and to evaluate them within an evolutionary frame, in order to obtain a better understanding of how the Wolbachia-host symbiosis evolved. The work here presented can be organized in three major sections: i) the sequencing and analysis of the genome of the filarial nematode Dirofilaria immitis and of its symbiotic Wolbachia strain, wDi; ii) the sequencing of the genome of Wolbachia endosymbiont of Litomosoides sigmodontis, and the phylogenomic reconstruction of the Wolbachia supergroups A-D; iii) a comparison of the genomes of 26 Wolbachia strains spanning the A to F supergroups. Here a schematic summary of the results is reported: 1. Dirofilaria immitis and the Wolbachia symbiont wDi show metabolic complementarity for fundamental pathways 2. The metabolic pathway for the synthesis of wDi membrane proteins is one evolving the fastest in the genome of the bacterium 3. Nematode Wolbachia belonging to supergroups C and D are monophyletic, indicating that a single transition to mutualism likely occurred during the evolution of Wolbachia 4. Wolbachia strains of the C supergroup show genomic features that are unique in the genus, such as a much higher level of synteny compared to the rest of Wolbachia supergroups, and a newly generated pattern of GC skew curves, typically observed in free-living bacteria genomes 5. Wolbachia supergroups show conserved genomic features, which suggest genomic isolation among them.

The phylogenetic reconstruction has noted great development in recent decades. The development of computers and device for sequencing biopolymers have been an enormous amount od phylogenetic data from different sources and different types. The scientists are trying to reconstruct a comlet tree of life from these data. The phylogenetic supertree are theoretically this option because a supertree alow a combination of all information gathered so far – in contras to the phylogenetic trees. This thesis present the method of reconstruction supertrees using average konsensus method.

The Phylogenetic reconstruction has seen great development in the last 30 years. Computers have become more powerful and more generally accessible, and computer algorithms more sophisticated. It comes the effort of scientists to reconstruct the entire tree of life from a large amount of phylogenetic data. Just for this purpose are formed phylogenetic supertrees that allow the combination of all information gathered so far. The aim of this work is to find a method to construct supertree that will give correct results.

Phylogenetic diversity (PD) is a measure of species biodiversity quantified by how much of an evolutionary tree is spanned by a subset of species. In this thesis, we study optimization problems that aim to find species sets with maximum PD in different scenarios, and examine random extinction models under various assumptions to predict the PD of species that will still be present in the future. Optimizing PD with Dependencies is a combinatorial optimization problem in which species form an ecological network. Here, we are interested in selecting species sets of a given size that are ecologically viable and that maximize PD. The NP-hardness of this problem is proved and it is established which special cases of the problem are computationally easy and which are computationally hard. It is also shown that it is NP-complete to decide whether the feasible solution obtained by the greedy algorithm is optimal. We formulate the optimization problem as an integer linear program and find exact solutions to the largest food web currently in the empirical literature. In addition, we give a generalization of PD that can be used for example when we do not know the true evolutionary history. Based on this measure, an optimization problem is formulated. We discuss the complexity and the approximability properties of this problem. In the generalized field of bullets model (g-FOB), species are assumed to become extinct with possibly different probabilities, and extinction events are independent. We show that under this model the distribution of future phylogenetic diversity converges to a normal distribution as the number of species grows. When extinction probabilities are influenced by some binary character on the tree, the state-based field of bullets model (s-FOB) represents a more realistic picture. We compare the expected loss of PD under this model to that under the associated g-FOB model and find that the former is always greater than or equal to the latter. It is natural to further generalize the s-FOB model to allow more than one binary character to affect the extinction probabilities. The expected future PD obtained for the resulting trait-dependent field of bullets model (t-FOB) is compared to that for the associated g-FOB model and our previous result is generalized.

Tese de doutoramento apresentada à Faculdade de Ciências, Universidade do Porto
Os peixes anteriormente incluídos no género Chondrostoma (Cyprinidae: Leuciscinae) distribuem-se pelo Sul e Centro da Europa, desde o Oceano Atlântico até ao Mar Cáspio e desde o Mediterrâneo até ao Báltico. Estes peixes encontram-se ainda presentes na Ásia Menor, Cáucaso e Mesopotâmia. O número de espécies incluídas neste grupo tem variado de acordo com os autores, devido ao uso de diferentes critérios de diagnose (i.e. morfológicos e osteológicos ou moleculares). Os estudos anteriores sobre a filogenia molecular deste grupo tinham originado politomias não resolvidas, embora alguns ciados monofíléticos e estatisticamente robustos tenham sido identificados: toxostoma, lemmingii, polylepis, arcasii, nasus, soetta e genei. A presente tese propôs-se atingir um conjunto de objectivos numa multiplicidade de escalas. Numa escala macroevolutiva, propõs-se resolver com mais taxa e fragmentos de um número maior de genes a filogenia dos peixes tradicionalmente incluídos no género Chondrostoma. Ao mesmo tempo, procurou-se contribuir para a compreensão da diversificação deste grupo na Península Ibérica. Entre os principais resultados destacam-se os seguintes. Elaborou-se uma filogenia estatisticamente robusta dos peixes que constituíam o género Chondrostoma, que mostrou que as politomias anteriormente obtidas resultavam de amostragem insuficiente quanto ao número de taxa e/ou quantidade de fragmentos de ADN. O mapeamento dos caracteres morfológicos na filogenia obtida mostrou que caracteres anteriormente considerados como diagnósticos do género eram homoplásicos, pelo que se impôs uma revisão do género Chondrostoma. Desta revisão resultou a restrição do género Chondrostoma às espécies do grupo nasus e a criação dos novos géneros: Pseudochondrostoma, Parachondrostoma, Ibero chondrostoma, Áchondrostoma e Protochondrostoma (que correspondem respectivamente às linhagens polylepis, toxostoma, lemmingii, arcasii e genei). Os eventos ciado genéticos que deram origem à formação destas linhagens parecem ter tido lugar há cerca de 11 milhões de anos, excluindo portanto a hipótese da difusão e VII Resumo diversificação do grupo ter ocorrido durante a fase oligohalina do Mediterrâneo (Lago Maré) no fim do Messiniano. Fica assim excluída a aplicação para estes peixes do modelo de dispersão proposto por Bianco. Três dos seis géneros definidos neste trabalho são endémicos da Península Ibérica e um quarto género tem a maioria das espécies na península com uma pequena extensão em França. Estes dados e as cronologias estimadas para a diversificação dentro destes géneros sugerem que grande parte da radiação do grupo se deu na Península Ibérica muito antes do final do Miocénico parecendo, no que se refere a este grupo, que os contactos entre a península e o resto da Europa em períodos mais recentes foram muito reduzidos, limitando-se a trocas entre o nordeste de Espanha e a França. Na península os géneros Achondrostoma, Iberochondrostoma e Parachondrostoma têm distribuições disjuntas, mas adjacentes, que sugerem que processos de vicariância podem ter tido um papel fundamental na sua diferenciação. Pseudochondrostoma apresenta grande número de semelhanças (e.g. boca ínfera e dotada de estojo córneo, tamanho comparativamente grande, migrações pré-reprodutoras) com os géneros Parachondrostoma e Chondrostoma. Discute-se a possibilidade destas semelhanças resultarem de convergência ou de episódios de hibridação antigos. A análise fiíogenética do género Achondrostoma permitiu mostrar que este género inclui duas linhagens separadas já desde o Miocénico. A. arcasii revelou-se polifilético incluindo peixes das duas linhagens acima referidas, sendo urgente uma revisão da sua taxonomia. Esta informação fiíogenética combinada com dados morfológicos permitiu identificar uma nova espécie no sudoeste da área de distribuição do género Achondrostoma, endémica do distrito de Lisboa e considerada Criticamente Em Perigo {Achondrostoma occidentale). Por o nome se encontrar indisponível foi necessário renomear Achondrostoma macrolepidotum, para A. oligolepis. A análise da filogeografia profunda do género Iberochondrostoma levou à proposta de um modelo de especiação em que uma grande espécie central, Iberochondrostoma lemmingii, originou na sua periferia e em diferentes períodos geológicos, diversas espécies de distribuição mais restrita. Este modelo de especiação do tipo peripátrico, suportado por dados de ADN nuclear e mitocondrial, parece consistente com a história geológica da Península Ibérica no Terciário. Procedeu-se à análise filogeográfica das populações de uma das espécies deste género, /. lusitanicum, tendo-se identificado ESUs distintas que impõem a descrição, o mais breve VIII possível, de uma nova espécie e fornecem informações importantes para o delineamento de estratégias de conservação desta espécie Criticamente Em Perigo. O estudo do comportamento reprodutor de /. lusitanicum bem como o estudo do comportamento agonístico de Pseudochondrostoma polylepis forneceram elementos etológicos relevantes para a conservação destes ciprinídeos. Do mesmo modo, o estudo da expansão de Alburnus alburnus na Península ibérica, que se tem acelerado de forma muito acentuada nos últimos anos, traz igualmente informações importantes para o delineamento de estratégias de conservação dos ciprinídeos nativos, cujas potenciais interacções ecológicas com esta exótica se encontram totalmente inexploradas. O desenvolvimento deprimers que permitem amplificar eficazmente um fragmento de mais de 900 bases do gene nuclear da beta-actina foi fundamental em quase todos os estudos genéticos referidos acima. Para além de contribuir para os estudos filogenéticos e filo geográficos já referidos, permitiu esclarecer a natureza do ancestral paterno de Squalius albumoides uma espécie hibridogenética que resultou de cruzamentos entre fêmeas de Squalius pyrenaicus e machos filogeneticamente muito próximos, mas distintos, de Anaecypris hispânica. O facto de peixes dos géneros Alburnus e Squalius híbridarem facilmente e a proximidade filogenética entre Alburnus,, Anaecypris e uma das linhagens que integra S. albumoides leva a considerar com grande preocupação a expansão de Alburnus alburnus cujo grande potencial de hibridação com peixes do género Squalius é conhecido e que pode bibridar e descaracterizar várias espécies endémicas da península. Finalmente desenvolveu-se e validou-se um novo método que permite atribuir as diferentes bases presentes nos cromatogramas de ADN diplóide ou polipióide a cada uma das cadeias constitutivas tirando partido de artefactos da sequenciação induzidos na vizinhança de indels em heterozigotia. Este método é útil tanto na análise de múltiplos SNP's no mesmo fragmento, como na identificação das sequências de ADN presentes em híbridos e na distinção de vários típos de políplóides. ------ABSTRACT ------ Fishes formeríy included in the genus Chondrostoma (Cyprínidae: Leuciscinae) are distributed through South and Central Europe, from the Atlantic to the Caspian and from the Mediterranean to the Baltic. They are also found in Ásia Minor, the Caucasus and Mesopotamia. The number of species included in the group has varied according with the authors due to the use of different diagnostic críteria (í.e, morphological and osteological or molecular). Previous molecular phylogenetic studies of this group yielded unresolved polytomies although some monophyletic and statistically well supported clades were identifíed: toxostoma, lemmingii, polylepis, arcasii, nasus, soetta e genei, The present thesis aimed to achieve several objectives at various leveis. At a macroevolutionary scale this study attempted to solve the phyiogeny of the fish traditionally included ín Chondrostoma with a broader sample of taxa and more DNA fragments. At the same time the study aimed to clarify the diversification of this group in the Iberian Península. The main results can be summarized as follows. A statistically robust phyiogeny of the fish formeríy included in the genus Chondrostoma was obtained. The former polytomies were solved which indicates that they were likeiy due to insufficient taxon sampiing or scarcity of molecular data. Mapping of morphological characters on the inferred phyiogeny showed that several traits considered to be diagnostic of the genus were homoplasic. Thus a revison of the genus Chondrostoma was undertaken. This revision restricted the genus Chondrostoma to the nasus lineage and gave rise to the new genera Pseudochondrostoma, Par achondrostoma, Iberochondrostoma, Achondrostoma and Protochondrostoma (corresponding to the lineages polylepis, toxostoma, lemmingii, arcasii and genei, respectivefy). The cladogenetíc events that gave rise to these lineages seem to have occurred 11 million years ago, excluding the hypothesis of diffusion and diversification during the oligohaline Lago Maré phase of the Mediterranean, near the end of the Messinian. Thus the model of dispersai proposed by Bianco does not hold for this group of fish. XI Sitmmary Three of the six genera defmed in this work are endemic of the Iberian Península and another one has the majority of its species in the península, with a little extension to France. These data and the chronology estimated for the diversifícation inside these genera suggest that most of the radiation in this group occurred ín the Iberian Península long before the end of the Miocene. It seems that in this group the contacts between the península and the rest of Europe were, ín recent times, very scarce, and limited to connectíons between northeast Spain and France. In the Iberian Peninsula the genera Achondrostoma, Iberochondrostoma and Parachondrostoma have disjunct but adjacent distribution áreas which suggests that vicariant processes may have played a fundamenta! role in their differentiation. Pseudochondrostoma shows several resemblances with the genera Parachondrostoma and Chondrostoma (e.g. inferior mouth with a horny blade, comparably large size and pre-reproductive migrations) with the genera Parachondrostoma and Chondrostoma. The hypotheses of these resemblances being the result of convergent evolution or ancient hybridization events are discussed. The phylogenetic analysis of the genus Achondrostoma showed that this genus inciudes two lineages separated since the Miocene. A. arcasii is polyphyletic and includes físh from the two lineages referred above, thus a revision of its taxonomy is urgently needed. This phyíogenetic information, combined with morphological data, allowed the identification of a new species in the southwest of the distribution área of the genus Achondrostoma, Achondrostoma occidentale. This new species is endemic of the Lisbon district and it is considered Critically In Danger. As the name macrolepidotum was unavailable it was necessary to rename Achondrostoma macrolepidotum to A. oligolepis. The analysis of the deep phyiogeography of the genus Iberochondrostoma resulted in the proposal of a model of speciation in which a large central species, Iberochondrostoma lemmingii, originated in its períphery and at different geological times, diverse species with small distribution áreas. This peripatric speciation model is supported by mitochondrial and nuclear DNA and it seems consistent with the geological history of the Iberian Peninsula in the Tertiary. The phyíogeographic analysis of the populations of Iberochondrostoma lusitanicum aliowed the identification of distinct ESUs, which in turn impose the description of a new species, as soon as possible, as well as reveaiing importam informatíon to the conservation strategies of this Critically in Danger species. XII Summary The studies on the reproductive behaviour of / lusitanicum, and on the agonistic behaviour of Pseudochondrostoma polylepis revealed some ethologícal patterns relevant to the conservation of the species. It was also the case with the study on the expansíon of Albumus âlburnus in the Iberian Península, a species which has spread very quickly in recent years. The development of primers that allowed the amplifícation of a fragment of more than 900 base pairs of the nuclear beta actin gene was fundamental to almost ali genetic studies referred above. Beside its contribution to these studies, it allowed the recognition of the paternal ancestor of Squalius alburnoides an hybridogenetic species which resuited from crossings between S. pyrenaicus females and males phylogenetically very close to but distinct from Anaecypris hispânica. The fact that species of the genera Albumus and Squalius hybridize easily and the phylogenetic proximity between Alburnus, Anaecypris and one of the lineages that integrates S. alburnoides, brings great concerns about the expansion of Albumus âlburnus. The high potential of hybridization recorded between this last species and fishes of the genus Squalius is well known and may result in the genetic descaracterization of several species endemic to the Iberian Península. Finally, a new method was developed and validated, that allows the attribution of different bases present in the DNA chromatograms of diploid or polyploidy fishes to each one of the constitutive chains, taking advantage of artefacts of the sequencing process induced in the vicinity of heterozygous indels. This method is useful in the analysis of multiple SNP's in the same fragment, in the Identification of DNA sequences present in hybrids and in the distinction of several kinds of polyploids.

Philosophiae Doctor - PhD
The Ruschieae is the most diverse and speciose tribe within the large subfamily Ruschioideae (Aizoaceae), with approximately 71 genera and a distribution centred in the arid parts of the Greater Cape Floristic Region (GCFR) of South Africa. Recent phylogenetic analyses provided the first insights into generic relationships within the tribe, with a number of novel generic relationships discovered. The tribal phylogeny recovered 12 large clades, of which the Conophytum-clade was one the most morphologically diverse based on leaf and capsule characters. The Conophytum-clade is an early-diverging lineage of the Ruschieae and includes the following 10 genera: Cheiridopsis N.E.Br., Conophytum N.E.Br., Enarganthe N.E.Br., Ihlenfeldtia H.E.K.Hartmann, Jensenobotrya A.G.J.Herre, Namaquanthus L.Bolus, Octopoma N.E.Br., Odontophorus N.E.Br., Ruschianthus L.Bolus and Schlechteranthus Schwantes. The present study presents an expanded phylogenetic analysis of the Conophytum-clade, with the sampling of the majority of species in the genera and a representative sampling (56% of species) of the speciose genus Conophytum. Phylogenetic data for up to nine plastid gene regions (atpB–rbcL, matK, psbJ–petA, rpl16, rps16, trnD– trnT, trnL–F, trnQᶷᶷᶢ–rps16, trnS–trnG) were produced for each of the sampled species. The produced plastid data was analyses using maximum parsimony, maximum likelihood and Bayesian inference. The combined plastid phylogenetic analyses were used in combination with morphological, anatomical and palynological data to assess generic and subgeneric circumscriptions within the clade. Upon assessment of generic circumscriptions in the Conophytum-clade, the number of recognised genera in the clade decreased from ten to seven. Arenifera A.G.J.Herre, which had not been sampled in any phylogeny of the Ruschieae, and Octopoma were recovered as polyphyletic, with species placed in the Conophytum-clade, while the type species was placed in the xeromorphic clade of the tribal phylogeny. The species of Arenifera and Octopoma placed in the Conophytum-clade were subsequently included in Schlechteranthus upon assessment of generic circumscriptions between the taxa. Two morphological groupings were recognised within Schlechteranthus, one including the species of Schlechteranthus and the other including species previously recognised as Arenifera and Octopoma. These two morphological groupings were treated as subgenera, with the erection of the new subgenus Microphyllus R.F.Powell. A detailed taxonomic revision of subgenus Microphyllus is presented with a key to species, descriptions of the species (including a new species: S. parvus R.F.Powell & Klak), known geographical distributions and illustrations of the species. In addition to the changes mentioned above, the expanded sampling and phylogenetic analyses of the Conophytum-clade recovered Ihlenfeldtia and Odontophorus embedded in Cheiridopsis. The species of Ihlenfeldtia were recovered with species of heiridopsis subgenus Aequifoliae H.E.K.Hartmann, while the species of Odontophorus were recovered as polyphyletic within the Cheiridopsis subgenus Odontophoroides H.E.K.Hartmann clade. Cheiridopsis was subsequently expanded to include the species of Ihlenfeldtia and Odontophorus, with these species accommodated in the subgenera of Cheiridopsis. The phylogenetic placement and relationship of these species was supported by the shared capsule morphology. The expanded sampling of the clade did not resolve the phylogenetic relationship of the monotypic genera Enarganthe, Jensenobotrya, Namaquanthus and Ruschianthus, with these genera unresolved in the Conophytum-clade. These genera however, exhibit a unique combination of morphological characters, such as a glabrous leaf epidermis and variation in pollen exine and colpi structure, in contrast to the other genera of the clade. The assessment of the generic circumscription of these genera, based on the molecular, morphological, anatomical and palynological data suggested that the generic statuses of these monotypic genera should be maintained. The expanded phylogenetic sampling of the morphologically diverse and speciose genus Conophytum recovered the genus as monophyletic. This monophyly was supported by the unique floral type in Conophytum, with the fused petaloid staminodes forming a tube. None of the sectional classifications were recovered as monophyletic but the phylogenetic analyses did recover a few clades which more or less corresponded to the current sectional classification of the genus. A number of clades were also recovered which included species from a range of different sections. Diverse leaf and floral traits were shown to have evolved numerous times across the genus. This was particularly interesting with regards to the selected floral traits, as the phylogeny indicated a number of switches in floral morphologies across the genus. The floral diversity was assessed in complex species communities of Conophytum across the GCFR, where up to 11 species of Conophytum are found occurring sympatrically, and floral traits were shown to be different across the species within the communities. Pollination competition and adaptation were suggested as possible drivers of floral diversity in the genus, with differences in phenology, anthesis and floral morphology within the species complex communities. The unique floral type of Conophytum has enabled the species to develop a diverse range of specialised flowers, with a variety of structures, scents and colours, resulting in the diverse floral morphologies found across the genus. The complex Conophytum species communities included both closely as well as distantly related species, suggesting the soft papery capsules of Conophytum are wind dispersed. This adaptation to long distance seed dispersal resulted in a significantly higher phylogenetic diversity in Conophytum when compared to its sister genus, Cheiridopsis. A population genetics study of Conophytum also suggested that the capsules may be wind dispersed, with an indication of genetic connectivity between the geographically isolated populations of C. marginatum Lavis across the Bushmanland Inselberg Region. Although the capsules are dispersed by wind, the seeds are released from the hygrochastic capsules by runoff during rainfall events. The relationship between seed dispersal and runoff is evident from the genetic structure of populations of C. maughanii N.E.Br. and C. ratum S.A.Hammer that occur on the tops and the surrounding bases of the inselbergs, as the drainage pattern was found to directly influence population structure in these species. In addition, the AFLP analyses provided insight into the conservation of the flagship species C. ratum. The summit populations of this species were shown to sustain the populations at the base of the Gamsberg. This finding is especially important, as the distribution of the species is restricted to the Gamsberg inselberg, where mining has already commenced as of this year.
National Research Foundation (NRF)

In the light of for sustainable development, microalgal biodiesel, as a renewable and sustainable energy type, has enjoyed a surge in popularity. In fact, differently from the first generation biofuels, the use of microalgae to produce bioenergy does not involve the triggering of "food for fuel" competitivity and thus represents a sustainable mean to face significant concerns, such as wars and political instabilities deriving from oil reserves shortage. Morevoer, the high oil yields and less land use are the main advantages of microalgae. However, in order to make the current technology viable at the large-scale, several limitations should be overcome. In particular, biomass and lipid productivities should be further increased and all the downstream processes, from harvesting to lipid extraction, should be optimized. To these aims, high efforts involving high investments should be done in order to implement an intensive multidisciplinary research activity both at the laboratory and the industrial scale. The microalgae cultivation is the base of biofuel development and suitable genetic engineering strategies have to be developed in order to augment the microalgae oil content and their growth rate so that biofuels production could performed in a sustainable way. In particular, the creation of new microalgal strains intrinsically characterized by high lipid productivities as well as by a good tolerance to high CO2 levels is an ambitious goal which might be achieved only once their genome is known. The results presented in this thesis represent the first step needed to design a genetic approache which may eventually facilitate large-scale production of algae. The use of transgenic microalgae for the production of bioproducts represent an enormous economic and biotechnology promise, because algal production combines the simplicity and speed of haploid, single-cell genetics in an organism with elaborate biosynthetic potential, and with the associated economic benefit of using photosynthesis to drive product formation. As technology continues to be progressed and algae production industrialization continues to be improved, microalgae energy as the third generation biofuel will contribute their own strength to relieve the tense situation of resources. The contribution of the present work, to this general target can be briefly summarized as follows. The growth kinetics of C. sorokiniana has been investigated along with their corresponding lipid content, both batch and helical photobioreactors. The main results achieved during this activity are the knowledge of the effect of nitrogen concentration in solution on the growth rate and lipid content of C. sorokiniana. These informations represent the first step towards the development of a nitrogen based strategy for the optimization of lipid productivity of C. sorokiniana cultures. As far as the genetic characterization activity, the chloroplast and mitochondrial DNAs of two strains, i.e. C. sorokiniana and C. variabilis, respectively, have been sequenced for the first time in the literature. The obtained results allowed to perform a phylogenetic assessment involving different microalgae strains belonging to the Chlorella clade. Such results represent the first important step towards the development of genetic engineering strategies aimed to improve the current microalgae based systems for the production of biofuels and the capture of CO2.

The evolution of biological species depends on changes in genes. Among these changes are the gradual accumulation of DNA mutations, insertions and deletions, duplication of genes, movements of genes within and between chromosomes, gene losses and gene transfer. As two populations of the same species evolve independently, they will eventually become reproductively isolated and become two distinct species. The evolutionary history of a set of related species through the repeated occurrence of this speciation process can be represented as a tree-like structure, called a phylogenetic tree or a species tree. Since duplicated genes in a single species also independently accumulate point mutations, insertions and deletions, they drift apart in composition in the same way as genes in two related species. The divergence of all the genes descended from a single gene in an ancestral species can also be represented as a tree, a gene tree that takes into account both speciation and duplication events. In order to reconstruct the evolutionary history from the study of extant species, we use sets of similar genes, with relatively high degree of DNA similarity and usually with some functional resemblance, that appear to have been derived from a common ancestor. The degree of similarity among different instances of the “same gene” in different species can be used to explore their evolutionary history via the reconstruction of gene family histories, namely gene trees. Orthology refers specifically to the relationship between two genes that arose by a speciation event, recent or remote, rather than duplication. Comparing orthologous genes is essential to the correct reconstruction of species trees, so that detecting and identifying orthologous genes is an important problem, and a longstanding challenge, in comparative and evolutionary genomics as well as phylogenetics. A variety of orthology detection methods have been devised in recent years. Although many of these methods are dependent on generating gene and/or species trees, it has been shown that orthology can be estimated at acceptable levels of accuracy without having to infer gene trees and/or reconciling gene trees with species trees. Therefore, there is good reason to look at the connection of trees and orthology from a different angle: How much information about the gene tree, the species tree, and their reconciliation is already contained in the orthology relation among genes? Intriguingly, a solution to the first part of this question has already been given by Boecker and Dress [Boecker and Dress, 1998] in a different context. In particular, they completely characterized certain maps which they called symbolic ultrametrics. Semple and Steel [Semple and Steel, 2003] then presented an algorithm that can be used to reconstruct a phylogenetic tree from any given symbolic ultrametric. In this thesis we investigate a new characterization of orthology relations, based on symbolic ultramterics for recovering the gene tree. According to Fitch’s definition [Fitch, 2000], two genes are (co-)orthologous if their last common ancestor in the gene tree represents a speciation event. On the other hand, when their last common ancestor is a duplication event, the genes are paralogs. The orthology relation on a set of genes is therefore determined by the gene tree and an “event labeling” that identifies each interior vertex of that tree as either a duplication or a speciation event. In the context of analyzing orthology data, the problem of reconciling event-labeled gene trees with a species tree appears as a variant of the reconciliation problem where genes trees have no labels in their internal vertices. When reconciling a gene tree with a species tree, it can be assumed that the species tree is correct or, in the case of a unknown species tree, it can be inferred. Therefore it is crucial to know for a given gene tree whether there even exists a species tree. In this thesis we characterize event-labelled gene trees for which a species tree exists and species trees to which event-labelled gene trees can be mapped. Reconciliation methods are not always the best options for detecting orthology. A fundamental problem is that, aside from multicellular eukaryotes, evolution does not seem to have conformed to the descent-with-modification model that gives rise to tree-like phylogenies. Examples include many cases of prokaryotes and viruses whose evolution involved horizontal gene transfer. To treat the problem of distinguishing orthology and paralogy within a more general framework, graph-based methods have been proposed to detect and differentiate among evolutionary relationships of genes in those organisms. In this work we introduce a measure of orthology that can be used to test graph-based methods and reconciliation methods that detect orthology. Using these results a new algorithm BOTTOM-UP to determine whether a map from the set of vertices of a tree to a set of events is a symbolic ultrametric or not is devised. Additioanlly, a simulation environment designed to generate large gene families with complex duplication histories on which reconstruction algorithms can be tested and software tools can be benchmarked is presented.

Rapidly evolving RNA and ssDNA viruses represent a fascinating field characterized by a strict interconnection between the “speculative” study of viral evolution and its practical implications in everyday veterinary medicine. This thesis has been thought as a collection of manuscripts that aimed to investigate different aspects and levels of viral evolution while still maintaining a focus on practical repercussions. Even if different infectious diseases and etiological agents are considered, they are all functional to the study of different aspects and implications of rapid virus evolution. Considering the heterogeneous nature of the manuscripts, they have been organized according to a “crescent” scale, starting from the lowest scale of viral evolution and progressing to broader scales. The manuscript “Viral subpopulations in aMPV vaccines are unlikely to be responsible for reversion to virulence.” addresses a fine-level analysis of the population structure of the AMPV subtype B live attenuated vaccine and its potential role in the previously demonstrated phenomenon of reversion to virulence. The widespread administration of live attenuated vaccines, despite their obvious advantages in terms of reducing disease prevalence, clinical signs and economic losses, is associated with costs that are not limited to the risk of reversion to virulence or to direct economic costs. Based on a wide collection of Italian samples, “Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype’s disappearance” reports the impact of these vaccines in complicating the diagnostic process and, as a consequence, the interpretation of the epidemiological scenario in the absence of known vaccine markers. Obviously, updated knowledge of the strains currently circulating in a particular area is of great relevance for the implementation of proper control strategies. With this aim in mind, a field survey, which is published in “Field survey of Avian Metapneumovirus in Northern Italy”, was conducted on hundreds of Italian farms to estimate and characterize the AMPV strains circulating in our country. To further support frequent and extensive surveys, an assay that is able to detect, quantify and genotype the two AMPV subtypes currently circulating in Italy was developed and validated. Because economic constraints often represent a major limit, efforts were made to reduce the assay costs compared with other real-time RT-PCR methods while still guaranteeing comparable or superior performances (“A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B”). Unfortunately, the diagnosis of rapidly evolving RNA viruses is itself an arduous task that requires a continuous evaluation and updating of diagnostic tools, even in laboratory that receive samples from limited geographic areas. The manuscripts entitled “Observation of high recombination occurrence of Porcine reproductive and respiratory syndrome virus in field condition” and “Phylodynamic analysis of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy: action of selective pressures and interactions between different clades.” address the study of the molecular epidemiology of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy considering the evolutionary forces driving PRRSV evolution at the local scale (i.e., high substitution rate, recombination, interaction between different clades and action of selective pressures). The high heterogeneity of PRRSV in the national contest was then evaluated with respect to the challenges that it poses in the development and validation of RT-PCR- and qRT-PCR-based diagnostic methods (“Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus”) and assessing its impact on diagnostic accuracy (“The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances”). Similarly, “International trades, local spread and viral evolution: the case of Porcine circovirus type 2 (PCV2) strains heterogeneity in Italy” investigates the genetic variability of PCV2 within national borders and compares it with the knowledge of its molecular epidemiology available from other countries. This study provides evidence regarding the role of both “in loco” evolution and importation of different genotypes and strains from foreign countries in determining the Italian PCV2 genetic heterogeneity. The crescent amount of PCV2 sequences deposited in publically available databases has revealed its marked variability and challenged the current classification criteria. Nevertheless, at least a superficial knowledge of the PCV2 molecular epidemiology is mandatory for the planning and evaluation of control strategies. “Revisiting the Taxonomical classification of PCV2: still a real challenge” proposes new criteria for the classification of PCV2 into different genotypes. Our aim was to provide a scheme that both accounts for the constraint imposed by the biological proprieties of this virus and allows a rapid, practical and easy way to classify PCV2 strains even during routine diagnostic activity. Last, “Genetic characterisation of porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: an opportunity to reconstruct the history of PCV2 evolution” investigates more speculative issues inherent to the PCV2 origin. The discovery of a PCV2c genotype, which, to date, was believed to be extinct, in a feral pig population characterized by a peculiar population history and by a complex, and still partially known, relationship network with other PCV2-susceptible species opens exciting scenarios concerning the history and origin of PCV2
I virus a RNA e a ssDNA rappresentano un affascinante campo di ricerca caratterizzato da una stretta interconnessione fra lo studio di aspetti più “speculativi”, inerenti all’evoluzione virale, e le implicazioni che questi comportano nella pratica veterinaria. La presente tesi è stata concepita come una raccolta di articoli che hanno esplorato diversi aspetti e livelli dell’evoluzione virale senza per questo trascurarne le ripercussioni pratiche. Diverse malattie infettive ed agenti eziologici sono stati selezionati onde investigare i diversi aspetti e implicazioni della rapida evoluzione di questi virus. In considerazione della natura eterogena dei manoscritti prodotti, questi sono stati organizzati secondo una “scala crescente”, dal livello più basso dell’evoluzione virale, procedendo verso scale via via maggiori. L’elaborato “Viral subpopulations in aMPV vaccines are unlikely to be responsible for reversion to virulence” affronta un analisi ad alta risoluzione della presenza di sottopopolazioni virali nei vaccini vivi attenuati basati sul sottotipo B del metapneumovirus aviare e del loro potenziale ruolo nel determinare fenomeni di reversione a virulenza. La somministrazione su vasta scala di vaccini vivi attenuati, nonostante gli ovvi vantaggi in termini di riduzione della prevalenza virale, dei segni clinici e delle perdite economiche, è associata a costi che non sono limitati a quelli di tipo meramente pecuniario. Sulla base di un ampia raccolta di campioni italiani, l’articolo “Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype’s disappearance” descrive l’impatto di questi vaccini, in assenza di marker vaccinali noti, nel complicare il processo diagnostico e, conseguentemente, l’interpretazione dello scenario epidemiologico. Ovviamente, una conoscenza aggiornata degli stipiti virali circolanti in una particolare area è di fondamentale importanza per l’implementazione di adeguate misure di controllo. Con quest’obiettivo in mente, uno studio di campo , pubblicato in “Diffusione dell’infezione da metapneumovirus aviare in allevamenti di tacchini e broiler nel Nord Italia”, è stato condotto su centinaia di allevamenti al fine di stimare e caratterizzare i ceppi di AMPV circolanti nel nostro territorio. In aggiunta, al fine di favorire studi sempre più frequenti e estesi, è stato sviluppato un test diagnostico in grado di rilevare, quantificare e tipizzare i sottotipi di AMPV attualmente circolanti in Italia. In considerazione del fatto che le spese relative a questi test rappresentano spesso uno dei maggiori limiti all’attività diagnostica e di ricerca, si è cercato di ridurre i costi rispetto ad altre metodiche comunemente utilizzate, garantendo nel contempo le medesime o migliori performance diagnostiche(“A Sensitive, Reproducible, and Economic Real-Time Reverse Transcription PCR Detecting Avian Metapneumovirus Subtypes A and B”). Sfortunatamente la diagnosi dei virus a RNA, essendo caratterizzati da una rapida evoluzione, rappresenta di per se stessa un ardua sfida e richiede una continua dedizione alla rivalutazione e aggiornamento delle metodiche diagnostiche esistenti. Gli articoli Observation of high recombination occurrence of Porcine reproductive and respiratory syndrome virus in field condition” e “Phylodynamic analysis of Porcine reproductive and respiratory syndrome virus (PRRSV) in Italy: action of selective pressures and interactions between different clades.” sono incentrati sullo studio dell’epidemiologia molecolare del Porcine reproductive and respiratory syndrome virus (PRRSV) in Italia. In particolare sono state studiate le forze evolutive che ne modellano e condizionano l’evoluzione (i.e., alto tasso di sostituzione nucleotidica, interazione fra differenti clade e azione di diverse pressioni selettive). L’elevata eterogeneità genetica di PRRSV nel nostro territorio è stata poi analizzata alla luce delle sue ripercussioni nello sviluppo e validazione di metodiche diagnostiche basate sull’uso della RT-PCR e qRT-PCR (“Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus”) e ne è stato valutato l’impatto sull’accuratezza diagnostica (“The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances”). Similmente, “International trades, local spread and viral evolution: the case of Porcine circovirus type 2 (PCV2) strains heterogeneity in Italy”, indaga la variabilità genetica di PCV2 all’interno dei confine nazionali comparandola altresì con le informazioni di epidemiologia molecolare disponibili per altri Stati. Il presente studio ha permesso di fornire significative evidenze sul ruolo sia dei commerci internazionali che dell’evoluzione “in loco” nel determinare l’eterogeneità di PCV2 riscontrata in Italia. Il progressivo aumento delle sequenza di PCV2 depositate in database pubblici ne ha dimostrato la grande variabilità su scala mondiale e ha evidenziato i limiti dei criteri di classificazione attualmente in uso. Tuttavia, una conoscenza quantomeno superficiale dell’epidemiologia molecolare di PCV2 è di basilare importanza per la pianificazione e la valutazione delle strategie di controllo. “Revisiting the Taxonomical classification of PCV2: still a real challenge” propone dei nuovi criteri per la classificazione di questo virus in diversi genotipi. Il nostro intento è stato quello di fornire uno schema che, pur tenendo conto dei limiti imposti dalle caratteristiche biologiche del virus, permettesse una rapida, pratica e facile genotipizzazione dei ceppi di PCV2 e che potesse quindi rispondere alle esigenze imposte dalla routinaria l’attività diagnostica. Infine, “Genetic characterisation of porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: an opportunity to reconstruct the history of PCV2 evolution”, si confronta con il campo, più specualtivo, dell’origine di PCV2. La scoperta di un ceppo appartenente al genotipo PCV2c, sino ad oggi ritenuto estinto, in una popolazione di suini selvatici caratterizzata da una peculiare storia e da complesse, a ancora solo parzialmente conosciute, relazioni con altre specie suscettibili a questo patogeno, apre nuovi ed eccitanti scenari concernenti la storia e l’origine di PCV2

Background Identifying orthologous molecular markers that potentially resolve relationships at and below species level has been a major challenge in molecular phylogenetics over the past decade. Non-coding regions of nuclear low- or single-copy markers are a vast and promising source of data providing information for shallow-scale phylogenetics. Taking advantage of public transcriptome data from the One Thousand Plant Project (1KP), we developed a genome-scale mining strategy for recovering potentially orthologous single-copy markers to address low-scale phylogenetics. Our marker design targeted the amplification of intron-rich nuclear single-copy regions from genomic DNA. As a case study we used Hydrangea section Cornidia, one of the most recently diverged lineages within Hydrangeaceae (Cornales), for comparing the performance of three of these nuclear markers to other "fast" evolving plastid markers. Results Our data mining and filtering process retrieved 73 putative nuclear single-copy genes which are potentially useful for resolving phylogenetic relationships at a range of divergence depths within Cornales. The three assessed nuclear markers showed considerably more phylogenetic signal for shallow evolutionary depths than conventional plastid markers. Phylogenetic signal in plastid markers increased less markedly towards deeper evolutionary divergences. Potential phylogenetic noise introduced by nuclear markers was lower than their respective phylogenetic signal across all evolutionary depths. In contrast, plastid markers showed higher probabilities for introducing phylogenetic noise than signal at the deepest evolutionary divergences within the tribe Hydrangeeae (Hydrangeaceae). Conclusions While nuclear single-copy markers are highly informative for shallow evolutionary depths without introducing phylogenetic noise, plastid markers might be more appropriate for resolving deeper-level divergences such as the backbone relationships of the Hydrangeaceae family and deeper, at which non-coding parts of nuclear markers could potentially introduce noise due to elevated rates of evolution. The herein developed and demonstrated transcriptome based mining strategy has a great potential for the design of novel and highly informative nuclear markers for a range of plant groups and evolutionary scales.

Background Identifying orthologous molecular markers that potentially resolve relationships at and below species level has been a major challenge in molecular phylogenetics over the past decade. Non-coding regions of nuclear low- or single-copy markers are a vast and promising source of data providing information for shallow-scale phylogenetics. Taking advantage of public transcriptome data from the One Thousand Plant Project (1KP), we developed a genome-scale mining strategy for recovering potentially orthologous single-copy markers to address low-scale phylogenetics. Our marker design targeted the amplification of intron-rich nuclear single-copy regions from genomic DNA. As a case study we used Hydrangea section Cornidia, one of the most recently diverged lineages within Hydrangeaceae (Cornales), for comparing the performance of three of these nuclear markers to other 'fast' evolving plastid markers. Results Our data mining and filtering process retrieved 73 putative nuclear single-copy genes which are potentially useful for resolving phylogenetic relationships at a range of divergence depths within Cornales. The three assessed nuclear markers showed considerably more phylogenetic signal for shallow evolutionary depths than conventional plastid markers. Phylogenetic signal in plastid markers increased less markedly towards deeper evolutionary divergences. Potential phylogenetic noise introduced by nuclear markers was lower than their respective phylogenetic signal across all evolutionary depths. In contrast, plastid markers showed higher probabilities for introducing phylogenetic noise than signal at the deepest evolutionary divergences within the tribe Hydrangeeae (Hydrangeaceae). Conclusions While nuclear single-copy markers are highly informative for shallow evolutionary depths without introducing phylogenetic noise, plastid markers might be more appropriate for resolving deeper-level divergences such as the backbone relationships of the Hydrangeaceae family and deeper, at which non-coding parts of nuclear markers could potentially introduce noise due to elevated rates of evolution. The herein developed and demonstrated transcriptome based mining strategy has a great potential for the design of novel and highly informative nuclear markers for a range of plant groups and evolutionary scales.

Projecting environmental niche models through time is a common goal when studying species response to climatic change. Species distribution models (SDMs) are commonly used to estimate a species' niche from observed patterns of occurrence and environmental predictors. However, a species niche is also shaped by non-environmental factors--including biotic interactions and dispersal barrier—truncating SDM estimates. Though truncated SDMs may accurately predict present-day species niche, projections through time are often biased by environmental condition change. Modeling niche in a phylogenetic framework leverages a clade's shared evolutionary history to pull species estimates closer towards phylogenetic conserved values and farther away from species specific biases. We propose a new Bayesian model of phylogenetic niche implemented in R. Under our model, species SDM parameters are transformed into biologically interpretable continuous parameters of environmental niche optimum, breadth, and tolerance evolving under multivariate Brownian motion random walk. Through simulation analyses, we demonstrated model accuracy and precision that improved as phylogeny size increased. We also demonstrated our model on a clade of eastern United States Plethodontid salamanders by accurately estimating species niche, even when no occurrence data is present. Our model demonstrates a novel framework where niche changes can be studied forwards and backwards through time to understand ancestral ranges, patterns of environmental specialization, and niche in data deficient species.
Master of Science
As many species face increasing pressure in a changing climate, it is crucial to understand the set of environmental conditions that shape species' ranges--known as the environmental niche--to guide conservation and land management practices. Species distribution models (SDMs) are common tools that are used to model species' environmental niche. These models treat a species' probability of occurrence as a function of environmental conditions. SDM niche estimates can predict a species' range given climate data, paleoclimate, or projections of future climate change to estimate species range shifts from the past to the future. However, SDM estimates are often biased by non-environmental factors shaping a species' range including competitive divergence or dispersal barriers. Biased SDM estimates can result in range predictions that get worse as we extrapolate beyond the observed climatic conditions. One way to overcome these biases is by leveraging the shared evolutionary history amongst related species to "fill in the gaps". Species that are more closely phylogenetically related often have more similar or "conserved" environmental niches. By estimating environmental niche over all species in a clade jointly, we can leverage niche conservatism to produce more biologically realistic estimates of niche. However, currently a methodological gap exists between SDMs estimates and macroevolutionary models, prohibiting them from being estimated jointly. We propose a novel model of evolutionary niche called PhyNE (Phylogenetic Niche Evolution), where biologically realistic environmental niches are fit across a set of species with occurrence data, while simultaneously fitting and leveraging a model of evolution across a portion of the tree of life. We evaluated model accuracy, bias, and precision through simulation analyses. Accuracy and precision increased with larger phylogeny size and effectively estimated model parameters. We then applied PhyNE to Plethodontid salamanders from Eastern North America. This ecologically-important and diverse group of lungless salamanders require cold and wet conditions and have distributions that are strongly affected by climatic conditions. Species within the family vary greatly in distribution, with some species being wide ranging generalists, while others are hyper-endemics that inhabit specific mountains in the Southern Appalachians with restricted thermal and hydric conditions. We fit PhyNE to occurrence data for these species and their associated average annual precipitation and temperature data. We identified no correlations between species environmental preference and specialization. Pattern of preference and specialization varied among Plethodontid species groups, with more aquatic species possessing a broader environmental niche, likely due to the aquatic microclimate facilitating occurrence in a wider range of conditions. We demonstrated the effectiveness of PhyNE's evolutionarily-informed estimates of environmental niche, even when species' occurrence data is limited or even absent. PhyNE establishes a proof-of-concept framework for a new class of approaches for studying niche evolution, including improved methods for estimating niche for data-deficient species, historical reconstructions, future predictions under climate change, and evaluation of niche evolutionary processes across the tree of life. Our approach establishes a framework for leveraging the rapidly growing availability of biodiversity data and molecular phylogenies to make robust eco-evolutionary predictions and assessments of species' niche and distributions in a rapidly changing world.

Phylogenetic analysis is a central tool in studies of comparative genomics. When a new region of DNA is isolated and sequenced, researchers are often forced to throw away months of computation on an existing phylogeny of homologous sequences in order to incorporate this new sequence. The previously constructed trees are often discarded, and the researcher begins the search again from scratch. The jumpstarting algorithm uses trees from the prior search as a starting point for a new phylogenetic search. This technique drastically decreases search time for large data sets. This kind of analysis is necessary as researchers analyze tree of life size data sets.

In phylogenetic analysis one study the relationship between different species. By comparing DNA from two different species it is possible to get a numerical value representing the difference between the species. For a set of species, all pair-wise comparisons result in a dissimilarity matrix d.

In this thesis I present a few methods for constructing a phylogenetic tree from d. The common denominator for these methods is that they do not generate a tree, but instead give a connected graph. The resulting graph will be a tree, in areas where the data perfectly matches a tree. When d does not perfectly match a tree, the resulting graph will instead show the different possible topologies, and how strong support they have from the data.

Finally I have tested the methods both on real measured data and constructed test cases.

Evolutionary relationships are key to modern understanding of biological systems. Phylogenetic search is the means by which these relationships are inferred. Phylogenetic search is NP-Hard. As such it is necessary to employ heuristic methods. This work proposes new methods based on viewing the relationships between species as sets of partitions. These methods produce more parsimonious phylogenies than current methods.

One of the restrictions used in all of the works done on phylogenetic invariants for group based models has been that the group be abelian. In my thesis, I aim to generalize the method of invariants for group-based models of DNA sequence evolution to include nonabelian groups. By using a nonabelian group to act one the nucleotides, one could capture the structure of the symmetric model for DNA sequence evolution. If successful, this line of research would unify the two separated strands of active research in the area today: Allman and Rhodes’s invariants for the symmetric model and Strumfels and Sullivant’s toric ideals of phylogenetic invariants. Furthermore, I want to look at the statistical properties of polynomial invariants to get a better understanding of how they behave when used with real, “noisy” data.

We investigate the usability of the Phylogenetic Likelihood Library (PLL) in Bayesian phylogenetic tree inference using Sequential Monte Carlo (SMC) algorithms. This is done by implementing two different versions of the same algorithm with two different approaches of the use of PLL. The implementation using the main PLL API encountered performance issues that the lower level implementation did not. We conclude that it is possible to use PLL in SMC methods but it is unclear if the main API is suitable.
Vi undersöker om programspråksbiblioteket Phylogenetic Likelihood Library (PLL) kan användas för bayesiansk inferens av phylogenetiska träd med en sekventiell Monte Carlo-metod (SMC). Genom att implementera algoritmen med två olika delar av PLL:s programmeringsgränssnitt visar vi att det går att använda PLL för att implementera SMC-algoritmen men att det är oklart om det huvudsakliga programmeringsgränssnittet är lämpligt.

Phylogenetics is the study of evolutionary relationships between species. Phylogenetic trees have long been the standard object used in evolutionary biology to illustrate how a given set of species are related. There are some groups (including certain plant and fish species) for which the ancestral history contains reticulation events, caused by processes that include hybridization, lateral gene transfer, and recombination. For such groups of species, it is appropriate to represent their ancestral history by phylogenetic networks: rooted acyclic digraphs, where arcs represent lines of genetic inheritance and vertices of in-degree at least two represent reticulation events. This thesis is concerned with the efficiency, accuracy, and tractability of mathematical models for phylogenetic network methods. Three important and related measures for summarizing the dissimilarity in phylogenetic trees are the minimum number of hybridization events required to fit two phylogenetic trees onto a single phylogenetic network (the hybridization number), the (rooted) subtree prune and regraft distance (the rSPR distance) and the tree bisection and reconnection distance (the TBR distance) between two phylogenetic trees. The respective problems of computing these measures are known to be NP-hard, but also fixed-parameter tractable in their respective natural parameters. This means that, while they are hard to compute in general, for cases in which a parameter (here the hybridization number and rSPR/TBR distance, respectively) is small, the problem can be solved efficiently even for large input trees. Here, we present new analyses showing that the use of the “cluster reduction” rule – already defined for the hybridization number and the rSPR distance and introduced here for the TBR distance – can transform any O(f(p) · n)-time algorithm for any of these problems into an O(f(k) · n)-time one, where n is the number of leaves of the phylogenetic trees, p is the natural parameter and k is a much stronger (that is, smaller) parameter: the minimum level of a phylogenetic network displaying both trees. These results appear in [9]. Traditional “distance based methods” reconstruct a phylogenetic tree from a matrix of pairwise distances between taxa. A phylogenetic network is a generalization of a phylogenetic tree that can describe evolutionary events such as reticulation and hybridization that are not tree-like. Although evolution has been known to be more accurately modelled by a network than a tree for some time, only recently have efforts been made to directly reconstruct a phylogenetic network from sequence data, as opposed to reconstructing several trees first and then trying to combine them into a single coherent network. In this work, we present a generalisation of the UPGMA algorithm for ultrametric tree reconstruction which can accurately reconstruct ultrametric tree-child networks from the set of distinct distances between each pair of taxa. This result will also appear in [15]. Moreover, we analyse the safety radius of the NETWORKUPGMA algorithm and show that it has safety radius 1/2. This means that if we can obtain accurate estimates of the set of distances between each pair of taxa in an ultrametric tree-child network, then NETWORKUPGMA correctly reconstructs the true network.

This thesis introduces three new tools for studying the evolution of different organisms given the evolutionary, or phylogenetic, tree that relates them. First, we show how posterior state probabilities can be used for exploring phylogenetic uncertainty, through posterior entropy of ancestral states. Second, we derive an explicit formula for the expected number of substitutions on a branch in a phylogeny, given the pattern at the leaves. Algorithms were implemented, as part of the ATV software, to calculate these values. They are used, in conjunction with SplitsTree4, to assess variation in rates over sites and lineages, and to predict model violations. Third, we show how techniques for spline interpolation and function approximation can be applied to estimate functions defined on a tree. We also present an example of the usage of interpolations on phylogenetic trees, by analyzing continuous traits on a phylogenetic tree through fossil data sets.

In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome. Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system. Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition. My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.

Simulation remains a very important approach to testing the robustness and accuracy of phylogenetic inference methods. However, current simulation programs are limited, especially concerning realistic models for simulating insertions and deletions (indels). In this thesis I implement a new, portable and flexible application, named INDELible, which can be used to generate nucleotide, amino acid and codon sequence data by simulating indels (under several models of indel length distribution) as well as substitutions (under a rich repertoire of substitution models). In particular, I introduce a simulation study that makes use of one of INDELible’s many unique features to simulate data with indels under codon models that allow the nonsynonymous/synonymous substitution rate ratio to vary among sites and branches. This data is used to quantify, for the first time, the precise effects of indels and alignment errors on the false-positive rate and power of the widely used branch-site test of positive selection. Several alignment programs are used and assessed in this context. Through the simulation experiment, I show that insertions and deletions do not cause the test to generate excessive false positives if the alignment is correct, but alignment errors can lead to unacceptably high false positives. Previous selection studies that use inferior alignment programs are revisited to demonstrate the applicability of my results in real world situations. Further work uses simulated data from INDELible to examine the effects of tree-shape and branch length on the alignment accuracy of several alignment programs, and the impact of alignment errors on different methods of phylogeny reconstruction. In particular, analysis is performed to explore which programs avoid generating the kind of alignment errors that are most detrimental to the process of phylogeny reconstruction.

In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.

The order Thysanoptera (Insecta: Paraneoptera), commonly known as thrips, includes organisms that exhibit a wide range of social and feeding behaviors that are of particular interest in evolutionary studies. These studies within thrips have been inhibited by the lack of knowledge of thrips relationships. The recognized classification scheme strives to reflect evolutionary relationships and is based upon morphology. Molecular data is next as morphology alone is not enough to resolve relationships. Few molecular studies have been conducted and all were limited in their taxon sampling and genetic sampling. To provide a foundation of future evolutionary studies, the objectives of this study are to (1) test the monophyly of the suborders Terebrantia and Tubulifera, (2) test the monophyly of the families and decipher their relationships, and (3) test the monophyly of the recognized subfamilies. Phylogenies were reconstructed based upon 5299 bp, from five genetic loci: 18S ribosomal DNA, 28S ribosomal DNA, Histone 3 (H3), Tubulin-alpha (TubA) and cytochrome oxidase c subunit I (COI). 99 thrips species from seven of the nine families, all six subfamilies and 70 genera were sequenced. Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian analysis all strongly support a monophyletic Tubulifera and Terebrantia. Phlaeothripidae, Aeolothripidae, Melanthripidae and Thripidae are all monophyletic families. The relationship between Aeolothripidae and Merothripidae to the rest of Terebrantia is equivocal. Morphological and molecular data suggest Aeolothripidae or Merothripidae could be the basal lineage of Terebrantia. Four of the six subfamilies are recovered as monophyletic. The two largest subfamilies, Phlaeothripinae and Thripinae, are paraphyletic and require further study to understand relationships within them.

The boletes are macrofungi which have undergone extensive taxonomic revisions since the advent of molecular tools. To further our understanding of the boletes in peninsular Florida, we sequenced two common Floridian boletes, and analyzed them with molecular phylogenetic tools. Boletus rubricitrinus, a common Florida bolete often found in lawns under Quercus, and likely has a distribution that extends to Texas. Based on ITS and LSU sequences and morphological studies, this species belongs in the genus Pulchroboletus. As the holotype is in poor condition, an epitype is established here. A thorough description of macroscopic and microscopic features is also provided for the species. Fungi in the genus Phylloporus are lamellate boletes that occur worldwide, but primarily in the tropics. Phylloporus boletinoides is a species which was described from Florida, and is found growing near Pinus spp. Based on ITS, LSU, and RPB1 sequences, we establish the novel genus Pseudophylloporus, which is allied to Bothia and Solioccasus. Morphological data are also provided from our collections, and one from Belize. Based on molecular data and a review of bolete literature, the delimitation of this genus suggests that there are three distinct lineages of boletes that have a lamellate hymenium in the Boletaceae. These molecular and morphological data will be useful to further improve our understanding of bolete taxonomy.