Genetic variation and admixture of red-eared sliders (Trachemys scripta elegans) in the USA.

The most ubiquitous, abundant, and invasive turtle on Earth, Trachemys scripta elegans (TSE, "red-eared slider"), is one of four taxa in a clade that is native to the USA and adjacent Mexico (three subspecies of Trachemys scripta plus Trachemys gaigeae). The present range-wide study of this clade is based on 173 known-locality mtDNA sequences combined with ddRAD libraries for 43 samples emphasizing the western part of the range of TSE, its contact with that of T. gaigeae, and anthropogenic hybrids between TSE and T. s. scripta. The data presented here are the first to sample the TSE × T. s. scripta intergrade zone or TSE × T. s. scripta crosses from introduced turtles. In the western part of its range (New Mexico and Texas), most samples of TSE from the Pecos River have mtDNA haplotypes matching T. gaigeae. Structure analysis of SNPs from the ddRAD show evidence of genetic admixture between T. gaigeae and TSE in all included samples from the Rio Grande and Pecos River. These populations also exhibit T. gaigeae-like head stripes, i.e., a postorbital marking that does not reach the eye. The genetic and morphological data are thereby reconciled, as both suggest that these TSE are intergrades. We recommend that these populations continue to be considered TSE, despite the admixture with T. gaigeae. In the Eastern United States, some samples of the morphologically intermediate subspecies T. s. troostii are not genetically distinct from TSE and some samples share morphological characters and genetic affinities with T. s. scripta. Based on these observations we conclude that the taxon T. s. troostii represents intergrades between TSE and T. s. scripta and should not be considered a valid taxon. Near the already established part of the intergrade zone between TSE and T. s. scripta, TSE mtDNA haplotypes have naturally introgressed into typical-looking samples of T. s. scripta in Georgia. Hybrids between introduced TSE and T. s. scripta are also confirmed deeper within the natural range of T. s. scripta in South Carolina and Virginia. Given the examples of feral hybrids deep within its range shown here and elsewhere, the threat of genetic pollution of T. s. scripta by feral TSE is established.

Molecular phylogeny and patterns of diversification in syngnathid fishes.

The family Syngnathidae is a large and diverse clade of morphologically unique bony fishes, with 57 genera and 300 described species of seahorses, pipefishes, pipehorses, and seadragons. They primarily inhabit shallow coastal waters in temperate and tropical oceans, and are characterized by a fused jaw, male brooding, and extraordinary crypsis. Phylogenetic relationships within the Syngnathidae remain poorly resolved due to lack of generic taxon sampling, few diagnostic morphological characters, and limited molecular data. The phylogenetic placement of the threatened, commercially exploited seahorses remains a topic of intense interest, with conflicting topologies based on morphology and predominantly mitochondrial genetic data. In this study, we integrate eight nuclear and mitochondrial markers and 17 morphological characters to investigate the phylogenetic structure of the family Syngnathidae at the generic level. We include 91 syngnathid species representing 48 of the 57 recognized genera, all major ocean basins, and a broad array of temperate and tropical habitats including rocky and coral reefs, sand and silt, mangroves, seagrass beds, estuaries, and rivers. Maximum likelihood and Bayesian analyses of 5160bp from eight loci produced high congruence among alternate topologies, defining well-supported and sometimes novel clades. We present a hypothesis that confirms a deep phylogenetic split between lineages with trunk- or tail-brood pouch placement, and provides significant new insights into the morphological evolution and biogeography of this highly derived fish clade. Based on the fundamental division between lineages - the tail brooding "Urophori" and the trunk brooding "Gastrophori" - we propose a revision of Syngnathidae classification into only two subfamilies: the Nerophinae and the Syngnathinae. We find support for distinct principal clades within the trunk-brooders and tail-brooders, the latter of which include seahorses, seadragons, independent lineages of pipehorses, and clades that originated in southern Australia and the Western Atlantic. We suggest the seahorse genus Hippocampus is of Indo-Pacific origin and its sister clade is an unexpected grouping of several morphologically disparate Indo-Pacific genera, including the Pacific pygmy pipehorses. Taxonomic revision is required for multiple genera, particularly to reflect deep evolutionary splits in nominal lineages from the Atlantic versus the Indo-Pacific.

A phylogenomic analysis of turtles.

Molecular analyses of turtle relationships have overturned prevailing morphological hypotheses and prompted the development of a new taxonomy. Here we provide the first genome-scale analysis of turtle phylogeny. We sequenced 2381 ultraconserved element (UCE) loci representing a total of 1,718,154bp of aligned sequence. Our sampling includes 32 turtle taxa representing all 14 recognized turtle families and an additional six outgroups. Maximum likelihood, Bayesian, and species tree methods produce a single resolved phylogeny. This robust phylogeny shows that proposed phylogenetic names correspond to well-supported clades, and this topology is more consistent with the temporal appearance of clades and paleobiogeography. Future studies of turtle phylogeny using fossil turtles should use this topology as a scaffold for their morphological phylogenetic analyses.

Population Structure and Seasonal Migration of the Spotted Eagle Ray, Aetobatus narinari.

Few studies have reported on the fine-scale population genetics of batoid species in the Atlantic basin. Here, we investigate the genetic diversity and population structure of the spotted eagle ray, Aetobatus narinari, sampled in the northeastern and southwestern parts of the Gulf of Mexico and in the northwestern Caribbean Sea. Samples were collected from 286 individuals sampled across 3 geographic localities. Estimates of divergence based on the mitochondrial cytochrome b gene and 10 nuclear microsatellite loci reveal weak but significant genetic structure among A. narinari populations in this region. Analysis of molecular variance estimates based on both marker types indicate significant differentiation between Florida and Mexico populations, while comparisons with Cuba suggest high levels of gene flow with rays from both Mexico and Florida. Conflicting results were found from the different marker types when sexes were analyzed separately underscoring the importance of applying multiple marker types when making inferences about population structure and sex-biased dispersal. Results from Bayesian clustering analyses suggest rays may be migrating south out of the Gulf of Mexico and into the northwestern Caribbean Sea. Given the impacts of fisheries on this species, coupled with the lack of population genetic data available, these findings offer valuable information to aid with conservation management strategies.

Genetic analyses of historic and modern marbled murrelets suggest decoupling of migration and gene flow after habitat fragmentation.

The dispersal of individuals among fragmented populations is generally thought to prevent genetic and demographic isolation, and ultimately reduce extinction risk. In this study, we show that a century of reduction in coastal old-growth forests, as well as a number of other environmental factors, has probably resulted in the genetic divergence of marbled murrelets (Brachyramphus marmoratus) in central California, despite the fact that 7 per cent of modern-sampled murrelets in this population were classified as migrants using genetic assignment tests. Genetic differentiation appears to persist because individuals dispersing from northern populations contributed relatively few young to the central California population, as indicated by the fact that migrants were much less likely to be members of parent-offspring pairs than residents (10.5% versus 45.4%). Moreover, a recent 1.4 per cent annual increase in the proportion of migrants in central California, without appreciable reproduction, may have masked an underlying decline in the resident population without resulting in demographic rescue. Our results emphasize the need to understand the behaviour of migrants and the extent to which they contribute offspring in order to determine whether dispersal results in gene flow and prevents declines in resident populations.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: A retrospective cohort study of patients with and without COVID-19.

BACKGROUND: Most data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have been presented as case series without comparison to patients with other acute respiratory illnesses. METHODS: We examined emergency department patients between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared clinical presentation, diagnostics, treatment, and outcomes. FINDINGS: Among 316 patients, 33 tested positive for SARS-CoV-2; 31 without COVID-19 tested positive for another respiratory virus. Among patients with additional viral testing (27/33), no SARS-CoV-2 co-infections were identified. Compared to those who tested negative, patients with COVID-19 reported longer symptoms duration (median 7d vs. 3d, p < 0.001). Patients with COVID-19 were more often hospitalized (79% vs. 56%, p = 0.014). When hospitalized, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p < 0.001) and more often developed ARDS (23% vs. 3%, p < 0.001). Most comorbidities, medications, symptoms, vital signs, laboratories, treatments, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. FUNDING: National Center for Advancing Translational Sciences, National Heart Lung Blood Institute, National Institute of Allergy and Infectious Diseases, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: a comparison of patients with and without COVID-19.

BACKGROUND: Emerging data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have largely been presented as case series. Few studies have compared these clinical features and outcomes of COVID-19 to other acute respiratory illnesses. METHODS: We examined all patients presenting to an emergency department in San Francisco, California between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared demographics, comorbidities, symptoms, vital signs, and laboratory results including viral diagnostics using PCR and mNGS. Among those hospitalized, we determined differences in treatment (antibiotics, antivirals, respiratory support) and outcomes (ICU admission, ICU interventions, acute respiratory distress syndrome, cardiac injury). FINDINGS: In a cohort of 316 patients, 33 (10%) tested positive for SARS-CoV-2; 31 patients, all without COVID-19, tested positive for another respiratory virus (16%). Among patients with additional viral testing, no co-infections with SARS-CoV-2 were identified by PCR or mNGS. Patients with COVID-19 reported longer symptoms duration (median 7 vs. 3 days), and were more likely to report fever (82% vs. 44%), fatigue (85% vs. 50%), and myalgias (61% vs 27%); p<0.001 for all comparisons. Lymphopenia (55% vs 34%, p=0.018) and bilateral opacities on initial chest radiograph (55% vs. 24%, p=0.001) were more common in patients with COVID-19. Patients with COVID-19 were more often hospitalized (79% vs. 56%, p=0.014). Of 186 hospitalized patients, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p<0.001) and were more likely to develop ARDS (23% vs. 3%, p<0.001). Most comorbidities, home medications, signs and symptoms, vital signs, laboratory results, treatment, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. These findings enhance understanding of the clinical characteristics of COVID-19 in comparison to other acute respiratory illnesses. .

Characterizing source-sink dynamics with genetic parentage assignments.

Source-sink dynamics have been suggested to characterize the population structure of many species, but the prevalence of source-sink systems in nature is uncertain because of inherent challenges in estimating migration rates among populations. Migration rates are often difficult to estimate directly with demographic methods, and indirect genetic methods are subject to a variety of assumptions that are difficult to meet or to apply to evolutionary timescales. Furthermore, such methods cannot be rigorously applied to high-gene-flow species. Here, we employ genetic parentage assignments in conjunction with demographic simulations to infer the level of immigration into a putative sink population. We use individual-based demographic models to estimate expected distributions of parent-offspring dyads under competing sink and closed-population models. By comparing the actual number of parent-offspring dyads (identified from multilocus genetic profiles) in a random sample of individuals taken from a population to expectations under these two contrasting demographic models, it is possible to estimate the rate of immigration and test hypotheses related to the role of immigration on population processes on an ecological timescale. The difference in the expected number of parent-offspring dyads between the two population models was greatest when immigration into the sink population was high, indicating that unlike traditional population genetic inference models, the highest degree of statistical power is achieved for the approach presented here when migration rates are high. We used the proposed genetic parentage approach to demonstrate that a threatened population of Marbled Murrelets (Braclhyrarmphus marmotus) appears to be supplemented by a low level of immigration (approximately 2-6% annually) from other populations.

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.

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.

PERMANENT GENETIC RESOURCES: Isolation and characterization of 10 tetranucleotide microsatellite loci in an enigmatic East African bird, the spot-throat (Modulatrix stictigula).

We describe the isolation of 10 tetranucleotide microsatellites from the spot-throat using an enrichment protocol. All loci were highly variable with the number of alleles ranging from six to 20 and observed heterozygosity ranging from 0.410 to 0.940. Although all loci were in Hardy-Weinberg equilibrium, locus Mst95 showed significant homozygote excess in both surveyed populations, possibly a consequence of the presence of null alleles at this locus. These loci will be used to determine the extent to which spot-throat populations are isolated in order to help set conservation priorities for this ancient African lineage.

Isolation and characterization of 10 tetranucleotide microsatellite loci from the yellow-streaked greenbul (Phyllastrephus flavostriatus) and cross-species amplification in four closely related taxa.

We describe the isolation of 10 tetranucleotide microsatellite loci from the yellow-streaked greenbul using an enrichment protocol. All loci were highly variable with the number of alleles ranging from 8 to 13, and observed heterozygosity ranging from 0.652 to 0.870. All loci were in Hardy-Weinberg equilibrium; however, loci Pfl12 and Pfl54 showed significant linkage disequilibrium. All 10 loci successfully amplified and were polymorphic in at least one of four related Phyllastrephus species. These loci should prove to be widely applicable to studies of phylogeography, hybridization and paternity in African greenbuls.