Speciation in Howea Palms Occurred in Sympatry, Was Preceded by Ancestral Admixture, and Was Associated with Edaphic and Phenological Adaptation.

Howea palms are viewed as one of the most clear-cut cases of speciation in sympatry. The sister species Howea belmoreana and H. forsteriana are endemic to the oceanic Lord Howe Island, Australia, where they have overlapping distributions and are reproductively isolated mainly by flowering time differences. However, the potential role of introgression from Australian mainland relatives had not previously been investigated, a process that has recently put other examples of sympatric speciation into question. Furthermore, the drivers of flowering time-based reproductive isolation remain unclear. We sequenced an RNA-seq data set that comprehensively sampled Howea and their closest mainland relatives (Linospadix, Laccospadix), and collected detailed soil chemistry data on Lord Howe Island to evaluate whether secondary gene flow had taken place and to examine the role of soil preference in speciation. D-statistics analyses strongly support a scenario whereby ancestral Howea hybridized frequently with its mainland relatives, but this only occurred prior to speciation. Expression analysis, population genetic and phylogenetic tests of selection, identified several flowering time genes with evidence of adaptive divergence between the Howea species. We found expression plasticity in flowering time genes in response to soil chemistry as well as adaptive expression and sequence divergence in genes pleiotropically linked to soil adaptation and flowering time. Ancestral hybridization may have provided the genetic diversity that promoted their subsequent adaptive divergence and speciation, a process that may be common for rapid ecological speciation.

Clinical Pathology of Plastic Ingestion in Marine Birds and Relationships with Blood Chemistry.

Pollution of the environment with plastic debris is a significant and rapidly expanding threat to biodiversity due to its abundance, durability, and persistence. Current knowledge of the negative effects of debris on wildlife is largely based on consequences that are readily observed, such as entanglement or starvation. Many interactions with debris, however, result in less visible and poorly documented sublethal effects, and as a consequence, the true impact of plastic is underestimated. We investigated the sublethal effects of ingested plastic in Flesh-footed Shearwaters (Ardenna carneipes) using blood chemistry parameters as a measure of bird health. The presence of plastic had a significant negative effect on bird morphometrics and blood calcium levels and a positive relationship with the concentration of uric acid, cholesterol, and amylase. That we found blood chemistry parameters being related to plastic pollution is one of the few examples to date of the sublethal effects of marine debris and highlights that superficially healthy individuals may still experience the negative consequences of ingesting plastic debris. Moving beyond crude measures, such as reduced body mass, to physiological parameters will provide much needed insight into the nuanced and less visible effects of plastic.

Arbuscular mycorrhizal fungi promote coexistence and niche divergence of sympatric palm species on a remote oceanic island.

Microbes can have profound effects on their hosts, driving natural selection, promoting speciation and determining species distributions. However, soil-dwelling microbes are rarely investigated as drivers of evolutionary change in plants. We used metabarcoding and experimental manipulation of soil microbiomes to investigate the impact of soil and root microbes in a well-known case of sympatric speciation, the Howea palms of Lord Howe Island (Australia). Whereas H. forsteriana can grow on both calcareous and volcanic soils, H. belmoreana is restricted to, but more successful on, volcanic soil, indicating a trade-off in adaptation to the two soil types. We suggest a novel explanation for this trade-off. Arbuscular mycorrhizal fungi (AMF) are significantly depleted in H. forsteriana on volcanic soil, relative to both H. belmoreana on volcanic soil and H. forsteriana on calcareous soil. This is mirrored by the results of survival experiments, where the sterilization of natural soil reduces Howea fitness in every soil-species combination except H. forsteriana on volcanic soil. Furthermore, AMF-associated genes exhibit evidence of divergent selection between Howea species. These results show a mechanism by which divergent adaptation can have knock-on effects on host-microbe interactions, thereby reducing interspecific competition and promoting the coexistence of plant sister species.

Genomic characterization of two novel pathogenic avipoxviruses isolated from pacific shearwaters (Ardenna spp.).

BACKGROUND: Over the past 20 years, many marine seabird populations have been gradually declining and the factors driving this ongoing deterioration are not always well understood. Avipoxvirus infections have been found in a wide range of bird species worldwide, however, very little is known about the disease ecology of avian poxviruses in seabirds. Here we present two novel avipoxviruses from pacific shearwaters (Ardenna spp), one from a Flesh-footed Shearwater (A. carneipes) (SWPV-1) and the other from a Wedge-tailed Shearwater (A. pacificus) (SWPV-2). RESULTS: Epidermal pox lesions, liver, and blood samples were examined from A. carneipes and A. pacificus of breeding colonies in eastern Australia. After histopathological confirmation of the disease, PCR screening was conducted for avipoxvirus, circovirus, reticuloendotheliosis virus, and fungal agents. Two samples that were PCR positive for poxvirus were further assessed by next generation sequencing, which yielded complete Shearwaterpox virus (SWPV) genomes from A. pacificus and A. carneipes, both showing the highest degree of similarity with Canarypox virus (98% and 67%, respectively). The novel SWPV-1 complete genome from A. carneipes is missing 43 genes compared to CNPV and contains 4 predicted genes which are not found in any other poxvirus, whilst, SWPV-2 complete genome was deemed to be missing 18 genes compared to CNPV and a further 15 genes significantly fragmented as to probably cause them to be non-functional. CONCLUSION: These are the first avipoxvirus complete genome sequences that infect marine seabirds. In the comparison of SWPV-1 and -2 to existing avipoxvirus sequences, our results indicate that the SWPV complete genome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus genome isolated from avian or other natural host species, and that it likely should be considered a separate species.

Speciation with gene flow on Lord Howe Island.

Understanding the processes underlying the origin of species is a fundamental goal of biology. It is widely accepted that speciation requires an interruption of gene flow between populations: ongoing gene exchange is considered a major hindrance to population divergence and, ultimately, to the evolution of new species. Where a geographic barrier to reproductive isolation is lacking, a biological mechanism for speciation is required to counterbalance the homogenizing effect of gene flow. Speciation with initially strong gene flow is thought to be extremely rare, and few convincing empirical examples have been published. However, using phylogenetic, karyological, and ecological data for the flora of a minute oceanic island (Lord Howe Island, LHI), we demonstrate that speciation with gene flow may, in fact, be frequent in some instances and could account for one in five of the endemic plant species of LHI. We present 11 potential instances of species divergence with gene flow, including an in situ radiation of five species of Coprosma (Rubiaceae, the coffee family). These results, together with the speciation of Howea palms on LHI, challenge current views on the origin of species diversity.

Harsh environmental conditions promote cooperative behavior in an epiphytic fern.

Harsh, unpredictable environments are known to favor cooperative groups in animals. Whether plants exhibit similar relationships is unknown. Staghorn ferns (Platycerium bifurcatum, Polypodiaceae) are epiphytes that form cooperative groups which build communal water and nutrient 'nests' at the tops of trees, a habitat characterized by water and nutrient stress. We conducted field observations to test whether staghorn ferns continue to live in large, reproductively active groups after they become dislodged from the canopy and fall to the forest floor, where they are less limited by water and nutrient deprivation. To rule out the potentially confounding effects of light limitation on the forest floor, we also conducted a multi-year glasshouse experiment where we transplanted individual plants into soil and onto vertically oriented boards under standardized light conditions. Results from field observations showed that dislodged colonies formed smaller groups that reproduced less than epiphytic colonies. Results from the glasshouse experiment showed that even when growing in full sun, terrestrial individuals tended to remain solitary, while epiphytic individuals tended to recruit new individuals into colonies. Results also showed that plants growing in potting soil and exposed to full sunlight sporulated more heavily than plants growing epiphytically. However, localities that are characterized by both elevated soil and light resources are generally not available to staghorn ferns in the wild, perhaps with the exception of large, epiphytic colonies with well-developed nests at the top of tree canopies. Overall results indicate that the harsh environmental conditions at the tops of trees trigger the formation of colonies in staghorn ferns, similarly to group living animals.

Exploring the pathology of liver, kidney, muscle, and stomach of fledgling seabirds associated with plastic ingestion.

There remain significant gaps in knowledge about 'sub-lethal' impacts of plastic ingestion, particularly chronic impacts on cells, tissues, or organs. Few studies have applied traditional animal health tools, such as histopathology, to assess physiological damage to wildlife, with fewer still providing information on the dosage or exposure to plastics needed to elicit negative effects. Our study seeks to investigate a common hypothesis in plastic pollution research; that an increasing plastics burden will have an impact on an animal's health, examining two wild species with high levels of environmental exposure to plastic through their diet. Here we assess the histopathology of the muscle, upper digestive tract, liver and kidney of two seabird species that are known to be commonly exposed to plastic, comparing exposed and non-exposed individuals. Fledgling seabirds showed histopathological evidence of cumulative pressures such as starvation, disease, and endoparasite burden. However, we observed no evidence of chronic harm that could be explicitly linked to the plastics. We found one case of haemorrhage, reaffirming that large/sharp plastic foreign bodies may cause acute physical damage. Given the numerous interacting pressures on the health of fledging seabirds, including exposure to plastic, this study highlights the need to scrutinise plastic-animal interactions and research though a One Health lens.

Sympatric speciation in palms on an oceanic island.

The origin of species diversity has challenged biologists for over two centuries. Allopatric speciation, the divergence of species resulting from geographical isolation, is well documented. However, sympatric speciation, divergence without geographical isolation, is highly controversial. Claims of sympatric speciation must demonstrate species sympatry, sister relationships, reproductive isolation, and that an earlier allopatric phase is highly unlikely. Here we provide clear support for sympatric speciation in a case study of two species of palm (Arecaceae) on an oceanic island. A large dated phylogenetic tree shows that the two species of Howea, endemic to the remote Lord Howe Island, are sister taxa and diverged from each other well after the island was formed 6.9 million years ago. During fieldwork, we found a substantial disjunction in flowering time that is correlated with soil preference. In addition, a genome scan indicates that few genetic loci are more divergent between the two species than expected under neutrality, a finding consistent with models of sympatric speciation involving disruptive/divergent selection. This case study of sympatric speciation in plants provides an opportunity for refining theoretical models on the origin of species, and new impetus for exploring putative plant and animal examples on oceanic islands.

Plastics in regurgitated Flesh-footed Shearwater (Ardenna carneipes) boluses as a monitoring tool.

Plastic production and pollution of the environment with plastic items is rising rapidly and outpacing current mitigation measures. Success of mitigation actions can only be determined if progress can be measured reliably through incorporation of specific, measurable targets. Here we evaluate temporal changes in the amount and composition of plastic in boluses from Flesh-footed Shearwaters during 2002-2020 and assess their suitability for measuring progress against national and international commitments to reduce plastic pollution. Plastic in the shearwater boluses showed a generally decreasing pattern from 2002 to 2015 and increasing again to 2020. The colour and type of plastics in boluses was comparable to items recovered from live and necropsied birds, but a much smaller sample size (~35 boluses/year) was required to detect changes in plastic number and mass over time. We therefore suggest shearwater boluses are a low-effort, high-statistical power monitoring tool for quantifying progress against environmental policies in Australia.

Temporal trends and interannual variation in plastic ingestion by Flesh-footed Shearwaters (Ardenna carneipes) using different sampling strategies.

The world's oceans are under increasing pressure from anthropogenic activities, including significant and rapidly increasing inputs of plastic pollution. Seabirds have long been considered sentinels of ocean health, providing data on physical and chemical pollutants in their marine habitats. However, long-term data that can elucidate important patterns and changes in seabird exposure to marine pollutants are relatively limited but are urgently needed to identify and support effective policy measures to reduce plastic waste. Using up to 12 years of data, we examined the benefits and challenges of different approaches to monitoring plastic in seabirds, and the relationship between plastic and body size parameters. We found the mass and number of ingested plastics per bird varied by sample type, with lavage and road-kill birds containing less plastic (9.17-9.33 pieces/bird) than beach-washed or otherwise dead birds (27.62-32.22 pieces/bird). Beached birds therefore provide data for only a particular subset of the population, mostly individuals in poorer body condition, including those severely impacted by plastics. In addition, the mass and number of plastics in beached birds were more variable, therefore the sample sizes required to detect a change in plastic over time were significantly larger than for lavaged birds. The use of lavaged birds is rare in studies of plastic ingestion due to ethical and methodological implications, and we recommend future work on ingested plastics should focus on sampling this group to ensure data are more representative of a population's overall exposure to plastics.

Seabird breeding islands as sinks for marine plastic debris.

Seabirds are apex predators in the marine environment and well-known ecosystem engineers, capable of changing their terrestrial habitats by introducing marine-derived nutrients via deposition of guano and other allochthonous inputs. However, with the health of the world's oceans under threat due to anthropogenic pressures such as organic, inorganic, and physical pollutants, seabirds are depositing these same pollutants wherever they come to land. Using data from 2018 to 2020, we quantify how the Flesh-footed Shearwater (Ardenna carneipes) has inadvertently introduced physical pollutants to their colonies on Lord Howe Island, a UNESCO World Heritage site in the Tasman Sea and their largest breeding colony, through a mix of regurgitated pellet (bolus) deposition and carcasses containing plastic debris. The density of plastics within the shearwater colonies ranged between 1.32 and 3.66 pieces/m2 (mean ± SE: 2.18 ± 0.32), and a total of 688,480 (95% CI: 582,409-800,877) pieces are deposited on the island each year. Our research demonstrates that seabirds are a transfer mechanism for marine-derived plastics, reintroducing items back into the terrestrial environment, thus making seabird colonies a sink for plastic debris. This phenomenon is likely occurring in seabird colonies across the globe and will increase in severity as global plastic production and marine plastic pollution accelerates without adequate mitigation strategies.

Sympatric speciation in mountain roses (Metrosideros) on an oceanic island.

Shifts in flowering time have the potential to act as strong prezygotic reproductive barriers in plants. We investigate the role of flowering time divergence in two species of mountain rose (Metrosideros) endemic to Lord Howe Island, Australia, a minute and isolated island in the Tasman Sea. Metrosideros nervulosa and M. sclerocarpa are sister species and have divergent ecological niches on the island but grow sympatrically for much of their range, and likely speciated in situ on the island. We used flowering time and population genomic analyses of population structure and selection, to investigate their evolution, with a particular focus on the role of flowering time in their speciation. Population structure analyses showed the species are highly differentiated and appear to be in the very late stages of speciation. We found flowering times of the species to be significantly displaced, with M. sclerocarpa flowering 53 days later than M. nervulosa. Furthermore, the analyses of selection showed that flowering time genes are under selection between the species. Thus, prezygotic reproductive isolation is mediated by flowering time shifts in the species, and likely evolved under selection, to drive the completion of speciation within a small geographical area. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

How sympatric is speciation in the Howea palms of Lord Howe Island?

The two species of the palm genus Howea (Arecaceae) from Lord Howe Island, a minute volcanic island in the Tasman Sea, are now regarded as one of the most compelling examples of sympatric speciation, although this view is still disputed by some authors. Population genetic and ecological data are necessary to provide a more coherent and comprehensive understanding of this emerging model system. Here, we analyse data on abundance, juvenile recruitment, pollination mode and genetic variation and structure in both species. We find that Howea forsteriana is less abundant than Howea belmoreana. The genetic data based on amplified fragment length polymorphisms markers indicate similar levels of variation in the two species, despite the estimated census population size of H. belmoreana being three times larger than that of H. forsteriana. Genetic structure within species is low although some weak isolation by distance is detectable. Gene flow between species appears to be extremely limited and restricted to early-generation hybrids - only three admixed individuals, classified as F2s or first generation backcrosses to a parental species, were found among sampled palms. We conclude that speciation in Howea was indeed sympatric, although under certain strict definitions it may be called parapatric.

Detection of ultrafine plastics ingested by seabirds using tissue digestion.

Plastic debris is a major global threat to marine ecosystems and species. However, our knowledge of this issue may be incomplete due to a lack of a standardized method for quantifying ingested ultrafine particles (1 µm - 1 mm) in wildlife. This study provides the first quantification of ultrafine plastic in seabirds using chemical and biological digestion treatments to extract plastic items from seabird gizzards. The alkaline agent, potassium hydroxide, outperformed the enzyme corolase, based on cost and efficiency (e.g., digestion time). Ultrafine plastics were observed in 7.0% of Flesh-footed Shearwater (Ardenna carneipes) gizzards collected from Lord Howe Island, Australia and accounted for 3.6% of all plastic items recovered (13 out of 359 items). Existing methods for extracting ingested plastic from seabirds do not account for ultrafine particles, therefore our results indicate current seabird plastic loads, and the associated physical and biological impacts, are underestimated.

Ecological speciation in sympatric palms: 4. Demographic analyses support speciation of Howea in the face of high gene flow.

The idea that populations must be geographically isolated (allopatric) to evolve into separate species has persisted for a long time. It is now clear that new species can also diverge despite ongoing genetic exchange, but few accepted cases of speciation in sympatry have held up when scrutinized using modern approaches. Here, we examined evidence for speciation of the Howea palms of Lord Howe Island, Australia, in light of new genomic data. We used coalescence-based demographic models combined with double digest restriction site associated DNA sequencing of multiple individuals and provide support for previous claims by Savolainen et al. that speciation in Howea did occur in the face of gene flow.

Trace element concentrations in feathers of seven petrels (Pterodroma spp.).

Gadfly petrels (Pterodroma spp.) are one of the most threatened and poorly studied seabird groups, and as marine predators, are exposed to biomagnified and bioaccumulated chemical pollutants from their prey. We quantified trace element concentrations in breast feathers of seven petrel species that breed in the southern hemisphere to quantify current concentrations. Selenium (Se) concentrations were significantly lower in chicks than adults; this was not observed for zinc (Zn) or lead (Pb). Overall, the species examined here exhibited similar concentrations of Se, with Pb and Zn concentrations more variable among species. The mean Se concentration in adult birds exceeded those thought to be potentially deleterious, and three species had concentrations that were above the assumed threshold for Pb toxicity. Further investigation of potentially toxic trace elements in gadfly petrels is warranted.

Uncovering the sub-lethal impacts of plastic ingestion by shearwaters using fatty acid analysis.

Marine plastic pollution is increasing exponentially, impacting an expanding number of taxa each year across all trophic levels. Of all bird groups, seabirds display the highest plastic ingestion rates and are regarded as sentinels of pollution within their foraging regions. The consumption of plastic contributes to sub-lethal impacts (i.e. morbidity, starvation) in a handful of species. Additional data on these sub-lethal effects are needed urgently to better understand the scope and severity of the plastics issue. Here we explore the application of fatty acid (FA) analysis as a novel tool to investigate sub-lethal impacts of plastic ingestion on seabird body condition and health. Using gas chromatography-mass spectrometry, we identified 37 individual FAs within the adipose, breast muscle and liver of flesh-footed (Ardenna carneipes) and short-tailed (Ardenna tenuirostris) shearwaters. We found high amounts of FA 16:0, 18:0, 20:5n3 (eicosapentaenoic acid), 22:6n3 (docosahexaenoic acid) and 18:1n9 in both species; however, the overall FA composition of the two species differed significantly. In flesh-footed shearwaters, high amounts of saturated and mono-unsaturated FAs (needed for fast and slow release energy, respectively) in the adipose and muscle tissues were related to greater bird body mass. While total FAs were not related to the amount of plastic ingested in either species, these data are a valuable contribution to the limited literature on FAs in seabirds. We encourage studies to explore other analytical tools to detect these sub-lethal impacts of plastic.

Ecological speciation in sympatric palms: 3. Genetic map reveals genomic islands underlying species divergence in Howea.

Although it is now widely accepted that speciation can occur in the face of continuous gene flow, with little or no spatial separation, the mechanisms and genomic architectures that permit such divergence are still debated. Here, we examined speciation in the face of gene flow in the Howea palms of Lord Howe Island, Australia. We built a genetic map using a novel method applicable to long-lived tree species, combining it with double digest restriction site-associated DNA sequencing of multiple individuals. Based upon various metrics, we detected 46 highly differentiated regions throughout the genome, four of which contained genes with functions that are particularly relevant to the speciation scenario for Howea, specifically salt and drought tolerance.

Ingestion of marine debris by Wedge-tailed Shearwaters (Ardenna pacifica) on Lord Howe Island, Australia during 2005-2018.

Annual rates of plastic production have been increasing rapidly since the 1950s. Inadequate or improper disposal of plastic products has contributed to a significant increase in plastic debris in the world's oceans and a corresponding increase in the number of species negatively affected by this debris. Here we investigate trends in the type, amount, and colour of ingested plastic over time, and determine whether ingested plastic contributes to reduced health of Wedge-tailed Shearwaters (Ardenna pacifica) on Lord Howe Island, Australia. The results show no clear influence of ingested plastic on body condition, while trends in the prevalence, number, and mass of plastic items ingested per bird during 2005 and 2013-2018 were more variable. There was some evidence adult birds are selecting plastic by colour. Future monitoring of this pan-tropical seabird would provide a unique opportunity to gather data from multiple sites, concurrently.