Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Knowledge of the diversity and evolutionary histories of insects in South Africa’s fynbos biome lags far behind what is known of the plant groups that make up this global biodiversity hotspot. To address this imbalance, I undertook a molecular phylogenetic study of fynbos-endemic Cephalelini leafhoppers that specialise on restios in the family Restionaceae. My phylogenetic results did not recover the South African Cephalelini as monophyletic, nor did I find monophyly of described species, but several monophyletic clades of species were found within Cephalelini. Furthermore, phylogenetic dating suggested that the divergence between South African and Australian Cephalelini post-dates Gondwanan vicariance, implying intercontinental extreme long distance dispersal of these insects ca. 5-11 MYA. Diversification within the Cephalelini is also much more recent than that of the Restionaceae hosts on which they specialise, negating the possibility of coevolution between plants and insects. Rather, analysis of phylogenetic conservatism of host use reveals that Cephalelini evolution has tracked the evolution of their Restionaceae hosts and that closely related insects feed on the same plant host tribes. A finer scale of tracking of host evolution (such as at the clade or genus level) is expected when taking into account how highly specific I find Cephalelini host use to be, but its absence might be explained by the recent divergence of Cephalelini relative to the age of Restionaceae. Analysis of conservatism of host use was also carried out using a phylogeny of the Restionaceae, and revealed that, overall, Cephalelini host use and avoidance have no phylogenetic bias, indicating many empty potential niches for Cephalelini, or alternately that host use is governed by factors which are phylogenetically unconstrained. Lastly, I also analysed the evolution of specialisation of Cephalelini and find no trend towards increased specialisation within the group, which is contrary to what is expected of the evolution of herbivorous insects. Overall, this study presents the first evidence of intercontinental dispersal of insect fauna between South Africa and Australia and as such highlights an unconsidered factor in the accumulation of faunal diversity in the fynbos biome. I find Cephalelini to be highly specialised in their host preference, but this pattern only becomes apparent at the tribal host level in the evolution of Cephalelini. Although Cephalelini are highly specialised, I find no evidence of evolution towards increasing specialisation within the group. / AFRIKAANSE OPSOMMING: Ons kennis van die evolusie van fynbosinsekte is redelik beperk vergeleke met hoe veel ons weet van die plante wat die merkwaardige diversiteit van die fynbos-bioom uitmaak. In `n poging om hierdie wanbalans reg te stel het ek `n molekulêr-evolusionêre studie onderneem van die fynbos-endemiese blaarspringer-groep Cephalelini, wat op Restionaceae-gasheerplante spesialiseer. Ons het drie geen-areas geamplifiseer vir filogeniekonstruksie en dateringsdoeleindes: insek-kern H3, insek- mitokondriaal COI en insek-simbiont Sulcia 16S. Met behulp van filogenetiese analise is bevind dat die Suid-Afrikaanse Cephalelini nie `n monofiletiese groep is nie en dat beskryfde spesies ook nie monofileties is nie, maar verskeie monofiletiese spesie-groepe is wel gevind. `n Oorkruis-gevalideerde dateringsoefening dui aan dat divergensie tussen Suid-Afrikaanse en Australiaanse Cephalelini meer onlangs as die verbrokkeling van Gondwana plaasgevind het, wat impliseer dat daar uitruiling van insekte tussen die kontinente oor die afgelope 5-11 MJ plaasgevind het. Die diversiteit van Cephalelini het ook veel meer onlangs ontstaan as dié van hulle Restionacaea-gashere wat beteken dat ko-evolusie in die eng sin nie moontlik is nie. ‘n Ontleding van die evolusie van gasheer-keuse deur Cephalelini dui wel aan dat Cephalelini-evolusie die patron van hul Restionaceae-gashere volg en dat naverwante Cephalelini dieselfde stam van Restionaceae as gashere verkies. Die bevinding dat die Cephalelini hoogs gespesialiseerd is in hulle gasheer-keuses lei ‘n mens tot die verwagting dat die evolusie van Cephalelini meer getrou die evolusie van hulle gashere sal volg as wat wel die geval is (dalk op die vlak van groep of genus), maar die relatiewe jeugdigheid van die Cephalelini vergeleke met die ouderdom van die Restionaceae is moontlik die rede hiervoor. Analise van gasheer-keuse is ook uitgevoer op `n filogenie van die Restionaceae wat aantoon dat daar nie ‘n beperking van verwantskappe is tussen gashere wat verkies of verwerp word nie, wat daarop dui dat daar `n menigte nisse is wat oënskynlik onbenut is, of dat Cephalelini gasheer-keuse bepaal word deur `n Restionaceae-eienskappe wat nie filogeneties beperk is nie. Laastens het ek die evolusie van spesialisering deur Cephalelini ontleed, maar geen neiging tot toenemende spesialisering binne die groep gevind nie, wat onverwags is in die lig van vorige studies. As `n geheel bied hierdie studie die eerste bewys van die uitruiling van insek-fauna tussen die Suid-Afrikaanse fynbos-bioom en ander kontinente, wat `n onopgetekende invloed op die diversiteit van diere in die fynbos is. Cephalelini is `n hoogs gespesialiseerde groep insekte, maar uit `n filogenetiese perspektief word dit eers waargeneem op die vlak van gasheerstam. Ten spyte van hulle hoë vlak van spesialisering het ek geen bewys gevind van `n toename daarin binne die evolusie van die groep nie.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine producing regions in SA. To date, only three GFLV isolates have been completely sequenced internationally, and limited sequence information is available for SA GFLV isolates. In this study, the first full-length GFLV genome sequence from a South African isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for phylogenetic analysis and revealed that the SA isolates are separate from other sequenced GFLV isolates. Full-length sequences were also used to investigate putative intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2 between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different recombination analysis software packages, the most notable of the putative recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2 5’ UTR might have evolved from an interspecies recombination event between GFLVF13- type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA) associated with South African GFLV isolates was also investigated. In a collaborative study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants were screened for satRNAs. SatRNAs were present in only two plants, containing isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were determined. No significant sequence variation could be detected between the GFLV isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44 RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3, which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen from a common ancestor, which may have originated from an interspecies recombination event. The GFLV-SACH44 satRNA was found to be more closely related to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA clone was however abolished when co-inoculated with an ArMV helper virus, even though it is phylogenetically more closely related to ArMV satRNAs. The full-length satRNA clones were modified to be used as vectors for expression and/or silencing of foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial sequences downstream of the open reading frame of the satRNA. These constructs were cloned into a binary vector to allow for agro-infiltration into plants. Full-length cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA expression and silencing constructs had to be aborted. Attempts to understand this failure revealed that, among other point mutations, four frameshifts had occurred in the RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious. Strategies to correct the mutations are discussed. Once these mutations have been corrected this study can continue in evaluating the use of the satRNA component for expression and silencing analysis. / AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is. Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3, bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3 RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse, met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’ ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44 RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens ‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44 satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44 satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2 klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise gebruik kan word.

Στην παρούσα εργασία αξιολογήθηκε η χρήση των μικροδορυφορικών δεικτών για τη μελέτη της γενετικής δομής και των φυλογενετικών σχέσεων των φυσικών πληθυσμών της Atherina boyeri που προέρχονταν τόσο από θαλάσσιες όσο και λιμναίες/λιμνοθαλάσσιες περιοχές της Ελλάδας. Προηγούμενες μελέτες βασιζόμενες κυρίως σε μιτοχονδριακούς και RAPD δείκτες έχουν υποδείξει την πιθανή παρουσία τριών ομάδων πληθυσμών στην Atherina boyeri με τόσο υψηλές γενετικές αποστάσεις μεταξύ τους που θα μπορούσαν να τις καθιστούν ακόμα και διαφορετικά είδη. Οι ομάδες αυτές είναι: οι θαλάσσιοι πληθυσμοί τύπου Ι (μη εστιγμένοι),οι θαλάσσιοι πληθυσμοί τύπου ΙΙ (εστιγμένοι) και οι λιμναίοι/λιμνοθαλάσσιοι πληθυσμοί. Η ανάλυση στην παρούσα εργασία πραγματοποιήθηκε με χρήση 11 μικροδορυφορικών δεικτών που σχεδιάστηκαν από τους Milana et al. (2009). Οι μικροδορυφορικοί δείκτες θεωρούνται εξαιρετικό εργαλείο μελέτης των φυλογενετικών σχέσεων μεταξύ πρόσφατα διαχωρισμένων ειδών δεδομένου ότι είναι άφθονοι, πυρηνικοί, διάσπαρτοι στο γονιδίωμα, υψηλά πολυμορφικοί και ταχέως εξελισσόμενοι. Τα αποτελέσματα έδειξαν πολύ υψηλό βαθμό πολυμορφισμού στους υπό ανάλυση πληθυσμούς και μεγάλη γενετική διαφοροποίηση μεταξύ των ελληνικών πληθυσμών του είδους όπως αυτή εκφράζεται από τις διαφορές στις συχνότητες των αλληλομόρφων των μικροδορυφορικών δεικτών. Φαίνεται επίσης να επιβεβαιώνεται η διάκριση μεταξύ των δυο θαλάσσιων τύπων της Atherina boyeri, ωστόσο οι λιμναίοι/λιμνοθαλάσσιοι πληθυσμοί παρουσιάζουν τόσο διαφορετικό γενετικό πρότυπο που θεωρείται εξαιρετικά δύσκολο να συνιστούν μια ενιαία ομάδα πληθυσμών. Οι δείκτες αυτοί δεν ήταν δυνατό να χρησιμοποιηθούν στην ανάλυση όλων των διαθέσιμων πληθυσμών, κυρίως λόγω της παρουσίας μη ενισχυόμενων (null) αλληλομόρφων, καθώς επίσης και πολλαπλών ή/και ασθενών ζωνών, γεγονός που επίσης υποδεικνύει την ύπαρξη μεγάλων γενετικών διαφοροποιήσεων, που πιθανώς ξεπερνούν τα όρια του είδους. Προκείμενου να ξεπεραστούν τα ανωτέρω προβλήματα απαιτείται η βελτιστοποίησή τους ανά ομάδα πληθυσμών, μέσω α) αλλαγών στις συνθήκες των PCR αντιδράσεων και β) κλωνοποίησης και αλληλούχισης των δεικτών αυτών από άτομα των πληθυσμών στα οποία ήταν λειτουργικοί, ώστε να επιτευχθεί ο σχεδιασμός νέων ζευγών εκκινητών, ειδικών για επιμέρους ομάδες πληθυσμών. / The present study aims at the measure of the genetic differentiation and the resolution of the phylogenetic relationships among A.boyeri populations originating from lakes/lagoons and marine sites of Greece. Previously studies based on RAPD and mitochondrial markers suggest the existence of three forms of populations of A.boyeri which could represent three different species. These three types are: marine type I, which includes almost all marine populations (non-punctuated), excluding specimens collected from Preveza, Evoia and Kos, which form the marine type II (punctuated) and the “lagoon” type which consists of all the lagoon/lake populations. In the present study, eleven microsatellite markers designed by Milana et al. (2009), were used. Microsatellite markers are supposed to be great tools in phylogenetic studies among recently separated species because they are abundant, nuclear, dispersed around the genome, highly polymorphic and rapidly evolving. Our results showed very high degree of polymorphism in the analyzed populations and extended genetic differentiation among Greek populations of the species as expressed by differences in allele frequencies of the microsatellite markers. They also seems to confirm the distinction between the two marine types of A.boyeri, but the lagoon/lake populations present different allele paterns, pointing to the possible existence of differentiated groups among them. Some of the markers could not be used in the analysis of all the available populations. This is mainly attributed to the presence of null alleles for some of the populations and to scoring difficulties raising from the presence of multiple and/or weak amplicons. This also indicates the existence of great genetic variations, which possibly exceed species limit. To overcome the above difficulties, optimization per marker is required, including a) optimization of PCR conditions and b) cloning and sequencing of these markers from individuals of the population that were functional to achieve the design of new prime pairs, specific for each group of populations .

Brain size varies greatly among vertebrates. It has been proposed that the diversity of brain size is produced and maintained through a balance of adaptations to different types and levels of cognitive ability and constraints for adaptive evolution. Phylogenetic comparative studies have made major contributions to our understanding of brain size evolution. However, previous studies have nearly exclusively focused on mammalian and avian taxa and almost no attempts have been made to investigate brain size evolution in ectothermic vertebrates. In my thesis, I studied brain size evolution in two groups of fish with extreme diversity in ecology, morphology and life history, Cichlidae and Syngnathidae. Using phylogenetic comparative methods, I investigated four key questions in vertebrate brain size evolution; cognitive adaptation, sexual selection, phenotypic integration and energetic constraints. I have demonstrated i) that phenotypic integration can link functionally unrelated traits, and this may constrain independent evolution of each part involved or promote concerted evolution of an integrated whole, ii) that brain-body static allometry constrains the direction of brain size evolution, even though the static-allometry showed ability to evolve, allowing evolution of relative brain size under allometric constraints, iii) that the energetic constraints of development and maintenance of brain tissue is an important factor in forming the diversity in brain size in cichlids and syngnathids, both at macroevolutionary and microevolutionary time scales, and iv) that adaptation for feeding and female mating competition may have played key roles in the adaptive evolution of brain size in pipefishes and seahorses. To conclude, my thesis shows the strong benefit of using fish as a model system to study brain size evolution with a phylogenetic comparative framework.

Best-known as the family of the cultivated flax, Linum usitatissimum L., the Linaceae is a small but ecologically diverse family of flowering plants, with approximately 250 species distributed throughout the temperate and tropical latitudes of the world. This work is an investigation of the systematics and biogeography of the family and a portion of its largest genus, Linum, using molecular phylogenetic methods. I collected DNA sequences of rbcL and matK genes from 51 species of Linaceae, representing all 14 genera, and combined them with data from 24 other families of the order Malpighiales in phylogenetic analyses. Results strongly support the monophyly of Linaceae and subfamily Linoideae in their current circumscriptions, but subfamily Hugonioideae is poorly supported. Molecular dating analysis suggests that the temperate Linoideae diversified in the Eocene or Oligocene, while tropical Hugonioideae diversified later, during the Miocene, perhaps ruling out Gondwanan vicariance as an explanation for their Pantropical distribution. Hugonia and Linum, the largest genera in their respective subfamilies, are each found have multiple segregate genera nested within them, indicating potential need for taxonomic revision of each subfamily. In Linoideae, I further investigate the phylogeny of a lineage that includes the yellow-flowered Linum sections Cathartolinum, Linopsis, and Syllinum, and the segregate genera Cliococca, Hesperolinon, Radiola, and Sclerolinon, to provide a framework in which to assess character evolution, classification, and biogeography. With data from four chloroplast markers (matK, ndhF, trnK 3’ intron, trnL-F region) and the nuclear ITS, and extensive sampling from Linum section Linopsis from Eurasia, Africa, and the Americas, Hesperolinon and Sclerolinon are shown to be related to a lineage of Central American linums including L. mexicanum Kunth and L. guatemalense Benth., while Cliococca is affiliated with South American Linum. The phylogeny supportes previous hypotheses of the evolution of some taxonomically important characters, and several well-supported lineages are identified which correspond to previously proposed taxonomic groupings. Results also provide evidence for a single trans-Atlantic disjunction and independent Old and New World colonizations of the southern hemisphere in yellow-flowered Linum, occurring during the Miocene. / text

The entire stretch of the Ohio River is under fish consumption advisories due to contamination with polychlorinated biphenyls (PCBs). In this study, natural attenuation and biostimulation of PCBs and microbial communities responsible for PCB transformations were investigated in Ohio River sediments. Natural attenuation of PCBs was negligible in sediments, which was likely attributed to low temperature conditions during most of the year, as well as low amounts of available nitrogen, phosphorus, and organic carbon. Moreover, surface sediments were relatively oxidized, as indicated by the prevalence of aerobic bacteria such as beta- Proteobacteria, alpha-Proteobacteria, Sphingobacteria, and Nitrospira in 16S rRNA sediment clone libraries. On the other hand, several reductive dechlorinators were detected in sediments, including Dehalococcoides, Desulfitobacterium spp. which suggested that reductive dechlorination might be possible in sediments under certain biogeochemical conditions. Considerable amounts of PCBs were transformed by reductive dechlorination (80% in 177 days by pattern N) when sediments were maintained under anaerobic conditions, amended with nutrients and organic carbon, and incubated at 25 ºC in lab microcosms. Analysis of 16S rRNA clone libraries from these treatments revealed that Bacteroidetes, Chloroflexi and Firmicutes were enriched and Proteobacteria were depleted compared to clone libraries from treatment without organic amendments. Reductive dechlorination was decreased in sediments incubated at 10 and 40 ºC, and was not affected by FeSO4 amendments compared to unamended sediments incubated at 25 ºC. Transformations of PCB-153 were investigated in sediments under anaerobic, aerobic and sequential anaerobic and aerobic conditions. Transformations were only observed in treatments with an anaerobic phase, which occurred by reductive dechlorination by pattern N. Neither PCB-153 nor dechlorination products PCB-99 or PCB-47 were transformed under aerobic conditions. Analysis of 16S rRNA clone libraries revealed that Bacteoridetes, Chloroflexi, and Firmicutes were enriched under anaerobic conditions and Proteobacteria were enriched under aerobic conditions. Results from this study revealed that natural attenuation and biostimulation were not effective at removing PCBs from Ohio River sediments. Hence, other remediation methods will need to be employed to decrease PCB levels in this ecosystem.

Two separate phylogenetic studies were performed for two different taxonomic levels within Hymenoptera. The first study examined the utility of expressed sequence tags for resolving relationships among hymenopteran superfamilies. Transcripts were assembled from 14,000 sequenced clones for 6 disparate Hymenopteran taxa, averaging over 660 unique contigs per species. Orthology and gene determination were performed using modifications to a previously developed computerized pipeline and compared against annotated insect genomes. Sequences from additional taxa were added from public databases with a final dataset of 24 genes for 16 taxa. The concatenated dataset recovered a robust and well-supported topology; however, there was extreme incongruity among individual gene trees. Analyses of sequences indicated strong compositional and transition biases, particularly in the third codon positions. The use of filtered supernetworks aided visualization of the existing congruent phylogenetic signal that existed across the individual gene trees. Additionally, treeness triangle plots indicated a strong residual signal in several gene trees and across codon positions in the concatenated dataset. However, most analyses of the concatenated dataset recovered expected relationships, known from other independent analyses. Thus, ESTs provide a powerful source of information for phylogenetic analysis, but results are sensitive to low taxonomic sampling and missing data. The second study examined subfamilial relationships within the parasitoid family Braconidae, using over 4kb of sequence data for 139 taxa. Bayesian inference of the concatenated dataset recovered a robust phylogeny, particularly for early divergences within the family. There was strong evidence supporting two independent lineages within the family: one leading to the noncyclostomes and one leading to the cyclostomes. Ancestral state reconstructions were performed to test the theory of ectoparasitism as the ancestral condition for all taxa within the family. Results indicated an endoparasitic ancestor for the family and for the non-cyclostome lineage, with an early transition to ectoparasitism for the cyclostome lineage. However, reconstructions of some nodes were sensitive to outgroup coding and will also be impacted with increased biological knowledge.

L'objet de cette thèse de bioinformatique est de mieux comprendre l’ensemble des systèmes de régulation génique chez les bactéries. La disponibilité de centaines de génomes complets chez les bactéries ouvre la voie aux approches de génomique comparative et donc à l’étude de l’évolution des réseaux transcriptionnels bactériens. Dans un premier temps, nous avons implémenté et validé plusieurs méthodes de prédiction d’opérons sur base des génomes bactériens séquencés. Suite à cette étude, nous avons décidé d’utiliser un algorithme qui se base simplement sur un seuil sur la distance intergénique, à savoir la distance en paires de bases entre deux gènes adjacents. Notre évaluation sur base d’opérons annotés chez Escherichia coli et Bacillus subtilis nous permet de définir un seuil optimal de 55pb pour lequel nous obtenons respectivement 78 et 79% de précision. Deuxièmement, l’identification des motifs de régulation transcriptionnelle, tels les sites de liaison des facteurs de transcription, donne des indications de l’organisation de la régulation. Nous avons développé une méthode de recherche d’empreintes phylogénétiques qui consiste à découvrir des paires de mots espacés (dyades) statistiquement sur-représentées en amont de gènes orthologues bactériens. Notre méthode est particulièrement adaptée à la recherche de motifs chez les bactéries puisqu’elle profite d’une part des centaines de génomes bactériens séquencés et d’autre part les facteurs de transcription bactériens présentent des domaines Hélice-Tour-Hélice qui reconnaissent spécifiquement des dyades. Une évaluation systématique sur 368 gènes de E.coli a permis d’évaluer les performances de notre méthode et de tester l’influence de plus de 40 combinaisons de paramètres concernant le niveau taxonomique, l’inférence d’opérons, le filtrage des dyades spécifiques de E.coli, le choix des modèles de fond pour le calcul du score de significativité, et enfin un seuil sur ce score. L’analyse détaillée pour un cas d’étude, l’autorégulation du facteur de transcription LexA, a montré que notre approche permet d’étudier l’évolution des sites d’auto-régulation dans plusieurs branches taxonomiques des bactéries. Nous avons ensuite appliqué la détection d’empreintes phylogénétiques à chaque gène de E.coli, et utilisé les motifs détectés comme significatifs afin de prédire les gènes co-régulés. Au centre de cette dernière stratégie, est définie une matrice de scores de significativité pour chaque mot détecté par gène chez l’organisme de référence. Plusieurs métriques ont été définies pour la comparaison de paires de profils de scores de sorte que des paires de gènes ayant des motifs détectés significativement en commun peuvent être regroupées. Ainsi, l’ensemble des nos méthodes nous permet de reconstruire des réseaux de co-régulation uniquement à partir de séquences génomiques, et nous ouvre la voie à l’étude de l’organisation et de l’évolution de la régulation transcriptionnelle pour des génomes dont on ne connaît rien. The purpose of my thesis is to study the evolution of regulation within bacterial genomes by using a cross-genomic comparative approach. Nowadays, numerous genomes have been sequenced facilitating in silico analysis in order to detect groups of functionally related genes and to predict the mechanism of their relative regulation. In this project, we combined prediction of operons and regulons in order to reconstruct the transcriptional regulatory network for a bacterial genome. We have implemented three methods in order to predict operons from a bacterial genome and evaluated them on hundreds of annotated operons of Escherichia coli and Bacillus subtilis. It turns out that a simple distance-based threshold method gives good results with about 80% of accuracy. The principle of this method is to classify pairs of adjacent genes as “within operon” or “transcription unit border”, respectively, by using a threshold on their intergenic distance: two adjacent genes are predicted to be within an operon if their intergenic distance is smaller than 55bp. In the second part of my thesis, I evaluated the performances of a phylogenetic footprinting approach based on the detection of over-represented spaced motifs. This method is particularly suitable for (but not restricted to) Bacteria, since such motifs are typically bound by factors containing a Helix-Turn-Helix domain. We evaluated footprint discovery in 368 E.coli K12 genes with annotated sites, under 40 different combinations of parameters (taxonomical level, background model, organism-specific filtering, operon inference, significance threshold). Motifs are assessed both at the level of correctness and significance. The footprint discovery method proposed here shows excellent results with E. coli and can readily be extended to predict cis-acting regulatory signals and propose testable hypotheses in bacterial genomes for which nothing is known about regulation. Moreover, the predictive power of the strategy, and its capability to track the evolutionary divergence of cis-regulatory motifs was illustrated with the example of LexA auto-regulation, for which our predictions are remarkably consistent with the binding sites characterized in different taxonomical groups. A next challenge was to identify groups of co-regulated genes (regulons), by regrouping genes with similar motifs, in order to address the challenging domain of the evolution of transcriptional regulatory networks. We tested different metrics to detect putative pairs of co-regulated genes. The comparison between predicted and annotated co-regulation networks shows a high positive predictive value, since a good fraction of the predicted associations correspond to annotated co-regulations, and a low sensitivity, which may be due to the consequence of highly connected transcription factors (global regulator). A regulon-per-regulon analysis indeed shows that the sensitivity is very weak for these transcription factors, but can be quite good for specific transcription factors. The originality of this global strategy is to be able to infer a potential network from the sole analysis of genome sequences, and without any prior knowledge about the regulation in the considered organism.

In the pre-genomic era, the relationships among species and their evolutionary histories were often determined by examining the fossil records. In the genomic era, these relationships are identified by analysing the genetic data, which also enables us to take a close-up view of the differences between the individual samples. Nevertheless, these relationships are often described by a tree-like structure or a network. In this thesis, we investigate some of the models that are used to describe these relationships. This thesis can be divided into two main parts. The first part focuses on investigating the theoretical properties of several neutral tree models that are often considered in phylogenetics and population genetics studies, such as the Yule–Harding model, the proportional to distinguishable arrangements and the Kingman coalescent models. In comparison to the first part, the other half of the thesis is more computationally oriented: we focus on developing and implementing methods of calculating gene tree probabilities of given species networks, and simulating genealogies within species networks.

Subalpine ecosystems are experiencing rapid changes in snow pack, temperature, and precipitation regime as a result of anthropogenic climate forcing. These changes in climate can have a profound effect on subalpine ecosystem structure and functioning, which may ultimately feed back to climate change. In this study, I examined the response of the subalpine meadow plant communities at the Rocky Mountain Biological Laboratory to natural and simulated climate change. First, I looked at whether changes in growing season precipitation or temperature regime would have the larger effect on subalpine ecosystem carbon flux. In a simulated warming experiment, changes in growing season precipitation had a tenfold larger effect on cumulative carbon flux than did the warming treatment. Along a natural climatic and elevational gradient, precipitation stimulates carbon uptake, particularly at higher elevations. Given projected decreases in summer precipitation in the high elevation Rockies, we predict a 20% decrease in carbon uptake from subalpine meadows. Second, I compared the taxonomic, phylogenetic and functional structure of plant communities along an elevational gradient to infer which climatic and biotic factors influence community assembly at each elevation. Floral and phenology traits become overdispersed at high elevation, mirroring phylogenetic relatedness, and suggesting pressure to diversify to attract pollinators during the abbreviated growing season. At the same time, leaf functional traits become clustered at high elevation, indicating multiple opposing assembly mechanisms in subalpine communities. Finally, I studied the natural history of sagebrush, Artemisia tridentate ssp. vaseyana, at its elevational range limit in subalpine meadows. In particular, I focused on the importance of warming and species interactions in elevational advance of the species. I found that facilitation by neighboring forbs was critical for sagebrush seedling survival, decreasing mortality by 75%. Seedling mortality was overwhelmingly due to desiccation of seedlings; therefore, neighboring forbs moderate temperature and water stress for seedlings. Despite the extremely limited growing season at high elevation caused by subfreezing temperatures, subalpine ecosystem structure and function are closely tied to water availability during the growing season. Therefore, improved predictions of future precipitation regimes over the Rocky Mountains will be our best tool for conservation of these fragile habitats.