Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

Ecological divergence combined with ancient allopatry in lizard populations from a small volcanic island.

Population divergence and speciation are often explained by geographical isolation, but may also be possible under high gene flow due to strong ecology-related differences in selection pressures. This study combines coalescent analyses of genetic data (11 microsatellite loci and 1 Kbp of mtDNA) and ecological modelling to examine the relative contributions of isolation and ecology to incipient speciation in the scincid lizard Chalcides sexlineatus within the volcanic island of Gran Canaria. Bayesian multispecies coalescent dating of within-island genetic divergence of northern and southern populations showed correspondence with the timing of volcanic activity in the north of the island 1.5-3.0 Ma ago. Coalescent estimates of demographic changes reveal historical size increases in northern populations, consistent with expansions from a volcanic refuge. Nevertheless, ecological divergence is also supported. First, the two morphs showed non-equivalence of ecological niches and species distribution modelling associated the northern morph with mesic habitat types and the southern morph with xeric habitat types. It seems likely that the colour morphs are associated with different antipredator strategies in the different habitats. Second, coalescent estimation of gene copy migration (based on microsatellites and mtDNA) suggest high rates from northern to southern morphs demonstrating the strength of ecology-mediated selection pressures that maintain the divergent southern morph. Together, these findings underline the complexity of the speciation process by providing evidence for the combined effects of ecological divergence and ancient divergence in allopatry.

Genetic characterization, at the mitochondrial and nuclear DNA levels, of five Canary Island dog breeds.

Many studies presenting genetic analysis of dog breeds have been conducted without the inclusion of island dog breeds, although isolation can be one of the main factors in their origin. Here we report the genetic analysis at the nuclear and mitochondrial DNA levels of five Canary Island dog breeds (Canarian Warren Hound, Canary Island Mastiff, Garafiano Shepherd, La Palma Rat-Hunter and El Hierro Wolfhound) to fill this gap and, at the same time, genetically characterize these breeds. We identified 168 alleles in autosomal microsatellites and 16 mitochondrial haplotypes. Observed and expected heterozygosities ranged from 0.556 to 0.783 and from 0.737 to 0.943 respectively. Furthermore, three haplotypes were newly described and exclusive to a particular breed (A17+ in the Canary Island Mastiff; A33+ in the Canarian Warren Hound; Bi in the La Palma Rat-Hunter). The outcome of our analyses also revealed different breed histories consistent with historical documents and hypothetical origin designations. Although mtDNA haplotypes showed poor breed discriminating power, autosomal markers allowed a clear clustering of each single population. We expect that our results, together with further analyses, will help to make the population histories of island dog breeds clearer.

Microsatellite loci isolation in the Canarian common chaffinch (Fringilla coelebs) and their utility in other Canarian finches.

The taxonomic classification of the common chaffinch (Fringilla coelebs) that inhabits the Canary Islands has been under debate for decades, mainly due to the absence of nuclear DNA analyses. In this study we describe the isolation and characterization of ten microsatellite loci (AAAG, AAAT and GT) from a La Palma specimen using an enrichment protocol. Two loci were monomorphic in the populations analysed (La Gomera and La Palma), but the remaining ones presented 2 or more alleles, with an average of 11.63 alleles per locus and an average observed heterozygosity of 0.735 (n = 44). All loci were tested for their utility in other Canarian populations and other finch species.

Highly polymorphic microsatellite loci for the Gran Canarian skink (Chalcides sexlineatus) and their applicability in other Canarian Chalcides.

The scincid lizard Chalcides sexlineatus is endemic to the island of Gran Canaria, within which it shows a pronounced phylogeographical pattern. Understanding the maintenance of this pattern requires knowledge of nuclear DNA structuring. Eleven polymorphic tetranucleotide microsatellite loci were isolated from a library constructed from genomic DNA enriched for AAAG repeat sequence. The number of alleles per locus ranged from 12 to 19, and expected heterozygosities spanned from 0.738 to 0.933 (n = 30). The majority of these could be cross-amplified in other endemic Canarian Chalcides. The loci will be used to investigate gene flow between the principal mitochondrial clades within C. sexlineatus.

Intraspecific evolution of Canary Island Plecotine bats, based on mtDNA sequences.

Island differentiation and relationships with congenerics were investigated in the endemic Canary Island bat Plecotus teneriffae, based on approximately 1 kb of mtDNA from the 16S rRNA and cytochrome b genes. P. teneriffae had closer affinities with P. austriacus than with P. auritus. Levels of differentiation between Canary Islands were quite high relative to Pipistrelle-like bats, consistent with philopatric behaviour in the Plecotus genus. Cladogenesis within P. teneriffae appears to have occurred after the emergence of the islands of El Hierro and La Palma during the Pleistocene. An intraspecific network shows that haplotypes from the younger islands of La Palma and El Hierro are connected to the Tenerife haplotype by a similarly large number of mutational steps. This suggests that they were both colonised at a similar time from the much older island of Tenerife. The other Plecotine bat species, Barbastellus barbastellus shows close affinities with B. barbastellus from mainland Spain, with levels of mtDNA divergence being comparable with intraspecific variation within other mammal species.

Phylogeography of pipistrelle-like bats within the Canary Islands, based on mtDNA sequences.

Evolution of three Canary Island Vespertilionid bat species, Pipistrellus kuhlii, Pipistrellus maderensis, and Hypsugo savii was studied by comparison of approximately 1 kbp of mtDNA (from cytochrome b and 16S rRNA genes) between islands. mtDNA reveals that both P. kuhlii and P. maderensis exist in sympatry on Tenerife (and possibly other islands). Their morphological similarity explains why their co-occurrence had not been detected previously. Levels of sequence divergence are quite low within P. maderensis. Haplotypes were either identical or separated by /=12 mutational steps) indicating colonization of the latter from the former sometime during the last approximately 1.2 Ma, with low subsequent gene flow. Unlike P. maderensis the El Hierro population alone appears to represent an ESU. The H. savii haplotypes detected in Gran Canaria and Tenerife are identical or separated by 1 mutational step.

The Atlas mountains as a biogeographical divide in North-West Africa: evidence from mtDNA evolution in the Agamid lizard Agama impalearis.

Since the early Miocene there have been several physical events within NW Africa that are likely to have had a major impact on its faunal diversity. Phylogeographical studies will shed new light on the biogeography of the region. We analysed mitochondrial DNA diversity in the agamid lizard Agama impalearis (also called A. bibronii) based on sequences from mitochondrial genes with very different evolutionary rates (16S rRNA and ND2). Well-supported topologies of rooted maximum parsimony trees (with a Laudakia outgroup) and unrooted haplotype networks indicated two major clades with similar branch lengths. These clades have non-overlapping distributions representing respective areas to the North and West and South and East of the Atlas mountain chain and each could be given full species recognition. Nested clade analyses indicate that historical and possible present-day allopatry account for the primary phylogeographic pattern. Further evidence is provided by the estimated timing of cladogenesis, based on calibration of evolutionary rates in the ND2 gene of another continental Agamid. Sequence divergence between clades corresponds to 8.5-9.4mya, coinciding with the main period of orogenic uplift of the Atlas. Additional evidence of cladogenesis by allopatric fragmentation is also detected within the North/West Atlas clade, although contiguous range expansion is the most predominant explanation of more recent phylogeographic effects in this species. Miocene vicariance mediated by the Atlas may provide a general explanation of intra- and interspecific biogeographical patterns in NW African species.

Phylogeography of Cape Verde Island skinks (Mabuya).

The Cape Verde Islands are of volcanic origin with most having appeared between the early Miocene and mid-Pleistocene. They contain six known species of Mabuya skinks. Phylogeographical relationships within and among the relatively widespread taxa M. stangeri, M. spinalis and M. delalandii were inferred, based on approximately 1 kbp of the cytochrome b gene (mitochondrial DNA). Reciprocal monophyly of M. spinalis and M. stangeri was established, which may have arisen from an early Pliocene/late Miocene cladogenetic event. Considerable between-island sequence divergence was detected among M. spinalis, which appears to have colonized the older islands (Sal and Boavista) first. Much lower sequence divergence was found in M. delalandii, indicating a more recent range expansion. Here, evidence points to colonization of the younger islands of Brava and Fogo soon after appearance. There are similarities between some of the described patterns and those seen in lizards from the Canary Islands.