The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine, the hihi.

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or "SNP chips", enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50 K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10 WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536 hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species.

Polygenic basis for adaptive morphological variation in a threatened Aotearoa | New Zealand bird, the hihi (Notiomystis cincta).

To predict if a threatened species can adapt to changing selective pressures, it is crucial to understand the genetic basis of adaptive traits, especially in species historically affected by severe bottlenecks. We estimated the heritability of three hihi (Notiomystis cincta) morphological traits known to be under selection (nestling tarsus length, body mass and head-bill length) using 523 individuals and 39 699 single nucleotide polymorphisms (SNPs) from a 50 K Affymetrix SNP chip. We then examined the genetic architecture of the traits via chromosome partitioning analyses and genome-wide association scans (GWAS). Heritabilities estimated using pedigree relatedness or genomic relatedness were low. For tarsus length, the proportion of genetic variance explained by each chromosome was positively correlated with its size, and more than one chromosome explained significant variation for body mass and head-bill length. Finally, GWAS analyses suggested many loci of small effect contributing to trait variation for all three traits, although one locus (an SNP within an intron of the transcription factor HEY2) was tentatively associated with tarsus length. Our findings suggest a polygenic nature for the morphological traits, with many small effect size loci contributing to the majority of the variation, similar to results from many other wild populations. However, the small effective population size, polygenic architecture and already low heritabilities suggest that both the total response and rate of response to selection are likely to be limited in hihi.

Assemblage of Focal Species Recognizers-AFSR: A technique for decreasing false indications of presence from acoustic automatic identification in a multiple species context.

Passive acoustic monitoring (PAM) coupled with automated species identification is a promising tool for species monitoring and conservation worldwide. However, high false indications of presence are still an important limitation and a crucial factor for acceptance of these techniques in wildlife surveys. Here we present the Assemblage of Focal Species Recognizers-AFSR, a novel approach for decreasing false positives and increasing models' precision in multispecies contexts. AFSR focusses on decreasing false positives by excluding unreliable sound file segments that are prone to misidentification. We used MatlabHTK, a hidden Markov models interface for bioacoustics analyses, for illustrating AFSR technique by comparing two approaches, 1) a multispecies recognizer where all species are identified simultaneously, and 2) an assemblage of focal species recognizers (AFSR), where several recognizers that each prioritise a single focal species are then summarised into a single output, according to a set of rules designed to exclude unreliable segments. Both approaches (the multispecies recognizer and AFSR) used the same sound files training dataset, but different processing workflow. We applied these recognisers to PAM recordings from a remote island colony with five seabird species and compared their outputs with manual species identifications. False positives and precision improved for all the five species when using AFSR, achieving remarkable 0% false positives and 100% precision for three of five seabird species, and < 6% false positives, and >90% precision for the other two species. AFSR' output was also used to generate daily calling activity patterns for each species. Instead of attempting to withdraw useful information from every fragment in a sound recording, AFSR prioritises more trustworthy information from sections with better quality data. AFSR can be applied to automated species identification from multispecies PAM recordings worldwide.

Gene Fusions Derived by Transcriptional Readthrough are Driven by Segmental Duplication in Human.

Gene fusion occurs when two or more individual genes with independent open reading frames becoming juxtaposed under the same open reading frame creating a new fused gene. A small number of gene fusions described in detail have been associated with novel functions, for example, the hominid-specific PIPSL gene, TNFSF12, and the TWE-PRIL gene family. We use Sequence Similarity Networks and species level comparisons of great ape genomes to identify 45 new genes that have emerged by transcriptional readthrough, that is, transcription-derived gene fusion. For 35 of these putative gene fusions, we have been able to assess available RNAseq data to determine whether there are reads that map to each breakpoint. A total of 29 of the putative gene fusions had annotated transcripts (9/29 of which are human-specific). We carried out RT-qPCR in a range of human tissues (placenta, lung, liver, brain, and testes) and found that 23 of the putative gene fusion events were expressed in at least one tissue. Examining the available ribosome foot-printing data, we find evidence for translation of three of the fused genes in human. Finally, we find enrichment for transcription-derived gene fusions in regions of known segmental duplication in human. Together, our results implicate chromosomal structural variation brought about by segmental duplication with the emergence of novel transcripts and translated protein products.

Little Adaptive Potential in a Threatened Passerine Bird.

Threatened species face numerous threats, including future challenges triggered by global change. A possible way to cope with these challenges is through adaptive evolution, which requires adaptive potential. Adaptive potential is defined as the genetic variance needed to respond to selection and can be assessed either on adaptive traits or fitness [1]. However, a lack of high-quality data has made it difficult to rigorously test adaptive potential in threatened species, leading to controversy over its magnitude [1-3]. Here we assess the adaptive potential of a threatened New Zealand passerine (the hihi, Notiomystis cincta) based on two populations: (1) the sole remaining natural population, on the island of Te Hauturu-o-Toi, and (2) a reintroduced population with a long-term dataset (intensively monitored for 20 years) based on the island of Tiritiri Matangi. We use molecular information (reduced representation genome sequencing, on both populations), as well as long-term phenotypic and fitness data from the Tiritiri Matangi population, to find (1) a lack of molecular genetic diversity at a genome-wide level in both populations, (2) low heritability of traits under selection and (3) negligible additive genetic variance of fitness in the Tiritiri Matangi population. In combination, these results support a lack of adaptive potential in this threatened species. We discuss our findings within the context of other passerines and methods for assessing adaptive potential, as well as the impact of these results on conservation practice, for the hihi and species of conservation concern in general.