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.

Whole snake genomes from eighteen families of snakes (Serpentes: Caenophidia) and their applications to systematics.

We present genome assemblies for 18 snake species representing 18 families (Serpentes: Caenophidia): Acrochordus granulatus, Aparallactus werneri, Boaedon fuliginosus, Calamaria suluensis, Cerberus rynchops, Grayia smithii, Imantodes cenchoa, Mimophis mahfalensis, Oxyrhabdium leporinum, Pareas carinatus, Psammodynastes pulverulentus, Pseudoxenodon macrops, Pseudoxyrhopus heterurus, Sibynophis collaris, Stegonotus admiraltiensis, Toxicocalamus goodenoughensis, Trimeresurus albolabris, and Tropidonophis doriae. From these new genome assemblies, we extracted thousands of loci commonly used in systematic and phylogenomic studies on snakes, including target-capture datasets composed of ultraconserved elements (UCEs) and anchored hybrid enriched loci (AHEs), as well as traditional Sanger loci. Phylogenies inferred from the two target-capture loci datasets were identical with each other and strongly congruent with previously published snake phylogenies. To show the additional utility of these non-model genomes for investigative evolutionary research, we mined the genome assemblies of two New Guinea island endemics in our dataset (S. admiraltiensis and T. doriae) for the ATP1a3 gene, a thoroughly researched indicator of resistance to toad toxin ingestion by squamates. We find that both these snakes possess the genotype for toad toxin resistance despite their endemism to New Guinea, a region absent of any toads until the human-mediated introduction of Cane Toads in the 1930s. These species possess identical substitutions that suggest the same bufotoxin resistance as their Australian congenerics (Stegonotus australis and Tropidonophis mairii) which forage on invasive Cane Toads. Herein, we show the utility of short-read high-coverage genomes, as well as improving the deficit of available squamate genomes with associated voucher specimens.

Multi-vendor evaluation of artificial intelligence as an independent reader for double reading in breast cancer screening on 275,900 mammograms.

BACKGROUND: Double reading (DR) in screening mammography increases cancer detection and lowers recall rates, but has sustainability challenges due to workforce shortages. Artificial intelligence (AI) as an independent reader (IR) in DR may provide a cost-effective solution with the potential to improve screening performance. Evidence for AI to generalise across different patient populations, screening programmes and equipment vendors, however, is still lacking. METHODS: This retrospective study simulated DR with AI as an IR, using data representative of real-world deployments (275,900 cases, 177,882 participants) from four mammography equipment vendors, seven screening sites, and two countries. Non-inferiority and superiority were assessed for relevant screening metrics. RESULTS: DR with AI, compared with human DR, showed at least non-inferior recall rate, cancer detection rate, sensitivity, specificity and positive predictive value (PPV) for each mammography vendor and site, and superior recall rate, specificity, and PPV for some. The simulation indicates that using AI would have increased arbitration rate (3.3% to 12.3%), but could have reduced human workload by 30.0% to 44.8%. CONCLUSIONS: AI has potential as an IR in the DR workflow across different screening programmes, mammography equipment and geographies, substantially reducing human reader workload while maintaining or improving standard of care. TRIAL REGISTRATION: ISRCTN18056078 (20/03/2019; retrospectively registered).

Evolution of the albumin protein family in reptiles.

The albumin family of proteins consists of vitamin-D binding protein/group-specific component (GC), serum albumin (ALB), alpha-fetoprotein (AFP), and afamin (AFM), which are responsible for transporting many ligands throughout the body. The albumin family proteins are physiologically and medically important, but our understanding of their functions and applications is hindered by the dearth of information regarding these proteins' evolutionary relationships and functions in non-mammalian lineages. In this study we investigate the evolution of the albumin family proteins in reptiles, using bioinformatic methods to survey available reptile genomes and transcriptomes for albumin family proteins and phylogenetically characterize their relationships. We reinforce the established evolutionary relationships among the albumin protein family in reptiles, however, they are variable in their number of domains, overall genetic sequence, and synteny. We find a novel absence of the physiologically important ALB in squamates and identify two distinct lineages of AFP, one in mammals and another in reptiles. Our study provides a comparative genomic framework for further studies identifying lineage-specific gene expansions that may compensate for the lack of serum albumin in squamates.

Habitat preference modulates trans-oceanic dispersal in a terrestrial vertebrate.

The importance of long-distance dispersal (LDD) in shaping geographical distributions has been debated since the nineteenth century. In terrestrial vertebrates, LDD events across large water bodies are considered highly improbable, but organismal traits affecting dispersal capacity are generally not taken into account. Here, we focus on a recent lizard radiation and combine a summary-coalescent species tree based on 1225 exons with a probabilistic model that links dispersal capacity to an evolving trait, to investigate whether ecological specialization has influenced the probability of trans-oceanic dispersal. Cryptoblepharus species that occur in coastal habitats have on average dispersed 13 to 14 times more frequently than non-coastal species and coastal specialization has, therefore, led to an extraordinarily widespread distribution that includes multiple continents and distant island archipelagoes. Furthermore, their presence across the Pacific substantially predates the age of human colonization and we can explicitly reject the possibility that these patterns are solely shaped by human-mediated dispersal. Overall, by combining new analytical methods with a comprehensive phylogenomic dataset, we use a quantitative framework to show how coastal specialization can influence dispersal capacity and eventually shape geographical distributions at a macroevolutionary scale.

The dynamic evolutionary history of genome size in North American woodland salamanders.

The genus Plethodon is the most species-rich salamander genus in North America, and nearly half of its species face an uncertain future. It is also one of the most diverse families in terms of genome sizes, which range from 1C = 18.2 to 69.3 pg, or 5-20 times larger than the human genome. Large genome size in salamanders results in part from accumulation of transposable elements and is associated with various developmental and physiological traits. However, genome sizes have been reported for only 25% of the species of Plethodon (14 of 55). We collected genome size data for Plethodon serratus to supplement an ongoing phylogeographic study, reconstructed the evolutionary history of genome size in Plethodontidae, and inferred probable genome sizes for the 41 species missing empirical data. Results revealed multiple genome size changes in Plethodon: genomes of western Plethodon increased, whereas genomes of eastern Plethodon decreased, followed by additional decreases or subsequent increases. The estimated genome size of P. serratus was 21 pg. New understanding of variation in genome size evolution, along with genome size inferences for previously unstudied taxa, provide a foundation for future studies on the biology of plethodontid salamanders.

Sequence capture and next-generation sequencing of ultraconserved elements in a large-genome salamander.

Amidst the rapid advancement in next-generation sequencing (NGS) technology over the last few years, salamanders have been left behind. Salamanders have enormous genomes-up to 40 times the size of the human genome-and this poses challenges to generating NGS data sets of quality and quantity similar to those of other vertebrates. However, optimization of laboratory protocols is time-consuming and often cost prohibitive, and continued omission of salamanders from novel phylogeographic research is detrimental to species facing decline. Here, we use a salamander endemic to the southeastern United States, Plethodon serratus, to test the utility of an established protocol for sequence capture of ultraconserved elements (UCEs) in resolving intraspecific phylogeographic relationships and delimiting cryptic species. Without modifying the standard laboratory protocol, we generated a data set consisting of over 600 million reads for 85 P. serratus samples. Species delimitation analyses support recognition of seven species within P. serratus sensu lato, and all phylogenetic relationships among the seven species are fully resolved under a coalescent model. Results also corroborate previous data suggesting nonmonophyly of the Ouachita and Louisiana regions. Our results demonstrate that established UCE protocols can successfully be used in phylogeographic studies of salamander species, providing a powerful tool for future research on evolutionary history of amphibians and other organisms with large genomes.

Combined next-generation sequencing and morphology reveal fine-scale speciation in Crocodile Skinks (Squamata: Scincidae: Tribolonotus).

Next-generation sequencing has vast potential to revolutionize the fields of phylogenetics and population genetics through its ability to collect genomic scale data sets of thousands of orthologous loci. Despite this potential, other types of data (e.g. morphology, ecology) remain important, particularly for studies endeavouring to delimit species. Here, we integrate next-generation sequencing with morphology to examine divergence between populations of Tribolonotus pseudoponceleti on the islands of Buka and Bougainville in the Solomon Archipelago. We used the Ion Torrent PGM to collect over 648 Mbp of sequence data for 12 samples, representing 1526 loci recovered from all samples, and 3342 were recovered from at least six samples. Genetic structure analyses strongly support the distinctiveness of these two populations, and Bayes factor delimitations decisively select speciation between Buka and Bougainville. Principal components and discriminant function analyses reveal concordant morphological divergence. Finally, demographic analyses via diffusion approximation and approximate Bayesian computation prefer a complex model of mid-Pleistocene divergence with migration, and a later decrease or cessation of migration and population size shift, suggesting a scenario in which migration was enabled by Pleistocene merging of these two islands, and limited when isolated by higher sea levels. Further analysis of four Sanger sequenced loci in IMa2 had limited power to distinguish among models including and excluding migration, but resulted in similar population size and divergence time estimates, although with much broader confidence intervals. This study represents a framework for how next-generation sequencing and morphological data can be combined and leveraged towards validating putative species and testing demographic scenarios for speciation.

Four new species of coccidia (Apicomplexa: Eimeriidae) from Owen Stanley Skinks, Papuascincus stanleyanus (Sauria: Scincidae), from Papua New Guinea.

Between September and November 1991, 12 Owen Stanley skinks, Papuascincus stanleyanus (Booulenger) were collected from various localities on Papua New Guinea and examined for coccidians. Six (50%) were found to harbour four eimerians that we describe here as new. Oocysts of Eimeria burseyi sp. n. were elongate to ellipsoidal with a bilayered wall and measured (length x width, L x W) 36.0 x 24.0 microm, with a L/W ratio of 1.5. Both micropyle and oocyst residuum were absent, but a polar granule was present. Oocysts of Eimeria goldbergi sp. n. were ellipsoidal, with a bilayered wall, and measured 21.4 x 16.1 microm; L/W ratio was 1.3. Both micropyle and oocyst residuum were absent, but a single or fragmented polar granule was present. Oocysts of Eimeria boulengeri sp. n. were spheroidal to slightly subspheroidal, with a thin, single-layered wall that readily collapses, and measured 16.0 microm, L/W ratio was 1.0. Both micropyle and oocyst residuum were absent, but usually one (sometimes two) polar granule(s) were present. Oocysts of Eimeria niuginiensis sp. n. were oblong to tapered with a bilayered wall, and measured 20.0 x 13.1 microm; L/W ratio was 1.5. A micropyle, oocyst residuum and polar granule were absent. To our knowledge, these represent the only coccidians ever described from P. stanleyanus.

A new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from the Solomon ground skink, Sphenomorphus solomonis (Boulenger) (Sauria: Scincidae) from Papua New Guinea.

Between September 1990 and November 1991, 19 Sphenomorphus spp. skinks, including nine S. jobiense, three S. simus, and seven Solomon ground skinks, S. solomonis (Boulenger), were collected from Madang and Morobe Provinces, Papua New Guinea (PNG), and examined for coccidia. A single S. solomonis was found to be infected with a new species of Eimeria Schneider, 1875. Oöcysts of Eimeria perkinsae n. sp. are ellipsoidal with a smooth, colourless, bi-layered wall, measure 18.6 × 14.7 µm, and have a length/width (L/W) ratio of 1.3; both micropyle and oöcyst residuum are absent, but a fragmented polar granule is present. Sporocysts are ovoidal, 8.9 × 6.4 µm, L/W 1.4; neither Stieda, sub-Stieda or para-Stieda bodies are present; a sporocyst residuum consisted of a loose cluster of granules dispersed between sporozoites. Sporozoites are comma-shaped with spheroidal anterior and posterior refractile bodies. This represents the first report of coccidia from this skink genus.

Establishing a Validation Infrastructure for Imaging-Based Artificial Intelligence Algorithms Before Clinical Implementation.

With promising artificial intelligence (AI) algorithms receiving FDA clearance, the potential impact of these models on clinical outcomes must be evaluated locally before their integration into routine workflows. Robust validation infrastructures are pivotal to inspecting the accuracy and generalizability of these deep learning algorithms to ensure both patient safety and health equity. Protected health information concerns, intellectual property rights, and diverse requirements of models impede the development of rigorous external validation infrastructures. The authors propose various suggestions for addressing the challenges associated with the development of efficient, customizable, and cost-effective infrastructures for the external validation of AI models at large medical centers and institutions. The authors present comprehensive steps to establish an AI inferencing infrastructure outside clinical systems to examine the local performance of AI algorithms before health practice or systemwide implementation and promote an evidence-based approach for adopting AI models that can enhance radiology workflows and improve patient outcomes.

Phylogeny and phylogeography of Mantophryne (Anura: Microhylidae) reveals cryptic diversity in New Guinea.

New Guinea is one of five high biodiversity wilderness areas, and frog diversity is exceptionally large, with more than 400 species described to date. The microhylid frog genus Mantophryne is endemic to New Guinea and consists of four species, three of which have narrow geographic distributions and a fourth, M. lateralis, with a broad range that spans the eastern half of the island. Here, we sequence 104 Mantophryne samples for three mitochondrial and three nuclear loci to reconstruct the first phylogeny of the genus and to examine spatial patterns of diversity within M. lateralis. Results indicate that the wide-ranging M. lateralis is composed of at least nine geographically separated and well-supported lineages that represent putative species. Biogeographic analysis suggests that Mantophryne evolved on the eastern Papuan peninsula with subsequent dispersal westward, as well as overwater dispersal events to the Louisiade and D'Entrecasteaux archipelagos.

A new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from Carlia spp. (Sauria: Scincidae) from Papua New Guinea.

A new species of Eimeria Schneider, 1875 from rainbow skinks, Carlia ailanpalai Zug and Carlia eothen Zug is described from specimens collected in Papua New Guinea (PNG). Oöcysts of Eimeria zugi n. sp. from one of one (100%) C. eothen are ellipsoidal to cylindroidal, with a smooth, colourless, bi-layered wall, measure 25.1 × 15.5 µm and have a length/width ratio of 1.6. The micropyle and the oöcyst residuum are absent, but a polar granule is present. The sporocysts are ovoidal to ellipsoidal and 10.3 × 7.1 µm in size and do not contain Stieda, sub-Stieda or para-Stieda bodies; and the sporocyst residuum is composed of a compact mass of large globules. The sporozoites are elongate, 12.8 × 2.9 µm in size, and contain anterior and posterior refractile bodies with a nucleus between them. This is the ninth species of coccidium described from skinks from PNG, and the new species described herein is apparently endemic to the skink genus Carlia (Gray).

Two new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from emerald tree skinks, Lamprolepis smaragdina (Lesson) (Sauria: Scincidae) from Papua New Guinea and the Philippines.

Two new species of Eimeria Schneider, 1875, from emerald tree skinks, Lamprolepis smaragdina (Lesson) are described from specimens collected in Papua New Guinea (PNG) and the Philippines. Oöcysts of Eimeria nuiailan n. sp. from the only L. smaragdina from PNG are ovoidal, with a smooth, colourless, bi-layered wall, measure 23.7 × 19.1 µm, and have a length/width (L/W) ratio of 1.3; both micropyle and oöcyst residuum are absent, but a fragmented polar granule is present. Sporocysts are ovoidal to ellipsoidal, 11.9 × 7.0 µm, L/W 1.7, and the wall is composed of two valves joined by a longitudinal suture; neither Stieda nor sub-Stieda bodies are present; a sporocyst residuum is present as a compact mass of granules. Sporozoites are elongate, 14.6 × 2.6 µm, and contain anterior and posterior refractile bodies with a nucleus between them. Oöcysts of Eimeria auffenbergi n. sp. from L. smaragdina collected in the Philippines are ovoidal, with a smooth, colourless, bi-layered wall, measure 19.9 × 15.8 µm, L/W 1.3; both micropyle and oöcyst residuum are absent, but one to four polar granules are present. Sporocysts are ovoidal to ellipsoidal, 10.3 × 5.8 µm, L/W 1.8, and the wall is composed of two valves joined by a longitudinal suture; neither Stieda nor sub-Stieda bodies are present; a sporocyst residuum is composed of dispersed granules.

The effects of sampling on delimiting species from multi-locus sequence data.

As a fundamental unit in biology, species are used in a wide variety of studies, and their delimitation impacts every subfield of the life sciences. Thus, it is of utmost importance that species are delimited in an accurate and biologically meaningful way. However, due to morphologically similar, cryptic species, and processes such as incomplete lineage sorting, this is far from a trivial task. Here, we examine the accuracy and sensitivity to sampling strategy of three recently developed methods that aim to delimit species from multi-locus DNA sequence data without a priori assignments of samples to putative species. Specifically, we simulate data at two species tree depths and a variety of sampling strategies ranging from five alleles per species and five loci to 20 alleles per species and 100 loci to test (1) Structurama, (2) Gaussian clustering, and (3) nonparametric delimitation. We find that Structurama accurately delimits even relatively recently diverged (greater than 1.5 N generations) species when sampling 10 or more loci. We also find that Gaussian clustering delimits more deeply divergent species (greater than 2.5 N generations) relatively well, but is not sufficiently sensitive to delimit more recently diverged species. Finally, we find that nonparametric delimitation performs well with 25 or more loci if gene trees are known without error, but performs poorly with estimated gene genealogies, frequently over-splitting species and mis-assigning samples. We thus suggest that Structurama represents a powerful tool for use in species delimitation. It should be noted, however, that intraspecific population structure may be delimited using this or any of the methods tested herein. We argue that other methods, such as other species delimitation methods requiring a priori putative species assignments (e.g. SpeDeSTEM, Bayesian species delimitation), and other types of data (e.g. morphological, ecological, behavioral) be incorporated in conjunction with these methods in studies attempting to delimit species.

Phylogeny, historical biogeography and body size evolution in Pacific Island Crocodile skinks Tribolonotus (Squamata; Scincidae).

Competition heavily influences the structure of island communities, particularly in species-rich areas. If ecologically similar lineages come into contact following dispersal, selection may favor rapid evolutionary change; if constraints prevent such change, lineage extinction may result. One mechanism for relieving competition among newly sympatric species is the evolution of body size differences, such as through character displacement or size assortment. The Crocodile skinks of the genus Tribolonotus exhibit a threefold variation in body size, and several species occur in sympatry. We use 2252 bp of DNA sequence data spanning two mitochondrial (cyt b and ND2) and three nuclear (C-mos, Rhodopsin and Phosducin) gene regions to reconstruct the phylogeny of Tribolonotus, use it to examine the biogeography of the genus, and test for size assortment or character displacement. We find evidence that Tribolonotus originated on either Greater Bougainville or in New Guinea, and subsequently colonized surrounding islands via multiple colonization events. Our ancestral state reconstructions support multiple instances of parallel and independent change in body size within Tribolonotus. Additionally, we find no evidence for size assortment and conflicting evidence for character displacement, which we argue suggests that character displacement, combined with ecological differences between New Guinean species (T. gracilis and T. novaeguineae), best explains the evolution of body size in the genus Tribolonotus.

A new species of Cyrtodactylus Gray (Gekkonidae: Squamata) from Manus Island, and extended description and range extension for Cyrtodactylus sermowaiensis (De Rooij).

Systematic investigations of vertebrate faunas from the islands of Melanesia are revealing high levels of endemism, dynamic biogeographic histories, and in some cases surprisingly long evolutionary histories of insularity. The bent-toed geckos in the Cyrtodactylus sermowaiensis Group mainly occur in northern New Guinea and nearby islands, however a further isolated population occurs on Manus Island in the Admiralty Archipelago approximately 300 km to the north of New Guinea. Here we first present a review of the genetic diversity, morphological variation and distribution of Cyrtodactylus sermowaiensis from northern New Guinea. Genetic structure and distributional records within Cyrtodactylus sermowaiensis broadly overlap with underlying Terranes in northern New Guinea, suggesting divergence on former islands that have been obscured by the infill and uplift of sedimentary basins since the late Pleistocene. Based on a combination of genetic and morphological differentiation we then describe the population from Manus Island as a new species, Cyrtodactylus crustulus sp. nov. This new species emphasises the high biological endemism and conservation significance of the Admiralty Islands, and especially Manus Island.

Four new species of Plasmodium from New Guinea lizards: integrating morphology and molecules.

New Guinea is one of the most biodiverse regions of the world, particularly in terms of the herpetofauna present, yet surprisingly little is known about the parasites that infect these organisms. A survey of diverse scinid and agamid lizard hosts from this country showed a diversity of malaria parasites infecting these hosts. We combined morphological and morphometric observations of the parasites (primarily gametocytes) along with DNA sequence data from the mitochondrial cytochrome b and cytochrome oxidase I genes and here describe 4 new species of Plasmodium, i.e. Plasmodium minuoviride n. sp., Plasmodium koreafense n. sp., Plasmodium megalotrypa n. sp., and Plasmodium gemini n. sp. A fifth species, Plasmodium lacertiliae Thompson and Hart 1946, is redescribed based on new observations of hosts and localities and additional molecular data. This combined morphological and molecular approach is advised for all future descriptions of new malaria parasite species, particularly in light of situations where every life-history stage is not available.

Newly discovered young CORE-SINEs in marsupial genomes.

Although recent mammalian genome projects have uncovered a large part of genomic component of various groups, several repetitive sequences still remain to be characterized and classified for particular groups. The short interspersed repetitive elements (SINEs) distributed among marsupial genomes are one example. We have identified and characterized two new SINEs from marsupial genomes that belong to the CORE-SINE family, characterized by a highly conserved "CORE" domain. PCR and genomic dot blot analyses revealed that the distribution of each SINE shows distinct patterns among the marsupial genomes, implying different timing of their retroposition during the evolution of marsupials. The members of Mar3 (Marsupialia 3) SINE are distributed throughout the genomes of all marsupials, whereas the Mac1 (Macropodoidea 1) SINE is distributed specifically in the genomes of kangaroos. Sequence alignment of the Mar3 SINEs revealed that they can be further divided into four subgroups, each of which has diagnostic nucleotides. The insertion patterns of each SINE at particular genomic loci, together with the distribution patterns of each SINE, suggest that the Mar3 SINEs have intensively amplified after the radiation of diprotodontians, whereas the Mac1 SINE has amplified only slightly after the divergence of hypsiprimnodons from other macropods. By compiling the information of CORE-SINEs characterized to date, we propose a comprehensive picture of how SINE evolution occurred in the genomes of marsupials.

Multiple origins of green blood in New Guinea lizards.

Several species of lizards from the megadiverse island of New Guinea have evolved green blood. An unusually high concentration of the green bile pigment biliverdin in the circulatory system of these lizards makes the blood, muscles, bones, tongue, and mucosal tissues bright green in color, eclipsing the crimson color from their red blood cells. This is a remarkable physiological feature because bile pigments are toxic physiological waste products of red blood cell catabolism and, when chronically elevated, cause jaundice in humans and all other vertebrates. Although these lizards offer a promising system to examine the evolution of extraordinary physiological characteristics, little is known about the phylogenetic relationships of green-blooded lizards or the evolutionary origins of green blood. We present the first extensive phylogeny for green-blooded lizards and closely related Australasian lizards using thousands of genomic regions to examine the evolutionary history of this unusual trait. Maximum likelihood ancestral character state reconstruction supports four independent origins of green blood. Our results lay the phylogenetic foundation necessary to determine the role, if any, of natural selection in shaping this enigmatic physiological trait as well as understanding the genetic, proteomic, and biochemical basis for the lack of jaundice in those species that have independently evolved green blood.