Phylogeography, genetic diversity, and population structure of Nile crocodile populations at the fringes of the southern African distribution.

Nile crocodiles are apex predators widely distributed in sub-Saharan Africa that have been viewed and managed as a single species. A complex picture of broad and fine-scale phylogeographic patterns that includes the recognition of two species (Crocodylus niloticus and Crocodylus suchus), and the structuring of populations according to river basins has started to emerge. However, previous studies surveyed a limited number of samples and geographical regions, and large areas of the continent remained unstudied. This work aimed at a fine scale portrait of Nile crocodile populations at the fringes of their geographic distribution in southern Africa. Wild and captive individuals were sampled across four major river systems (Okavango, Lower Kunene, Lower Shire and Limpopo) and the KwaZulu-Natal region. A multi-marker approach was used to infer phylogeographic and genetic diversity patterns, including new and public mitochondrial data, and a panel of 11 nuclear microsatellites. All individuals belonged to a phylogenetic clade previously associated with the C. niloticus species, thus suggesting the absence of C. suchus in southern Africa. The distribution of mitochondrial haplotypes indicated ancestral genetic connectivity across large areas, with loss of diversity along the north-south axis. Genetic variation partitioned the populations primarily into western and eastern regions of southern Africa, and secondarily into the major river systems. Populations were partitioned into five main groups corresponding to the Lower Kunene, the Okavango, the Lower Shire, and the Limpopo rivers, and the KwaZulu-Natal coastal region. All groups show evidence of recent bottlenecks and small effective population sizes. Long-term genetic diversity is likely to be compromised, raising conservation concern. These results emphasize the need for local genetic assessment of wild populations of Nile crocodiles to inform strategies for management of the species in southern Africa.

New polymorphic microsatellite loci revealed for the dusky shark Carcharhinus obscurus through Ion Proton double-digest RAD sequencing.

The non-model shark species, dusky shark Carcharhinus obscurus, is a bio-economically and recreationally important shark in many areas of its range. Despite of the fishery importance of C. obscurus few genetic resources are currently available for the species. Here, we report on the isolation of eight novel microsatellite loci from C. obscurus using a double-digest restriction site associated DNA (RAD) sequencing approach on the Ion Proton semiconductor platform (ddRADseq-ion). We characterised the loci in 26 individuals and all loci were polymorphic, exhibiting 5-10 alleles (average 6.6), and observed and expected heterozygosities of 0.385-0.962 and 0.479-0.847, respectively. We found that all pairs of loci were in linkage equilibrium and conformed to Hardy-Weinberg expectations. The loci reported in this study are only the second set of microsatellite loci ever characterized for C. obscurus and will be valuable for molecular ecology studies for this vulnerable species.

Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure.

BACKGROUND: Next generation sequencing (NGS) allows the detection of minor variant HIV drug resistance mutations (DRMs). However data from new NGS platforms after Prevention-of-Mother-to-Child-Transmission (PMTCT) regimen failure are limited. OBJECTIVE: To compare major and minor variant HIV DRMs with Illumina MiSeq and Life Technologies Ion Personal Genome Machine (PGM) in infants infected despite a PMTCT regimen. STUDY DESIGN: We conducted a cross-sectional study of NGS for detecting DRMs in infants infected despite a zidovudine (AZT) and Nevirapine (NVP) regimen, before initiation of combination antiretroviral therapy. Sequencing was performed on PCR products from plasma samples on PGM and MiSeq platforms. Bioinformatic analyses were undertaken using a codon-aware version of the Smith-Waterman mapping algorithm and a mixture multinomial error filtering statistical model. RESULTS: Of 15 infants, tested at a median age of 3.4 months after birth, 2 (13%) had non-nucleoside reverse transcriptase inhibitor (NNRTI) DRMs (K103N and Y181C) by bulk sequencing, whereas PGM detected 4 (26%) and MiSeq 5 (30%). NGS enabled the detection of additional minor variant DRMs in the infant with K103N. Coverage and instrument quality scores were higher with MiSeq, increasing the confidence of minor variant calls. CONCLUSIONS: NGS followed by bioinformatic analyses detected multiple minor variant DRMs in HIV-1 RT among infants where PMTCT failed. The high coverage of MiSeq and high read quality improved the confidence of identified DRMs and may make this platform ideal for minor variant detection.