Pervasive relaxed selection in termite genomes.

Genetic changes that enabled the evolution of eusociality have long captivated biologists. More recently, attention has focussed on the consequences of eusociality on genome evolution. Studies have reported higher molecular evolutionary rates in eusocial hymenopteran insects compared with their solitary relatives. To investigate the genomic consequences of eusociality in termites, we analysed nine genomes, including newly sequenced genomes from three non-eusocial cockroaches. Using a phylogenomic approach, we found that termite genomes have experienced lower rates of synonymous substitutions than those of cockroaches, possibly as a result of longer generation times. We identified higher rates of non-synonymous substitutions in termite genomes than in cockroach genomes, and identified pervasive relaxed selection in the former (24-31% of the genes analysed) compared with the latter (2-4%). We infer that this is due to reductions in effective population size, rather than gene-specific effects (e.g. indirect selection of caste-biased genes). We found no obvious signature of increased genetic load in termites, and postulate efficient purging of deleterious alleles at the colony level. Additionally, we identified genomic adaptations that may underpin caste differentiation, such as genes involved in post-translational modifications. Our results provide insights into the evolution of termites and the genomic consequences of eusociality more broadly.

The spotted parrotfish genome provides insights into the evolution of a coral reef dietary specialist (Teleostei: Labridae: Scarini: Cetoscarus ocellatus).

With over 600 valid species, the wrasses (family Labridae) are among the largest and most successful families of the marine teleosts. They feature prominently on coral reefs where they are known not only for their impressive diversity in colouration and form but also for their functional specialisation and ability to occupy a wide variety of trophic guilds. Among the wrasses, the parrotfishes (tribe Scarini) display some of the most dramatic examples of trophic specialisation. Using abrasion-resistant biomineralized teeth, parrotfishes are able to mechanically extract protein-rich micro-photoautotrophs growing in and among reef carbonate material, a dietary niche that is inaccessible to most other teleost fishes. This ability to exploit an otherwise untapped trophic resource is thought to have played a role in the diversification and evolutionary success of the parrotfishes. In order to better understand the key evolutionary innovations leading to the success of these dietary specialists, we sequenced and analysed the genome of a representative species, the spotted parrotfish (Cetoscarus ocellatus). We find significant expansion, selection and duplications within several detoxification gene families and a novel poly-glutamine expansion in the enamel protein ameloblastin, and we consider their evolutionary implications. Our genome provides a useful resource for comparative genomic studies investigating the evolutionary history of this highly specialised teleostean radiation.

The genome of a globally invasive passerine, the common myna, Acridotheres tristis.

In an era of global climate change, biodiversity conservation is receiving increased attention. Conservation efforts are greatly aided by genetic tools and approaches, which seek to understand patterns of genetic diversity and how they impact species health and their ability to persist under future climate regimes. Invasive species offer vital model systems in which to investigate questions regarding adaptive potential, with a particular focus on how changes in genetic diversity and effective population size interact with novel selection regimes. The common myna (Acridotheres tristis) is a globally invasive passerine and is an excellent model species for research both into the persistence of low-diversity populations and the mechanisms of biological invasion. To underpin research on the invasion genetics of this species, we present the genome assembly of the common myna. We describe the genomic landscape of this species, including genome wide allelic diversity, methylation, repeats, and recombination rate, as well as an examination of gene family evolution. Finally, we use demographic analysis to identify that some native regions underwent a dramatic population increase between the two most recent periods of glaciation, and reveal artefactual impacts of genetic bottlenecks on demographic analysis.

Phylogeography of the Sunda pangolin, Manis javanica: Implications for taxonomy, conservation management and wildlife forensics.

The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD-seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the 'out of Borneo hypothesis', whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range-wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.

Tracing the introduction of the invasive common myna using population genomics.

The common myna (Acridotheres tristis) is one of the most invasive bird species in the world, yet its colonisation history is only partly understood. We identified the introduction history and population structure, and quantified the genetic diversity of myna populations from the native range in India and introduced populations in New Zealand, Australia, Fiji, Hawaii, and South Africa, based on thousands of single nucleotide polymorphism markers in 814 individuals. We were able to identify the source population of mynas in several invasive locations: mynas from Fiji and Melbourne, Australia, were likely founded by individuals from a subpopulation in Maharashtra, India, while mynas in Hawaii and South Africa were likely independently founded by individuals from other localities in India. Our findings suggest that New Zealand mynas were founded by individuals from Melbourne, which, in turn, were founded by individuals from Maharashtra. We identified two genetic clusters among New Zealand mynas, divided by New Zealand's North Island's axial mountain ranges, confirming previous observations that mountains and thick forests may form barriers to myna dispersal. Our study provides a foundation for other population and invasion genomic studies and provides useful information for the management of this invasive species.

Social network analysis reveals context-dependent kin relationships in wild sulphur-crested cockatoos Cacatua galerita.

A preference to associate with kin facilitates inclusive fitness benefits, and increased tolerance or cooperation between kin may be an added benefit of group living. Many species exhibit preferred associations with kin; however, it is often hard to disentangle active preferences from passive overlap, for example caused by limited dispersal or inheritance of social position. Many parrots exhibit social systems consisting of pair-bonded individuals foraging in variably sized fission-fusion flocks within larger communal roosts of hundreds of individuals. Previous work has shown that, despite these fission-fusion dynamics, individuals can exhibit long-term preferred foraging associations outside their pair bonds. Yet the underlying drivers of these social preferences remain largely unknown. In this study, we use a network approach to examine the influence of kinship on social associations and interactions in wild, communally roosting sulphur-crested cockatoos, Cacatua galerita. We recorded roost co-membership, social associations and interactions in 561 individually marked birds across three neighbouring roosts. We then collected genetic samples from 205 cockatoos, and conducted a relationship analysis to construct a kinship network. Finally, we tested correlations between kinship and four social networks: association, affiliative, low-intensity aggression and high-intensity aggression. Our result showed that while roosting groups were clearly defined, they showed little genetic differentiation or kin structuring. Between roost movement was high, with juveniles, especially females, repeatedly moving between roosts. Both within roosting communities, and when visiting different roosts, individuals preferentially associated with kin. Supporting this, individuals were also more likely to allopreen kin. However, contrary to expectation, individuals preferred to direct aggression towards kin, with this effect only observed when individuals shared roost membership. By measuring social networks within and between large roosting groups, we could remove potential effects of passive spatial overlap on kin structuring. Our study reveals that sulphur-crested cockatoos actively prefer to associate with kin, both within and between roosting groups. By examining this across different interaction types, we further demonstrate that sulphur-crested cockatoos exhibit behavioural and context-dependent interaction rules towards kin. Our results help reveal the drivers of social association in this species, while adding to the evidence for social complexity in parrots.

Genomic analysis reveals strong population structure in the Giant Sydney Crayfish (Euastacus spinifer (Heller, 1865)).

Australia is home to over 140 species of freshwater crayfish (Decapoda: Parastacidae), representing a centre of diversity for this group in the Southern Hemisphere. Species delimitation in freshwater crayfish is difficult because many species show significant variation in colouration and morphology. This is particularly evident in the genus Euastacus, which exhibits large variations in colour and spination throughout its putative range. To understand this variation, we investigated the genetic diversity, population structure, phylogeny, and evolutionary timescale of the Giant Sydney Crayfish (Euastacus spinifer (Heller, 1865)). Our data set is sampled from over 70 individuals from across the ∼600 km range of the species, and includes a combination of two mitochondrial markers and more than 7000 single-nucleotide polymorphisms (SNPs) from the nuclear genome. Data were also obtained for representatives of the close relative, Euastacus vesper McCormack and Ahyong, 2017. Genomic SNP analyses revealed strong population structure, with multiple distinct populations showing little evidence of gene flow or migration. Phylogenetic analyses of mitochondrial data revealed similar structure between populations. Taken together, our analyses suggest that E. spinifer, as currently understood, represents a species complex, of which E. vesper is a member. Molecular clock estimates place the divergences within this group during the Pleistocene. The isolated and highly fragmented populations identified in our analyses probably represent relict populations of a previously widespread ancestral species. Periodic flooding events during the Pleistocene are likely to have facilitated the movement of these otherwise restricted freshwater crayfish within and between drainage basins, including the Murray-Darling and South East Coast Drainages. We present evidence supporting the recognition of populations in the southern parts of the range of E. spinifer as one or two separate species, which would raise the number of species within the E. spinifer complex to at least three. Our results add to the growing body of evidence that many freshwater crayfish exhibit highly fragmented, range-restricted distributions. In combination with the life-history traits of these species, the restricted distributions exacerbate the threats already placed on freshwater crayfish, which are among the five most endangered animal groups globally.

Correction: Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

Advances in sequencing technologies have revolutionized wildlife conservation genetics. Analysis of genomic data sets can provide high-resolution estimates of genetic structure, genetic diversity, gene flow, and evolutionary history. These data can be used to characterize conservation units and to effectively manage the genetic health of species in a broad evolutionary context. Here we utilize thousands of genome-wide single-nucleotide polymorphisms (SNPs) and mitochondrial DNA to provide the first genetic assessment of the Australian red-tailed black-cockatoo (Calyptorhynchus banksii), a widespread bird species comprising populations of varying conservation concern. We identified five evolutionarily significant units, which are estimated to have diverged during the Pleistocene. These units are only partially congruent with the existing morphology-based subspecies taxonomy. Genetic clusters inferred from mitochondrial DNA differed from those based on SNPs and were less resolved. Our study has a range of conservation and taxonomic implications for this species. In particular, we provide advice on the potential genetic rescue of the Endangered and restricted-range subspecies C. b. graptogyne, and propose that the western C. b. samueli population is diagnosable as a separate subspecies. The results of our study highlight the utility of considering the phylogeographic relationships inferred from genome-wide SNPs when characterizing conservation units and management priorities, which is particularly relevant as genomic data sets become increasingly accessible.

Expediting the sampling, decalcification, and forensic DNA analysis of large elephant ivory seizures to aid investigations and prosecutions.

The illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action.

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species.

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome-wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red-tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site-associated DNA marker approach (DArTseq™ ). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq-based method to produce reliable genome-wide SNP data from traditional museum specimens.

Correction: A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

[This corrects the article DOI: 10.1371/journal.pone.0198565.].

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as 'vulnerable' due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala's ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala's survival in the wild.

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future 'rapid species identification tests' can be trialed.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.