Ultraconserved Elements Research Articles

Mitochondrial genomes and thousands of ultraconserved elements resolve the taxonomy and historical biogeography of the Euphonia and Chlorophonia finches (Passeriformes: Fringillidae)

Abstract Relationships of the Neotropical finches in the genera Euphonia and Chlorophonia (Fringillidae: Euphoniinae) have been clarified by recent molecular studies, but species-level relationships within this group have not been thoroughly addressed. In this study, we sampled specimens representing every recognized species of these genera, in addition to 2 outgroup taxa, and used target enrichment to sequence thousands of ultraconserved element (UCE) loci, as well as mitochondrial DNA reconstructed from off-target reads, from each specimen to infer these relationships. We constructed both concatenation and coalescent-based estimates of phylogeny from this dataset using matrices of varying levels of completeness, and we generated a time-scaled ultrametric tree using a recently published fossil-based external calibration. We found uniformly strong support for a monophyletic subfamily Euphoniinae and genus Chlorophonia, but a paraphyletic Euphonia across UCEs and mitochondrial genomes. Otherwise, our inferred relationships were largely concordant with previous studies. Our time-tree indicated a stem divergence time of 13.8 million years ago for this lineage, followed by a relatively young crown age of only 7.1 myr. Reconstructions of biogeographic history based on this tree suggest a South American origin for crown Euphoniinae, possibly resulting from a transoceanic dispersal event from the Eastern Hemisphere, followed by 2 dispersal events into the Caribbean and as many as 6 invasions of North America coinciding with recent estimates of the age at which the Isthmus of Panama had completely formed. We recommend splitting Euphonia and resurrecting the genus Cyanophonia for the 3 blue-hooded species more closely related to Chlorophonia. Based on our results, we suspect that there is undescribed species-level diversity in at least one, possibly many, widespread and phenotypically diverse species.

Ancient DNA From Museum Specimens and Next Generation Sequencing Help Resolve the Controversial Evolutionary History of the Critically Endangered Puebla Deer Mouse

Key insights into the evolutionary history of recently extinct or critically endangered species can be obtained through analysis of genomic data collected using high-throughput sequencing and ancient DNA from museum specimens, particularly where specimens are rare. For instance, the evolutionary history of the critically endangered Puebla deer mouse, Peromyscus mekisturus, remains unclear due to discordance between morphological and molecular phylogenetic analyses. However, previous molecular analyses were based on PCR and Sanger sequencing of only a few mitochondrial genes. Here, we used ancient DNA from historical museum specimens followed by target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and whole mitochondrial genomes to test the validity of the previous phylogenetic placement of P. mekisturus. Based on UCEs and mitogenomes, our results revealed that P. mekisturus forms a well-supported distinct lineage outside the clade containing all other members of the Peromyscus melanophrys group. Additionally, the mitogenome phylogeny further supports the placement of P. mekisturus as the sister species of the genus Reithrodontomys. This conflicts with the previous mtDNA phylogenetic reconstruction, in which P. mekisturus was nested within the species P. melanophrys. Our study demonstrates that high-throughput sequencing of ancient DNA, appropriately controlling for contamination and degradation, can provide a robust resolution of the evolutionary history and taxonomic status of species for which few or no modern genetic samples exist. In light of our results and pending further analysis with denser taxon sampling and the addition of morphological data, a re-evaluation of the taxonomy and conservation management plans of P. mekisturus is needed to ensure that the evolutionary distinctiveness of this species is recognized in future conservation efforts.

Sitticine jumping spiders: phylogeny, classification, and chromosomes (Araneae, Salticidae, Sitticini).

The systematics of sitticine jumping spiders is reviewed, with a focus on the Palearctic and Nearctic regions, in order to revise their generic classification, clarify the species of one region (Canada), and study their chromosomes. A genome-wide molecular phylogeny of 23 sitticine species, using more than 700 loci from the arachnid Ultra-Conserved Element (UCE) probeset, confirms the Neotropical origins of sitticines, whose basal divergence separates the new subtribeAillutticina (a group of five Neotropical genera) from the subtribe Sitticina (five genera of Eurasia and the Americas). The phylogeny shows that most Eurasian sitticines form a relatively recent and rapid radiation, which we unite into the genus Attulus Simon, 1868, consisting of the subgenera Sitticus Simon, 1901 (seven described species), Attulus (41 described species), and Sittilong Prószyński, 2017 (one species). Five species of Attulus occur natively in North America, presumably through dispersals back from the Eurasian radiation, but an additional three species were more recently introduced from Eurasia. Attus palustris Peckham & Peckham, 1883 is considered to be a full synonym of Euophrys floricola C. L. Koch, 1837 (not a distinct subspecies). Attus sylvestris Emerton, 1891 is removed from synonymy and recognized as a senior synonym of Sitticus magnus Chamberlin & Ivie, 1944. Thus, the five native Attulus in North America are Attulus floricola, A. sylvestris, A. cutleri, A. striatus, and A. finschi. The other sitticines of Canada and the U.S.A. are placed in separate genera, all of which arose from a Neotropical radiation including Jollas Simon, 1901 and Tomis F.O.Pickard-Cambridge, 1901: (1) Attinella Banks, 1905 (A. dorsata, A. concolor, A. juniperi), (2) Tomis (T. welchi), and (3) Sittisax Prószyński, 2017 (S. ranieri). All Neotropical and Caribbean “Sitticus” are transferred to either Jollas (12 species total) or Tomis (14 species). Attinella (three species) and Tomis are both removed from synonymy with Sitticus; the synonymy of Sitticus cabellensis Prószyński, 1971 with Pseudattulus kratochvili Caporiacco, 1947 is restored; Pseudattulus Caporiacco, 1947 is synonymized with Tomis. Six generic names are newly synonymized with Attulus and one with Attinella. Two Neotropical species are described as new, Jollas cupreussp. nov. and Tomis manabitasp. nov. Forty-six new combinations are established and three are restored. Three species synonymies are restored, one is new, and two are rejected. Across this diversity of species is a striking diversification of chromosome complements, with X-autosome fusions occurring at least four times to produce neo-Y sex chromosome systems (X1X2Y and X1X2X3Y), some of which (Sittisax ranieri and S. saxicola) are sufficiently derived as to no longer preserve the simple traces of ancestral X material. The correlated distribution of neo-Y and a base autosome number of 28 suggests that neo-Y origins occurred preferentially in lineages with the presence of an extra pair of autosomes.

Phylogeography of the Variable Antshrike (Thamnophilus caerulescens), a South American passerine distributed along multiple environmental gradients

The Neotropics show a wealth of distributional patterns shared by many co-distributed species. A distinctive pattern is the so-called “circum-Amazonian distribution,” which is observed in species that do not occur in Amazonia but rather along a belt of forested habitats spanning south and east of Amazonia, the Andean foothills, and often into the Venezuelan Coastal Range and the Tepuis. Although this pattern is widespread across animals and plants, its underlying biogeographic mechanisms remain poorly understood. The Variable Antshrike (Thamnophilus caerulescens) is a sexually dimorphic suboscine passerine that exhibits extreme plumage variation and occurs along the southern portion of the circum-Amazonian belt. We describe broad-scale phylogeographic patterns of T. caerulescens and assess its demographic history using DNA sequences from the mitochondrion and ultraconserved elements (UCEs). We identified three genomic clusters: a) northern Atlantic Forest; b) southeastern Cerrado and central-southern Atlantic Forest, and c) Chaco and Andes. Our results were consistent with Pleistocene divergence followed by gene flow, mainly between the latter two clusters. There were no genetic signatures of rapid population expansions or bottlenecks. The population from the northern Atlantic Forest was the most genetically divergent group within the species. The demographic history of T. caerulescens was probably affected by series of humid and dry periods throughout the Quaternary that generated subtle population expansions and contractions allowing the intermittent connection of habitats along the circum-Amazonian belt. Recognizing the dynamic history of climate-mediated forest expansions, contractions, and connections during the South American Pleistocene is central toward a mechanistic understanding of circum-Amazonian distributions.

Systematics, biogeography, and diversification of Scytalopus tapaculos (Rhinocryptidae), an enigmatic radiation of Neotropical montane birds

Abstract We studied the phylogeny, biogeography, and diversification of suboscine passerines in the genus Scytalopus (Rhinocryptidae), a widespread, species-rich, and taxonomically challenging group of Neotropical birds. We analyzed nuclear (exons, regions flanking ultraconserved elements) and mitochondrial (ND2) DNA sequence data for a taxonomically and geographically comprehensive sample of specimens collected from Costa Rica to Patagonia and Brazil. We found that Scytalopus is a monophyletic group sister to Eugralla and consists of 3 main clades roughly distributed in (1) the Southern Andes, (2) eastern Brazil, and (3) the Tropical Andes and Central America. The clades from the Southern Andes and eastern Brazil are sister to each other. Despite their confusing uniformity in plumage coloration, body shape, and overall appearance, rates of species accumulation through time in Scytalopus since the origin of the clade in the Late Miocene are unusually high compared with those of other birds, suggesting rapid non-adaptive diversification in the group. We attribute this to their limited dispersal abilities making them speciation-prone and their occurrence in a complex landscape with numerous barriers promoting allopatric differentiation. Divergence times among species and downturns in species accumulation rates in recent times suggest that most speciation events in Scytalopus predate climatic oscillations of the Pleistocene. Our analyses identified various cases of strong genetic structure within species and lack of monophyly of taxa, flagging populations which likely merit additional study to clarify their taxonomic status. In particular, detailed analyses of species limits are due in S. parvirostris, S. latrans, S. speluncae, the S. atratus complex, and the Southern Andes clade.

A first phylogenomic hypothesis for Eulophidae (Hymenoptera, Chalcidoidea)

ABSTRACT Eulophidae is a hyper-diverse family of chalcidoid wasps with 324 genera, about 5300 described species and probably thousands of others to be described. Until now, the absence of unequivocal morphological apomorphies and the low resolution provided by the handful of Sanger sequenced genes have hampered the reconstruction of phylogenetic relationships within the family. Here, we used ultra-conserved elements and their flanking regions to resolve relationships among 84 species of eulophids included in 63 genera representing all subfamilies and most tribes, plus 15 outgroups. Our analyses recover all traditional Eulophidae subfamilies and tribes with high support and globally agree with the traditional classification of the family. Our results confirm that Eulophinae + Tetrastichinae is the sister group of (Opheliminae + Entiinae) + Entedoninae. At the generic level, our analyses provide high support for intergeneric relationships for which morphology and Sanger markers previously failed to provide resolution. Our results also confirm that Trisecodes does not group with Eulophidae and may not belong to this family; however, its correct classification still awaits a large-scale phylogenomic hypothesis for Chalcidoidea. This work opens new avenues towards a better understanding of the evolutionary history, biogeography and evolution of host–parasitoid associations in this hyper-diverse family of chalcidoid wasps.

Whole genome phylogeny of Gallus: introgression and data-type effects

BackgroundPrevious phylogenetic studies that include the four recognized species of Gallus have resulted in a number of distinct topologies, with little agreement. Several factors could lead to the failure to converge on a consistent topology, including introgression, incomplete lineage sorting, different data types, or insufficient data.MethodsWe generated three novel whole genome assemblies for Gallus species, which we combined with data from the published genomes of Gallus gallus and Bambusicola thoracicus (a member of the sister genus to Gallus). To determine why previous studies have failed to converge on a single topology, we extracted large numbers of orthologous exons, introns, ultra-conserved elements, and conserved non-exonic elements from the genome assemblies. This provided more than 32 million base pairs of data that we used for concatenated maximum likelihood and multispecies coalescent analyses of Gallus.ResultsAll of our analyses, regardless of data type, yielded a single, well-supported topology. We found some evidence for ancient introgression involving specific Gallus lineages as well as modest data type effects that had an impact on support and branch length estimates in specific analyses. However, the estimated gene tree spectra for all data types had a relatively good fit to their expectation given the multispecies coalescent.ConclusionsOverall, our data suggest that conflicts among previous studies probably reflect the use of smaller datasets (both in terms of number of sites and of loci) in those analyses. Our results demonstrate the importance of sampling large numbers of loci, each of which has a sufficient number of sites to provide robust estimates of gene trees. Low-coverage whole genome sequencing, as we did here, represents a cost-effective means to generate the very large data sets that include multiple data types that enabled us to obtain a robust estimate of Gallus phylogeny.

Opening the door to greater phylogeographic inference in Southeast Asia: Comparative genomic study of five codistributed rainforest bird species using target capture and historical DNA.

Indochina and Sundaland are biologically diverse, interconnected regions of Southeast Asia with complex geographic histories. Few studies have examined phylogeography of bird species that span the two regions because of inadequate population sampling. To determine how geographic barriers/events and disparate dispersal potential have influenced the population structure, gene flow, and demographics of species that occupy the entire area, we studied five largely codistributed rainforest bird species: Arachnothera longirostra, Irena puella, Brachypodius atriceps, Niltava grandis, and Stachyris nigriceps. We accomplished relatively thorough sampling and data collection by sequencing ultraconserved elements (UCEs) using DNA extracted from modern and older (historical) specimens. We obtained a genome‐wide set of 753–4,501 variable loci and 3,919–18,472 single nucleotide polymorphisms. The formation of major within‐species lineages occurred within a similar span of time (0.5–1.5 mya). Major patterns in population genetic structure are largely consistent with the dispersal potential and habitat requirements of the study species. A population break across the Isthmus of Kra was shared only by the two hill/submontane insectivores (N. grandis and S. nigriceps). Across Sundaland, there is little structure in B. atriceps, which is a eurytopic and partially frugivorous species that often utilizes forest edges. Two other eurytopic species, A. longirostra and I. puella, possess highly divergent populations in peripheral Sunda Islands (Java and/or Palawan) and India. These species probably possess intermediate dispersal abilities that allowed them to colonize new areas, and then remained largely isolated subsequently. We also observed an east–west break in Indochina that was shared by B. atriceps and S. nigriceps, species with very different habitat requirements and dispersal potential. By analyzing high‐throughput DNA data, our study provides an unprecedented comparative perspective on the process of avian population divergence across Southeast Asia, a process that is determined by geography, species characteristics, and the stochastic nature of dispersal and vicariance events.

Streamlining universal single-copy orthologue and ultraconserved element design: A case study in Collembola.

Genomic data sets are increasingly central to ecological and evolutionary biology, but far fewer resources are available for invertebrates. Powerful new computational tools and the rapidly decreasing cost of Illumina sequencing are beginning to change this, enabling rapid genome assembly and reference marker extraction. We have developed and tested a practical workflow for developing genomic resources in nonmodel groups with real-world data on Collembola (springtails), one of the most dominant soil animals on Earth. We designed universal molecular marker sets, single-copy orthologues (BUSCOs) and ultraconserved elements (UCEs), using three existing and 11 newly generated genomes. Both marker types were tested in silico via marker capture success and phylogenetic performance. The new genomes were assembled with Illumina short reads and 9,585-14,743 protein-coding genes were predicted with ab initio and protein homology evidence. We identified 1,997 benchmarking universal single-copy orthologues (BUSCOs) across 14 genomes and created and assessed a custom BUSCO data set for extracting single-copy genes. We also developed a new UCE probe set containing 46,087 baits targeting 1,885 loci. We successfully captured 1,437-1,865 BUSCOs and 975-1,186 UCEs across 14 genomes. Phylogenomic reconstructions using these markers proved robust, giving new insight on deep-time collembolan relationships. Our study demonstrates the feasibility of generating thousands of universal markers from highly efficient whole-genome sequencing, providing a valuable resource for genome-scale investigations in evolutionary biology and ecology.

Investigating the utility of traditional and genomic multi-locus datasets to resolve relationships in Lipaugus and Tijuca (Cotingidae)

Rapid diversification limits our ability to resolve evolutionary relationships and examine diversification history, as in the case of the Neotropical cotingas. Here we present an analysis with complete taxon sampling for the cotinga genera Lipaugus and Tijuca, which include some of the most range-restricted (e.g., T. condita) and also the most widespread and familiar (e.g., L. vociferans) forest birds in the Neotropics. We used two datasets: (1) Sanger sequencing data sampled from eight loci in 34 individuals across all described taxa and (2) sequence capture data linked to 1,079 ultraconserved elements and conserved exons sampled from one or two individuals per species. Phylogenies estimated from the Sanger sequencing data failed to resolve three nodes, but the sequence capture data produced a well-supported tree. Lipaugus and Tijuca formed a single, highly supported clade, but Tijuca species were not sister and were embedded within Lipaugus. A dated phylogeny confirmed Lipaugus and Tijuca diversified rapidly in the Miocene. Our study provides a detailed evolutionary hypothesis for Lipaugus and Tijuca and demonstrates that increasing genomic sampling can prove instrumental in resolving the evolutionary history of recent radiations.

Phylogenomic Species Delimitation, Taxonomy, and ‘Bird Guide’ Identification for the Neotropical Ant Genus Rasopone (Hymenoptera: Formicidae)

Abstract Rasopone Schmidt and Shattuck is a poorly known lineage of ants that live in Neotropical forests. Informed by phylogenetic results from thousands of ultraconserved elements (UCEs) and mitochondrial DNA barcodes, we revise the genus, providing a new morphological diagnosis and a species-level treatment. Analysis of UCE data from many Rasopone samples and select outgroups revealed non-monophyly of the genus. Monophyly of Rasopone was restored by transferring several species to the unrelated genus Mayaponera Schmidt and Shattuck. Within Rasopone, species are morphologically very similar, and we provide a ‘bird guide’ approach to identification rather than the traditional dichotomous key. Species are arranged by size in a table, along with geographic range and standard images. Additional diagnostic information is then provided in individual species accounts. We recognize a total of 15 named species, of which the following are described as new species: R. costaricensis, R. cryptergates, R. cubitalis, R. guatemalensis, R. mesoamericana, R. pluviselva, R. politognatha, R. subcubitalis, and R. titanis. An additional 12 morphospecies are described but not formally named due to insufficient material. Rasopone panamensis (Forel, 1899) is removed from synonymy and elevated to species. The following species are removed from Rasopone and made new combinations in Mayaponera: M. arhuaca (Forel, 1901), M. becculata (Mackay and Mackay, 2010), M. cernua (Mackay and Mackay, 2010), M. conicula (Mackay and Mackay, 2010), M. longidentata (Mackay and Mackay, 2010), and M. pergandei (Forel, 1909).

Analysis of ultraconserved elements supports African origins of narrow-mouthed frogs

Narrow-mouthed frogs (Anura: Microhylidae) are globally distributed and molecular data suggest the rapid evolution of multiple subfamilies shortly after their origin. Despite recent progress, several subfamilial relationships remain unexplored using phylogenomic data. We analysed 1,796 nuclear ultraconserved elements, a total matrix of 400,664 nucleotides, from representatives of most microhylid subfamilies. Summary method species-tree and maximum likelihood analyses unambiguously supported Hoplophryninae as the earliest diverging microhylid and confirm Chaperininae as a junior synonym of Microhylinae. Given the emerging consensus that subfamilies from mainland Africa diverged early, microhylids have likely occupied the continent for more than 66 million years.

Phylogenomic analysis of trichomycterid catfishes (Teleostei: Siluriformes) inferred from ultraconserved elements

The family Trichomycteridae is one of the most diverse groups of freshwater catfishes in South and Central America with eight subfamilies, 41 genera and more than 300 valid species. Its members are widely distributed throughout South America, reaching Costa Rica in Central America and are recognized by extraordinary anatomical specializations and trophic diversity. In order to assess the phylogenetic relationships of Trichomycteridae, we collected sequence data from ultraconserved elements (UCEs) of the genome from 141 specimens of Trichomycteridae and 12 outgroup species. We used a concatenated matrix to assess the phylogenetic relationships by Bayesian inference (BI) and maximum likelihood (ML) searches and a coalescent analysis of species trees. The results show a highly resolved phylogeny with broad agreement among the three distinct analyses, providing overwhelming support for the monophyletic status of subfamily Trichomycterinae including Ituglanis and Scleronema. Previous relationship hypotheses among subfamilies are strongly corroborated, such as the sister relationship between Copionodontinae and Trichogeninae forming a sister clade to the remaining trichomycterids and the intrafamilial clade TSVSG (Tridentinae-Stegophilinae-Vandelliinae-Sarcoglanidinae-Glanapteryginae). Monophyly of Glanapteryginae and Sarcoglanidinae was not supported and the enigmatic Potamoglanis is placed outside Tridentinae.

The phylogeny of Empis and Rhamphomyia (Diptera, Empididae) investigated using UCEs including an over 150 years old museum specimen

The genera Empis Linneus, 1758 and Rhamphomyia Meigen, 1822 (Empidoidea, Empididae Latreille, 1809) are two large genera of flies commonly named dagger flies. They are widely distributed in the world with most species described from the Palearctic Region. Empis comprises about 810 described species and Rhamphomyia comprises about 610 described species, together they represent one third of the known species diversity in Empididae. Two recent studies on the phylogeny of the two genera using Sanger sequencing on a few genetic markers, did not support monophyly of them. In this study high throughput sequencing of target enriched molecular data of ultraconserved elements or UCEs was used to investigate the phylogenetic relationships of included representatives of the genera. This method has proven useful on old and dry museum specimens with high amounts of degraded DNA, which was also tested herein. For this purpose, a commercially synthesized bait kit has previously been developed for Diptera which this study was the first one to test. Three out of nine old and dry museum specimens were successfully sequenced, one with an age of at least 154 years. Higher DNA concentration yielded a greater number of reads. Analyses conducted in the study confirmed that both Empis and Rhamphomyia are non-monophyletic.

Global domination by crazy ants: phylogenomics reveals biogeographical history and invasive species relationships in the genus Nylanderia (Hymenoptera: Formicidae)

AbstractNylanderia (Emery) is one of the world's most diverse ant genera, with 123 described species worldwide and hundreds more undescribed. Fifteen globetrotting or invasive species have widespread distributions and are often encountered outside their native ranges. A molecular approach to understanding the evolutionary history and to revision of Nylanderia taxonomy is needed because historical efforts based on morphology have proven insufficient to define major lineages and delimit species boundaries, especially where adventive species are concerned. To address these problems, we generated the first genus‐wide genomic dataset of Nylanderia using ultraconserved elements (UCEs) to resolve the phylogeny of major lineages, determine the age and origin of the genus, and describe global biogeographical patterns. Sampling from seven biogeographical regions revealed a Southeast Asian origin of Nylanderia in the mid‐Eocene and four distinct biogeographical clades in the Nearctic, the Neotropics, the Afrotropics/Malagasy region, and Australasia. The Nearctic and Neotropical clades are distantly related, indicating two separate dispersal events to the Americas between the late Oligocene and early Miocene. We also addressed the problem of misidentification that has characterized species‐level taxonomy in Nylanderia as a result of limited morphological variation in the worker caste by evaluating the integrity of species boundaries in six of the most widespread Nylanderia species. We sampled across ranges of species in the N. bourbonica complex (N. bourbonica (Forel) + N. vaga (Forel)), the N. fulva complex (N. fulva (Mayr) + N. pubens (Forel)), and the N. guatemalensis complex (N. guatemalensis (Forel) + N. steinheili (Forel)) to clarify their phylogenetic placement. Deep splits within these complexes suggest that some species names – specifically N. bourbonica and N. guatemalensis – each are applied to multiple cryptic species. In exhaustively sampling Nylanderia diversity in the West Indies, a ‘hot spot’ for invasive taxa, we found five adventive species among 22 in the region; many remain morphologically indistinguishable from one another, despite being distantly related. We stress that overcoming the taxonomic impediment through the use of molecular phylogeny and revisionary study is essential for conservation and invasive species management.

Studies of mice deleted for Sox3 and uc482: relevance to X-linked hypoparathyroidism.

Hypoparathyroidism is genetically heterogeneous and characterized by low plasma calcium and parathyroid hormone (PTH) concentrations. X-linked hypoparathyroidism (XLHPT) in two American families is associated with interstitial deletion-insertions involving deletions of chromosome Xq27.1 downstream of SOX3 and insertions of predominantly non-coding DNA from chromosome 2p25.3. These could result in loss, gain, or movement of regulatory elements, which include ultraconserved element uc482, which could alter SOX3 expression. To investigate this, we analysed SOX3 expression in EBV-transformed lymphoblastoid cells from three affected males, three unaffected males, and four carrier females from one XLHPT family. SOX3 expression was similar in all individuals, indicating that the spatiotemporal effect of the interstitial deletion-insertion on SOX3 expression postulated to occur in developing parathyroids did not manifest in lymphoblastoids. Expression of SNTG2, which is duplicated and inserted into the X chromosome, and ATP11C, which is moved telomerically, were also similarly expressed in all individuals. Investigation of male hemizygous (Sox3−/Y and uc482−/Y) and female heterozygous (Sox3+/− and uc482+/−) knockout mice, together with wild-type littermates (male Sox3+/Y and uc482+/Y, and female Sox3+/+ and uc482+/+), revealed Sox3−/Y, Sox3+/−, uc482−/Y, and uc482+/− mice to have normal plasma biochemistry, compared to their respective wild-type littermates. When challenged with a low calcium diet, all mice had hypocalcaemia, and elevated plasma PTH concentrations and alkaline phosphatase activities, and Sox3−/Y, Sox3+/−, uc482−/Y, and uc482+/− mice had similar plasma biochemistry, compared to wild-type littermates. Thus, these results indicate that absence of Sox3 or uc482 does not cause hypoparathyroidism and that XLHPT likely reflects a more complex mechanism.

Parachute geckos free fall into synonymy: Gekko phylogeny, and a new subgeneric classification, inferred from thousands of ultraconserved elements

Recent phylogenetic studies of gekkonid lizards have revealed unexpected, widespread paraphyly and polyphyly among genera, unclear generic boundaries, and a tendency towards the nesting of taxa exhibiting specialized, apomorphic morphologies within geographically widespread “generalist” clades. This is especially true in Australasia, where monophyly of Gekko proper has been questioned with respect to phenotypically ornate flap-legged geckos of the genus Luperosaurus, the Philippine false geckos of the genus Pseudogekko, and even the elaborately “derived” parachute geckos of the genus Ptychozoon. Here we employ sequence capture targeting 5060 ultraconserved elements (UCEs) to infer phylogenomic relationships among 42 representative ingroup gekkonine lizard taxa. We analyze multiple datasets of varying degrees of completeness (10, 50, 75, 95, and 100 percent complete with 4715, 4051, 3376, 2366, and 772 UCEs, respectively) using concatenated maximum likelihood and multispecies coalescent methods. Our sampling scheme addresses four persistent systematic questions in this group: (1) Are Luperosaurus and Ptychozoon monophyletic, and are any of these named species truly nested within Gekko? (2) Are prior phylogenetic estimates of Sulawesi’s L. iskandari as the sister taxon to Melanesian G. vittatus supported by our genome-scale dataset? (3) Is the high-elevation L. gulat of Palawan Island correctly placed within Gekko? (4) And, finally, where do the enigmatic taxa P. rhacophorus and L. browni fall in a higher-level gekkonid phylogeny? We resolve these issues; confirm with strong support some previously inferred findings (placement of Ptychozoon taxa within Gekko; the sister taxon relationship between L. iskandari and G. vittatus); resolve the systematic position of unplaced taxa (L. gulat, and L. browni); and transfer L. iskandari, L. gulat, L. browni, and all members of the genus Ptychozoon to the genus Gekko. Our unexpected and novel systematic inference of the placement of Ptychozoon rhacophorus suggests that this species is not grouped with Ptychozoon or even Luperosaurus (as previously expected) but may, in fact, be most closely related to several Indochinese species of Gekko. With our resolved and strongly supported phylogeny, we present a new classification emphasizing the most inclusive, original generic name (Gekko) for these ~60 taxa, arranged into seven subgenera.

A genome alignment of 120 mammals highlights ultraconserved element variability and placenta-associated enhancers.

BackgroundMultiple alignments of mammalian genomes have been the basis of many comparative genomic studies aiming at annotating genes, detecting regions under evolutionary constraint, and studying genome evolution. A key factor that affects the power of comparative analyses is the number of species included in a genome alignment.ResultsTo utilize the increased number of sequenced genomes and to provide an accessible resource for genomic studies, we generated a mammalian genome alignment comprising 120 species. We used this alignment and the CESAR method to provide protein-coding gene annotations for 119 non-human mammals. Furthermore, we illustrate the utility of this alignment by 2 exemplary analyses. First, we quantified how variable ultraconserved elements (UCEs) are among placental mammals. Leveraging the high taxonomic coverage in our alignment, we estimate that UCEs contain on average 4.7%–15.6% variable alignment columns. Furthermore, we show that the center regions of UCEs are generally most constrained. Second, we identified enhancer sequences that are only conserved in placental mammals. We found that these enhancers are significantly associated with placenta-related genes, suggesting that some of these enhancers may be involved in the evolution of placental mammal-specific aspects of the placenta.ConclusionThe 120-mammal alignment and all other data are available for analysis and visualization in a genome browser at https://genome-public.pks.mpg.de/and for download at https://bds.mpi-cbg.de/hillerlab/120MammalAlignment/.

RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons.

While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR/Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of “poison” cassette exons, which disrupt their host genes’ reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor suppressor activity. The essentiality and cancer relevance of poison exons likely contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.

Phylogenomic analysis of the beetle suborder Adephaga with comparison of tailored and generalized ultraconserved element probe performance

ABSTRACTAdephaga is the second largest suborder of beetles (Coleoptera) and they serve as important arthropod predators in both aquatic and terrestrial ecosystems. The suborder is divided into Geadephaga comprising terrestrial families and Hydradephaga for aquatic lineages. Despite numerous studies, phylogenetic relationships among the adephagan families and monophyly of the Hydradephaga itself remain in question. Here we conduct a comprehensive phylogenomic analysis of the suborder using ultraconserved elements (UCEs). This study presents the first in vitro test of a newly developed UCE probe set customized for use within Adephaga that includes both probes tailored specifically for the suborder, alongside generalized Coleoptera probes previously found to work in adephagan taxa. We assess the utility of the entire probe set, as well as comparing the tailored and generalized probes alone for reconstructing evolutionary relationships. Our analyses recovered strong support for the paraphyly of Hydradephaga with whirligig beetles (Gyrinidae) placed as sister to all other adephagan families. Geadephaga was strongly supported as monophyletic and placed sister to a clade composed of Haliplidae + Dytiscoidea. Monophyly of Dytiscoidea was strongly supported with relationships among the dytiscoid families resolved and strongly supported. Relationships among the subfamilies of Dytiscidae were strongly supported but largely incongruent with prior phylogenetic estimates for the family. The results of our UCE probe comparison showed that tailored probes alone outperformed generalized probes alone, as well as the full combined probe set (containing both types of probes), under decreased taxon sampling. When taxon sampling was increased, the full combined probe set outperformed both tailored probes and generalized probes alone. This study provides further evidence that UCE probe sets customized for a focal group result in a greater number of recovered loci and substantially improve phylogenomic analysis.