Octávio S. Paulo

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species

BackgroundThe process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly.ResultsIn Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies.ConclusionsMany current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another.

The persistence of Pliocene populations through the Pleistocene climatic cycles: evidence from the phylogeography of an Iberian lizard.

Ancient climatic fluctuations have caused changes in the demography and distribution of many species. The genetic differentiation between populations of the same species and of sister species is often attributed largely to the more recent Pleistocene fluctuations. Recent interpretations, which implicate earlier episodes, have proved controversial. We address the timing of genetic divergence in the Iberian lizard Lacerta schreiberi by studying the phylogeography of the cytochrome b sequence. The species has a remarkable morphological uniformity, yet our evidence suggests that earlier events in the Pliocene initiated the main divergence between populations. This interpretation implies that the different populations survived through the Pleistocene in separate localities. This conclusion is robust to different molecular clock calibrations. The persistence of earlier differentiation through the Pleistocene has wide implications for our understanding of Pleistocene refugia in this species and, by extension, to the biogeography of the whole region.

The role of vicariance vs. dispersal in shaping genetic patterns in ocellated lizard species in the western Mediterranean

The schism between North Africa and Southern Europe caused by the opening of the Strait of Gibraltar and the consequent refilling of the Mediterranean basin at the end of Messinian salinity crisis (MSC), 5.33 million years ago, has been advocated as the main event shaping biogeographical patterns in the western Mediterranean as exemplified by the distribution of species and subspecies and genetic variation within the ocellated lizard group. To reassess the role of the MSC, partial sequences of three mitochondrial DNA genes (cytochrome b, 12S and 16S ribosomal RNA) and two nuclear genes (β‐fibrinogen and C‐mos) from species of the ocellated lizard group were analysed. Three alternative hypotheses were tested: that divergence was initiated (i) by post‐MSC vicariance as the basin filled, (ii) when separate populations established either side of the strait by pre‐MSC overseas dispersal, and (iii) by post‐MSC overseas dispersal. The pattern and level of divergence detected clearly refute the post‐MSC vicariance hypothesis, and support a model of divergence initiated by earlier overseas dispersal. Indeed, our best estimate is that the basal Euro‐African divergence predates the MSC event by several million years. The estimated divergence times among the populations in former Miocene Mediterranean islands, the current Betic and Rifian mountains, from adjacent mainland populations suggest overseas dispersal for the former and overland dispersal, or perhaps vicariance, for the latter. These results suggest that the MSC may have played a much less important role in shaping the current western Mediterranean biogeographical patterns than might have been anticipated from the dramatic nature of the episode.

Using nested clade analysis to assess the history of colonization and the persistence of populations of an Iberian Lizard

The distribution of the lizard Lacerta schreiberi is likely to have been severely affected by the climatic cycles that have influenced the Iberian Peninsula. Information about the species ecology and Iberian physiogeography was used to generate specific hypotheses about episodes of colonization and subsequent population persistence. These hypotheses generated predictions about the distribution of genetic variation, which were tested using nested clade analysis (NCA) supplemented by analysis of molecular variance (amova). Two predictions were confirmed by NCA; that is those that specified multiple and allopatric refugia. However, the remaining three predictions were not corroborated by the analyses. Firstly, a simple analysis of the distribution of genetic variability failed to detect an expected difference in the pattern of colonization between the inland mountain system and the coastal region. Moreover, while NCA did detect the expected genetic pattern in southern coastal populations, it was explained in terms of long‐distance migration, which seems implausible because of the extent of unsuitable habitat. A more likely cause of the pattern is population fragmentation and a reduction in population size caused during the Holocene. Finally, NCA also failed to detect a northwestern population expansion, which is supported by other evidence. We conclude that NCA has a limited ability to detect range expansion led by individuals with more ancestral (interior) haplotypes.

Assemblathon 2: evaluating de novo

BackgroundThe process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly.ResultsIn Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies.ConclusionsMany current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another.

Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts

To improve phylogenetic resolution of the Colletotrichum gloeosporioides species complex we developed and tested the performance of a new set of primers for the Apn2/MAT locus with a case study of 22 isolates. These were isolated mainly from coffee plants and represent six divergent and well characterized species within the C. gloeosporioides complex. Following previous studies on this locus, we have generated sequence data from an expanded region (> 4600 bp), revealing increased phylogenetic informativeness when compared to other commonly used markers such as ITS, β-tub2 and GS. Within the Apn2/MAT locus the ApMAT marker alone was almost as informative in terms of phylogenetic resolution as a seven-gene concatenated dataset. Our results further revealed that gene-tree discordance may come to be a common issue in resolving evolutionary relationships in the C. gloeosporioides complex, highlighting the importance of multilocus approaches. The use of state-of-the-art data analysis techniques and a highly informative dataset as employed here may abate this issue and hopefully assist in disentangling the C. gloeosporioides complex.

Systematic and phylogeographical assessment of the Acanthodactylus erythrurus group (Reptilia: Lacertidae) based on phylogenetic analyses of mitochondrial and nuclear DNA.

We have used mitochondrial 12S rRNA, 16S rRNA and nuclear beta-fibrinogen (intron 7) sequences to investigate the phylogenetic and phylogeographic relationships between Acanthodactylus erythrurus group species (except for A. boueti). The phylogenetic analyses of the Acanthodactylus genus did not cluster A. guineensis and A. savignyi with the remaining species of the group (A. blanci, A. lineomaculatus and A. erythrurus). Within the A. erythrurus group, the results of the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) showed a complex phylogeny with geographic structure, but it was not congruent with the present taxonomy. Some taxonomic units, such as A. blanci, A. lineomaculatus, A. e. atlanticus and A. e. belli did not form monophyletic genetic units. The application of a molecular clock suggested that the uplift of the Atlas Mountains in the mid-late Miocene and the reopening of the Strait of Gibraltar could be major biogeographic events responsible for the genetic differentiation in the group. Additionally, diverse micro-evolutionary patterns due to the recent contraction/expansion phases of the habitats in North Africa associated with the high dispersal capabilities of these lizards could be related to the complex phylogenetic patterns observed.

concatenator: sequence data matrices handling made easy

concatenator is a simple and user‐friendly software that implements two very useful functions for phylogenetics data analysis. It concatenates NEXUS files of several fragments in a single NEXUS file ready to be used in phylogenetics software, such as paup and mrbayes and it converts FASTA sequence data files to NEXUS and vice‐versa. Additionally, concatenated files can be prepared for partition tests in paup. It is freely available in http://cobig2.fc.ul.pt.

Phylogeography and demographic history of Lacerta lepida in the Iberian Peninsula: multiple refugia, range expansions and secondary contact zones

BackgroundThe Iberian Peninsula is recognized as an important refugial area for species survival and diversification during the climatic cycles of the Quaternary. Recent phylogeographic studies have revealed Iberia as a complex of multiple refugia. However, most of these studies have focused either on species with narrow distributions within the region or species groups that, although widely distributed, generally have a genetic structure that relates to pre-Quaternary cladogenetic events. In this study we undertake a detailed phylogeographic analysis of the lizard species, Lacerta lepida, whose distribution encompasses the entire Iberian Peninsula. We attempt to identify refugial areas, recolonization routes, zones of secondary contact and date demographic events within this species.ResultsResults support the existence of 6 evolutionary lineages (phylogroups) with a strong association between genetic variation and geography, suggesting a history of allopatric divergence in different refugia. Diversification within phylogroups is concordant with the onset of the Pleistocene climatic oscillations. The southern regions of several phylogroups show a high incidence of ancestral alleles in contrast with high incidence of recently derived alleles in northern regions. All phylogroups show signs of recent demographic and spatial expansions. We have further identified several zones of secondary contact, with divergent mitochondrial haplotypes occurring in narrow zones of sympatry.ConclusionsThe concordant patterns of spatial and demographic expansions detected within phylogroups, together with the high incidence of ancestral haplotypes in southern regions of several phylogroups, suggests a pattern of contraction of populations into southern refugia during adverse climatic conditions from which subsequent northern expansions occurred. This study supports the emergent pattern of multiple refugia within Iberia but adds to it by identifying a pattern of refugia coincident with the southern distribution limits of individual evolutionary lineages. These areas are important in terms of long-term species persistence and therefore important areas for conservation.

Multiple approaches to detect outliers in a genome scan for selection in ocellated lizards (Lacerta lepida) along an environmental gradient

Identification of loci with adaptive importance is a key step to understand the speciation process in natural populations, because those loci are responsible for phenotypic variation that affects fitness in different environments. We conducted an AFLP genome scan in populations of ocellated lizards (Lacerta lepida) to search for candidate loci influenced by selection along an environmental gradient in the Iberian Peninsula. This gradient is strongly influenced by climatic variables, and two subspecies can be recognized at the opposite extremes: L. lepida iberica in the northwest and L. lepida nevadensis in the southeast. Both subspecies show substantial morphological differences that may be involved in their local adaptation to the climatic extremes. To investigate how the use of a particular outlier detection method can influence the results, a frequentist method, DFDIST, and a Bayesian method, BayeScan, were used to search for outliers influenced by selection. Additionally, the spatial analysis method was used to test for associations of AFLP marker band frequencies with 54 climatic variables by logistic regression. Results obtained with each method highlight differences in their sensitivity. DFDIST and BayeScan detected a similar proportion of outliers (3–4%), but only a few loci were simultaneously detected by both methods. Several loci detected as outliers were also associated with temperature, insolation or precipitation according to spatial analysis method. These results are in accordance with reported data in the literature about morphological and life‐history variation of L. lepida subspecies along the environmental gradient.