Genome Assembly of Pyrocephalus nanus: A Step Toward the Genetic Conservation of the Endangered Little Vermilion Flycatcher of the Galapagos Islands.

Incredibly powerful whole genome studies of conservation genetics, evolution, and biogeography become possible for non-model organisms when reference genomes are available. Here, we report the sequence and assembly of the whole genome of the little vermilion flycatcher (Pyrocephalus nanus; family Tyrannidae), which is an endemic, endangered, and declining species of the Galapagos Islands. Using PacBio HiFi reads to assemble long contigs and Hi-C reads for scaffolding, we assembled a genome of 1.07 Gb comprising 267 contigs in 152 scaffolds, scaffold N50 74 M, contig N50 17.8 M, with 98.9% assigned to candidate chromosomal sequences and 99.72% of the BUSCO passeriformes 10,844 single-copy orthologs present. In addition, we used the novel HiFiMiTie pipeline to fully assemble and verify all portions of the mitochondrial genome from HiFi reads, obtaining a mitogenome of 17,151 bases, containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, two control regions, and a unique structure of control region duplication and repeats. These genomes will be a critical tool for much-needed studies of phylogenetics, population genetics, biogeography, and conservation genetics of Pyrocephalus and related genera. This genome and other studies that use it will be able to provide recommendations for conservation management, taxonomic improvement, and to understand the evolution and diversification of this genus within the Galapagos Islands.

Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

APPARENT PREVALENCE, DIVERSITY, AND ASSOCIATED LESIONS OF PERIORBITAL NEMATODES IN A POPULATION OF BARRED OWLS (STRIX VARIA) FROM NORTHERN CALIFORNIA, USA.

Over the last four decades, Barred Owls (Strix varia) have expanded their range to include much of western North America, including California. This expansion is suspected to have contributed to declining populations of a closely related species, the federally threatened Northern Spotted Owl (Strix occidentalis caurina). As a result, understanding potential health threats to Barred Owls has implications for Spotted Owl health and recovery. From 2016 to 2020, 69 Barred Owls were collected to determine the apparent prevalence of periorbital nematode infection, to identify the parasite species present, and to investigate the potential pathologic effects on their hosts. The nematodes were morphologically identified as Oxyspirura and Aprocta spp. On the basis of phylogenetic analyses, they were clearly divergent from published sequences of other species within these genera. Overall, 34 (49%) Barred Owls were infected with periorbital nematodes, with Oxyspirura sp. infections being much more common (94%) than Aprocta sp. (18%). Histopathology revealed varying severity of conjunctivitis in infected owls. Despite the frequency of infection and subsequent inflammation, parasite burden was not associated with reduced body weight in these owls. As a result, the potential health effect of these nematodes is unclear. Further taxonomic characterization is needed to determine potential novelty of these nematodes.

Prevalence and diversity of avian haemosporidian parasites across islands of Milne Bay Province, Papua New Guinea.

The taxonomically diverse and relatively understudied avifauna of Papua New Guinea's (PNG) island archipelagos provide a unique ecological framework for studying haemosporidian parasite differentiation and geographic structure. We implemented molecular and phylogenetic analyses of partial mitochondrial DNA sequences to assess the host distribution of 3 genera of vector-transmitted avian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) across a range of islands off the southeastern tip of PNG. We identified 40 new lineages of haemosporidians, including five lineages belonging to Leucocytozoon, a genus not previously described in this region. Leucocytozoon infections were only observed on the larger, human-inhabited islands. Lineages belonging to Haemoproteus were diverse and had broad geographic distribution. Compared to the mainland, Haemoproteus parasites on the smaller, more distant islands had greater host specificity and lower infection prevalence. The black sunbird (Leptocoma aspasia), a commonly caught species, was shown to be a rare host for Haemoproteus spp. infections. Moreover, although birds of the genus Pitohui harbor a neurotoxin (homobatrachotoxin), they demonstrated an infection prevalence comparable to other bird species. The islands of PNG display heterogeneous patterns of haemosporidian diversity, distribution and host-specificity and serve as a valuable model system for studying host-parasite-vector interactions.

Population connectivity across a highly fragmented distribution: Phylogeography of the Chalcophaps doves.

Chalcophaps is a morphologically conserved genus of ground-walking doves distributed from India to mainland China, south to Australia, and across the western Pacific to Vanuatu. Here, we reconstruct the evolutionary history of this genus using DNA sequence data from two nuclear genes and one mitochondrial gene, sampled from throughout the geographic range of Chalcophaps. We find support for three major evolutionary lineages in our phylogenetic reconstruction, each corresponding to the three currently recognized Chalcophaps species. Despite this general concordance, we identify discordant mitochondrial and nuclear ancestries in the subspecies C. longirostris timorensis, raising further questions about the evolutionary history of this Timor endemic population. Within each of the three species, we find evidence for isolation by distance or hierarchical population structure, indicating an important role for geography in the diversification of this genus. Despite being distributed broadly across a highly fragmented geographic region known as a hotspot for avian diversification, the Chalcophaps doves show modest levels of phenotypic and genetic diversity, a pattern potentially explained by strong population connectivity owing to high overwater dispersal capability.

Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on "toxin sponge" proteins.

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX "toxin sponge" protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

Genomic Variation and Recent Population Histories of Spotted (Strix occidentalis) and Barred (Strix varia) Owls.

Spotted owls (SOs, Strix occidentalis) are a flagship species inhabiting old-growth forests in western North America. In recent decades, their populations have declined due to ongoing reductions in suitable habitat caused by logging, wildfires, and competition with the congeneric barred owl (BO, Strix varia). The northern spotted owl (S. o. caurina) has been listed as "threatened" under the Endangered Species Act since 1990. Here, we use an updated SO genome assembly along with 51 high-coverage whole-genome sequences to examine population structure, hybridization, and recent changes in population size in SO and BO. We found that potential hybrids identified from intermediate plumage morphology were a mixture of pure BO, F1 hybrids, and F1 × BO backcrosses. Also, although SO underwent a population bottleneck around the time of the Pleistocene-Holocene transition, their population sizes rebounded and show no evidence of any historical (i.e., 100-10,000 years ago) population decline. This suggests that the current decrease in SO abundance is due to events in the past century. Finally, we estimate that western and eastern BOs have been genetically separated for thousands of years, instead of the previously assumed recent (i.e., <150 years) divergence. Although this result is surprising, it is unclear where the ancestors of western BO lived after the separation. In particular, although BO may have colonized western North America much earlier than the first recorded observations, it is also possible that the estimated divergence time reflects unsampled BO population structure within central or eastern North America.

Speciation and gene flow across an elevational gradient in New Guinea kingfishers.

Closely related species with parapatric elevational ranges are ubiquitous in tropical mountains worldwide. The gradient speciation hypothesis proposes that these series are the result of in situ ecological speciation driven by divergent selection across elevation. Direct tests of this scenario have been hampered by the difficulty inferring the geographic arrangement of populations at the time of divergence. In cichlids, sticklebacks and Timema stick insects, support for ecological speciation driven by other selective pressures has come from demonstrating parallel speciation, where divergence proceeds independently across replicated environmental gradients. Here, we take advantage of the unique geography of the island of New Guinea to test for parallel gradient speciation in replicated populations of Syma kingfishers that show extremely subtle differentiation across elevation and between historically isolated mountain ranges. We find that currently described high-elevation and low-elevation species have reciprocally monophyletic gene trees and form nuclear DNA clusters, rejecting this hypothesis. However, demographic modelling suggests selection has likely maintained species boundaries in the face of gene flow following secondary contact. We compile evidence from the published literature to show that although in situ gradient speciation in labile organisms such as birds appears rare, divergent selection and post-speciation gene flow may be an underappreciated force in the origin of elevational series and tropical beta diversity along mountain slopes.

Whole-Genome Analysis of Introgression Between the Spotted Owl and Barred Owl (Strix occidentalis and Strix varia, Respectively; Aves: Strigidae) in Western North America.

As the barred owl (Strix varia; Aves: Strigiformes: Strigidae) expands throughout western North America, hybridization between barred and spotted owls (Strix varia and S. occidentalis, respectively), if abundant, may lead to genetic swamping of the endangered spotted owl. We analyzed low-coverage, whole-genome sequence data from fifty-one barred and spotted owls to investigate recent introgression between these two species. Although we obtained genomic confirmation that these species can and do hybridize and backcross, we found no evidence of widespread introgression. Plumage characteristics of western S. varia that suggested admixture with S. occidentalis appear unrelated to S. occidentalis ancestry and may instead reflect local selection.

Complete mitochondrial genome sequences of the northern spotted owl (Strix occidentalis caurina) and the barred owl (Strix varia; Aves: Strigiformes: Strigidae) confirm the presence of a duplicated control region.

We report here the successful assembly of the complete mitochondrial genomes of the northern spotted owl (Strix occidentalis caurina) and the barred owl (S. varia). We utilized sequence data from two sequencing methodologies, Illumina paired-end sequence data with insert lengths ranging from approximately 250 nucleotides (nt) to 9,600 nt and read lengths from 100-375 nt and Sanger-derived sequences. We employed multiple assemblers and alignment methods to generate the final assemblies. The circular genomes of S. o. caurina and S. varia are comprised of 19,948 nt and 18,975 nt, respectively. Both code for two rRNAs, twenty-two tRNAs, and thirteen polypeptides. They both have duplicated control region sequences with complex repeat structures. We were not able to assemble the control regions solely using Illumina paired-end sequence data. By fully spanning the control regions, Sanger-derived sequences enabled accurate and complete assembly of these mitochondrial genomes. These are the first complete mitochondrial genome sequences of owls (Aves: Strigiformes) possessing duplicated control regions. We searched the nuclear genome of S. o. caurina for copies of mitochondrial genes and found at least nine separate stretches of nuclear copies of gene sequences originating in the mitochondrial genome (Numts). The Numts ranged from 226-19,522 nt in length and included copies of all mitochondrial genes except tRNAPro , ND6, and tRNAGlu . Strix occidentalis caurina and S. varia exhibited an average of 10.74% (8.68% uncorrected p-distance) divergence across the non-tRNA mitochondrial genes.

Northern Spotted Owl (Strix occidentalis caurina) Genome: Divergence with the Barred Owl (Strix varia) and Characterization of Light-Associated Genes.

We report here the assembly of a northern spotted owl (Strix occidentalis caurina) genome. We generated Illumina paired-end sequence data at 90× coverage using nine libraries with insert lengths ranging from ∼250 to 9,600 nt and read lengths from 100 to 375 nt. The genome assembly is comprised of 8,108 scaffolds totaling 1.26 × 109 nt in length with an N50 length of 3.98 × 106 nt. We calculated the genome-wide fixation index (FST) of S. o. caurina with the closely related barred owl (Strix varia) as 0.819. We examined 19 genes that encode proteins with light-dependent functions in our genome assembly as well as in that of the barn owl (Tyto alba). We present genomic evidence for loss of three of these in S. o. caurina and four in T. alba. We suggest that most light-associated gene functions have been maintained in owls and their loss has not proceeded to the same extent as in other dim-light-adapted vertebrates.

Evaluating hybridization capture with RAD probes as a tool for museum genomics with historical bird specimens.

Laboratory techniques for high-throughput sequencing have enhanced our ability to generate DNA sequence data from millions of natural history specimens collected prior to the molecular era, but remain poorly tested at shallower evolutionary time scales. Hybridization capture using restriction site-associated DNA probes (hyRAD) is a recently developed method for population genomics with museum specimens. The hyRAD method employs fragments produced in a restriction site-associated double digestion as the basis for probes that capture orthologous loci in samples of interest. While promising in that it does not require a reference genome, hyRAD has yet to be applied across study systems in independent laboratories. Here, we provide an independent assessment of the effectiveness of hyRAD on both fresh avian tissue and dried tissue from museum specimens up to 140 years old and investigate how variable quantities of input DNA affect sequencing, assembly, and population genetic inference. We present a modified bench protocol and bioinformatics pipeline, including three steps for detection and removal of microbial and mitochondrial DNA contaminants. We confirm that hyRAD is an effective tool for sampling thousands of orthologous SNPs from historic museum specimens to describe phylogeographic patterns. We find that modern DNA performs significantly better than historical DNA better during sequencing but that assembly performance is largely equivalent. We also find that the quantity of input DNA predicts %GC content of assembled contiguous sequences, suggesting PCR bias. We caution against sampling schemes that include taxonomic or geographic autocorrelation across modern and historic samples.

Reconstructing the molecular phylogeny of giant sengis (Macroscelidea; Macroscelididae; Rhynchocyon).

Giant sengis (Macroscelidea; Macroscelididae; Rhynchocyon), also known as giant elephant-shrews, are small-bodied mammals that range from central through eastern Africa. Previous research on giant sengi systematics has relied primarily on pelage color and geographic distribution. Because some species have complex phenotypic variation and large geographic ranges, we used molecular markers to evaluate the phylogeny and taxonomy of the genus, which currently includes four species: R. chrysopygus, R. cirnei (six subspecies), R. petersi (two subspecies), and R. udzungwensis. We extracted DNA from fresh and historical museum samples from all taxa except one R. cirnei subspecies, and we generated and analyzed approximately 4700 aligned nucleotides (2685 bases of mitochondrial DNA and 2019 bases of nuclear DNA) to reconstruct a molecular phylogeny. We genetically evaluate Rhynchocyon spp. sequences previously published on GenBank, propose that the captive R. petersi population in North American zoos is likely R. p. adersi, and suggest that hybridization among taxa is not widespread in Rhynchocyon. The DNA sample we have from the distinctive but undescribed giant sengi from the Boni forest of northern coastal Kenya is unexpectedly nearly identical to R. chrysopygus, which will require further study. Our analyses support the current morphology-based taxonomy, with each recognized species forming a monophyletic clade, but we propose elevating R. c. stuhlmanni to a full species.

Novel Picornavirus Associated with Avian Keratin Disorder in Alaskan Birds.

UNLABELLED: Avian keratin disorder (AKD), characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees (Poecile atricapillus) in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100%) AKD-affected individuals were positive, while only 2/9 (22%) control individuals were infected with poecivirus. Two northwestern crows (Corvus caurinus) and two red-breasted nuthatches (Sitta canadensis) with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD. IMPORTANCE: Avian keratin disorder (AKD) is an increasingly common disease of wild birds. This disease, characterized by beak overgrowth, was first described in the late 1990s and has been spreading rapidly both geographically and in terms of host species affected. AKD decreases host fitness and can be fatal. However, the cause of the disease has remained elusive, and its impact on host populations is poorly understood. We found a novel and divergent picornavirus in 19/19 AKD-affected black-capped chickadees that we examined but in only 2/9 control cases. We also found this virus in 4 individuals of 2 other passerine species that exhibited symptoms consistent with AKD. Our data suggest that this novel picornavirus warrants further investigation as the causative agent of AKD.

Petrosaltator gen. nov., a new genus replacement for the North African sengi Elephantulus rozeti (Macroscelidea; Macroscelididae).

In 2003, a molecular phylogeny was published that examined the role of the Sahara Desert as a vicariant event in the evolution of sengis (also known as elephant-shrews.) The phylogeny included a single sample from the North African sengi, Elephantulus rozeti (Duvernoy, 1833), which was found to be more closely related to the sengi genus Petrodromus Peters, 1846 than to other Elephantulus. Here we independently test the monophyly of Elephantulus using an additional specimen of E. rozeti and similar phylogenetic analyses, and discuss additional morphological and behavioral data that support the phylogeny. We propose a revised taxonomy that reflects the current paraphyly of Elephantulus and the sister relationship of E. rozeti and Petrodromus, including a new genus name for the North African sengi, Petrosaltator rozeti gen. nov., nov. comb. We additionally define two tribes within the subfamily Macroscelidinae, the Macroscelidini (including Macroscelides, Petrodromus, and Petrosaltator), and the Elephantulini (including all other members of Elephantulus).

Phylogeography of the Vermilion Flycatcher species complex: Multiple speciation events, shifts in migratory behavior, and an apparent extinction of a Galápagos-endemic bird species.

The Vermilion Flycatcher (Pyrocephalus rubinus) is a widespread species found in North and South America and the Galápagos. Its 12 recognized subspecies vary in degree of geographic isolation, phenotypic distinctness, and migratory status. Some authors suggest that Galápagos subspecies nanus and dubius constitute one or more separate species. Observational reports of distinct differences in song also suggest separate species status for the austral migrant subspecies rubinus. To evaluate geographical patterns of diversification and taxonomic limits within this species complex, we carried out a molecular phylogenetic analysis encompassing 10 subspecies and three outgroup taxa using mitochondrial (ND2, Cyt b) and nuclear loci (ODC introns 6 through 7, FGB intron 5). We used samples of preserved tissues from museum collections as well as toe pad samples from museum skins. Galápagos and continental clades were recovered as sister groups, with initial divergence at ∼1mya. Within the continental clade, North and South American populations were sister groups. Three geographically distinct clades were recovered within South America. We detected no genetic differences between two broadly intergrading North American subspecies, mexicanus and flammeus, suggesting they should not be recognized as separate taxa. Four western South American subspecies were also indistinguishable on the basis of loci that we sampled, but occur in a region with patchy habitat, and may represent recently isolated populations. The austral migrant subspecies, rubinus, comprised a monophyletic mitochondrial clade and had many unique nuclear DNA alleles. In combination with its distinct song, exclusive song recognition behavior, different phenology, and an isolated breeding range, our data suggests that this taxon represents a separate species from other continental populations. Mitochondrial and nuclear genetic data, morphology, and behavior suggest that Galápagos forms should be elevated to two full species corresponding to the two currently recognized subspecies, nanus and dubius. The population of dubius is presumed to be extinct, and thus would represent the first documented extinction of a Galápagos-endemic bird species. Two strongly supported mitochondrial clades divide Galápagos subspecies nanus in a geographic pattern that conflicts with previous hypotheses that were based on plumage color. Several populations of nanus have recently become extinct or are in serious decline. Urgent conservation measures should seek to preserve the deep mitochondrial DNA diversity within nanus, and further work should explore whether additional forms should be recognized within nanus. Ancestral states analysis based on our phylogeny revealed that the most recent common ancestor of extant Vermilion Flycatcher populations was migratory, and that migratory behavior was lost more often than gained within Pyrocephalus and close relatives, as has been shown to be the case within Tyrannidae as a whole.

Bird-Window Collisions at a West-Coast Urban Park Museum: Analyses of Bird Biology and Window Attributes from Golden Gate Park, San Francisco.

Bird-window collisions are a major and poorly-understood generator of bird mortality. In North America, studies of this topic tend to be focused east of the Mississippi River, resulting in a paucity of data from the Western flyways. Additionally, few available data can critically evaluate factors such as time of day, sex and age bias, and effect of window pane size on collisions. We collected and analyzed 5 years of window strike data from a 3-story building in a large urban park in San Francisco, California. To evaluate our window collision data in context, we collected weekly data on local bird abundance in the adjacent parkland. Our study asks two overarching questions: first-what aspects of a bird's biology might make them more likely to fatally strike windows; and second, what characteristics of a building's design contribute to bird-window collisions. We used a dataset of 308 fatal bird strikes to examine the relationships of strikes relative to age, sex, time of day, time of year, and a variety of other factors, including mitigation efforts. We found that actively migrating birds may not be major contributors to collisions as has been found elsewhere. We found that males and young birds were both significantly overrepresented relative to their abundance in the habitat surrounding the building. We also analyzed the effect of external window shades as mitigation, finding that an overall reduction in large panes, whether covered or in some way broken up with mullions, effectively reduced window collisions. We conclude that effective mitigation or design will be required in all seasons, but that breeding seasons and migratory seasons are most critical, especially for low-rise buildings and other sites away from urban migrant traps. Finally, strikes occur throughout the day, but mitigation may be most effective in the morning and midday.

Genetic differentiation within a widespread "supertramp" taxon: Molecular phylogenetics of the Louisiade White-eye (Zosterops griseotinctus).

"Supertramp" species are highly specialized overwater dispersers, and are useful taxa for investigating the influence of dispersal ability on speciation and diversification in island settings. The Louisiade White-eye (Zosterops griseotinctus) is a widespread avian supertramp endemic to Papua New Guinea's offshore islands. We used maximum likelihood and Bayesian inference to reconstruct phylogenetic relationships based on 2 mitochondrial and 1 nuclear loci (1813 bp total) from 88 individuals representing all 4 named subspecies and the full breadth of the species' range. We found significant geographic and population genetic structure, and support for a major clade containing the coral islets of the central Louisiade Archipelago and outlying Nissan Island. We found evidence of metapopulation structure and gene flow within the Louisiade Archipelago clade, and relatively high genetic distinctiveness of outlying island populations, including the population on volcanically-defaunated Long Island. We reject a hypothesis of panmixia within the Louisiade White-eye despite their long-range dispersal ability, and find evidence of selection against dispersal ability in populations on high-elevation islands where disturbance is rare. Our study represents a rare intraspecies phylogeny of an avian supertramp, and sheds light on patterns of evolution in highly vagile island species.