Genomic architecture of autism from comprehensive whole-genome sequence annotation.

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.

Discovery of genomic variation across a generation.

Over the past 30 years (the timespan of a generation), advances in genomics technologies have revealed tremendous and unexpected variation in the human genome and have provided increasingly accurate answers to long-standing questions of how much genetic variation exists in human populations and to what degree the DNA complement changes between parents and offspring. Tracking the characteristics of these inherited and spontaneous (or de novo) variations has been the basis of the study of human genetic disease. From genome-wide microarray and next-generation sequencing scans, we now know that each human genome contains over 3 million single nucleotide variants when compared with the ~ 3 billion base pairs in the human reference genome, along with roughly an order of magnitude more DNA-approximately 30 megabase pairs (Mb)-being 'structurally variable', mostly in the form of indels and copy number changes. Additional large-scale variations include balanced inversions (average of 18 Mb) and complex, difficult-to-resolve alterations. Collectively, ~1% of an individual's genome will differ from the human reference sequence. When comparing across a generation, fewer than 100 new genetic variants are typically detected in the euchromatic portion of a child's genome. Driven by increasingly higher-resolution and higher-throughput sequencing technologies, newer and more accurate databases of genetic variation (for instance, more comprehensive structural variation data and phasing of combinations of variants along chromosomes) of worldwide populations will emerge to underpin the next era of discovery in human molecular genetics.

Single nucleotide polymorphisms (SNPs) provide unprecedented resolution of species boundaries, phylogenetic relationships, and genetic diversity in the mastiff bats (Molossus).

Mammals are one of the better known groups of animals, and in the Neotropics bats typically comprise about half of the mammalian species diversity. But, well resolved species-level phylogenies are still lacking for most taxa of bats. One broadly distributed genus is the mastiff bats, Molossus. Species within this genus are morphologically very similar, which results in a confusing and unstable taxonomy. In addition, low levels of genetic divergence among some clades make resolution of phylogenetic relationships difficult. Most authors recognize Molossus as being monophyletic, however, phylogenetic relationships within the genus remain poorly understood based on traditional Sanger sequencing of individual genes. We propose a more comprehensive framework based on large-scale genomic data derived from Next Generation Sequencing techniques to better understand evolutionary relationships within a group of closely related species with a rich taxonomic history. In this study, we utilized the NGS method of Genotype by Sequencing (GBS) to test the monophyly of the genus, understand evolutionary relationships within Molossus and investigate the genetic integrity of currently recognized species. Given that both de novo and reference genome pipelines are often used in the assembly of Single Nucleotide Polymorphism data from GBS, and that several tree inference methodologies have been proposed for SNP data, we test whether different alignments and phylogenetic approaches produce similar results. We also examined how the process of SNP identification and mapping can affect the consistency of the analyses. Our data provide the first high resolution phylogeny for the genus Molossus, bringing new insights into recognition of species boundaries and relationships among taxa. This study clarifies the taxonomy of Molossus and elevates the number of species in the genus from 11 to 14. We suggest the revalidation of the names M. nigricans, and M. fluminensis, which were synonymized under the name M. rufus; and M. bondae, previously synonymized under the name M. currentium. Different alignments and phylogenetic inferences produce consistent results, supporting use of SNP approach in addressing evolutionary questions on a macroevolutionary scale where the genetic distance among clades is low.

Functional Shifts in Bat Dim-Light Visual Pigment Are Associated with Differing Echolocation Abilities and Reveal Molecular Adaptation to Photic-Limited Environments.

Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.

The role of ecological factors in shaping bat cone opsin evolution.

Bats represent one of the largest and most striking nocturnal mammalian radiations, exhibiting many visual system specializations for performance in light-limited environments. Despite representing the greatest ecological diversity and species richness in Chiroptera, Neotropical lineages have been undersampled in molecular studies, limiting the potential for identifying signatures of selection on visual genes associated with differences in bat ecology. Here, we investigated how diverse ecological pressures mediate long-term shifts in selection upon long-wavelength (Lws) and short-wavelength (Sws1) opsins, photosensitive cone pigments that form the basis of colour vision in most mammals, including bats. We used codon-based likelihood clade models to test whether ecological variables associated with reliance on visual information (e.g. echolocation ability and diet) or exposure to varying light environments (e.g. roosting behaviour and foraging habitat) mediated shifts in evolutionary rates in bat cone opsin genes. Using additional cone opsin sequences from newly sequenced eye transcriptomes of six Neotropical bat species, we found significant evidence for different ecological pressures influencing the evolution of the cone opsins. While Lws is evolving under significantly lower constraint in highly specialized high-duty cycle echolocating lineages, which have enhanced sonar ability to detect and track targets, variation in Sws1 constraint was significantly associated with foraging habitat, exhibiting elevated rates of evolution in species that forage among vegetation. This suggests that increased reliance on echolocation as well as the spectral environment experienced by foraging bats may differentially influence the evolution of different cone opsins. Our study demonstrates that different ecological variables may underlie contrasting evolutionary patterns in bat visual opsins, and highlights the suitability of clade models for testing ecological hypotheses of visual evolution.

A recurrent SHANK3 frameshift variant in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is genetically complex with ~100 copy number variants and genes involved. To try to establish more definitive genotype and phenotype correlations in ASD, we searched genome sequence data, and the literature, for recurrent predicted damaging sequence-level variants affecting single genes. We identified 18 individuals from 16 unrelated families carrying a heterozygous guanine duplication (c.3679dup; p.Ala1227Glyfs*69) occurring within a string of 8 guanines (genomic location [hg38]g.50,721,512dup) affecting SHANK3, a prototypical ASD gene (0.08% of ASD-affected individuals carried the predicted p.Ala1227Glyfs*69 frameshift variant). Most probands carried de novo mutations, but five individuals in three families inherited it through somatic mosaicism. We scrutinized the phenotype of p.Ala1227Glyfs*69 carriers, and while everyone (17/17) formally tested for ASD carried a diagnosis, there was the variable expression of core ASD features both within and between families. Defining such recurrent mutational mechanisms underlying an ASD outcome is important for genetic counseling and early intervention.

Does evolution of echolocation calls and morphology in Molossus result from convergence or stasis?

Although many processes of diversification have been described to explain variation of morphological traits within clades that have obvious differentiation among taxa, not much is known about these patterns in complexes of cryptic species. Molossus is a genus of bats that is mainly Neotropical, occurring from the southeastern United States to southern Argentina, including the Caribbean islands. Molossus comprises some groups of species that are morphologically similar but phylogenetically divergent, and other groups of species that are genetically similar but morphologically distinct. This contrast allows investigation of unequal trait diversification and the evolution of morphological and behavioural characters. In this study, we assessed the role of phylogenetic history in a genus of bat with three cryptic species complexes, and evaluated if morphology and behavior are evolving concertedly. The Genotype by Sequence genomic approach was used to build a species-level phylogenetic tree for Molossus and to estimate the ancestral states of morphological and echolocation call characters. We measured the correlation of phylogenetic distances to morphological and echolocation distances, and tested the relationship between morphology and behavior when the effect of phylogeny is removed. Morphology evolved via a mosaic of convergence and stasis, whereas call design was influenced exclusively through local adaptation and convergent evolution. Furthermore, the frequency of echolocation calls is negatively correlated with the size of the bat, but other characters do not seem to be evolving in concert. We hypothesize that slight variation in both morphology and behaviour among species of the genus might result from niche specialization, and that traits evolve to avoid competition for resources in similar environments.

Cryptic diversity and range extension in the big-eyed bat genus Chiroderma (Chiroptera, Phyllostomidae).

Since the last systematic review of Chiroderma (big-eyed bats) more than two decades ago, we report on biodiversity surveys that expand the distribution and species diversity of this Neotropical genus. The Caribbean endemic species Chiroderma improvisum is documented for the first time from Nevis in the northern Lesser Antilles. A broader geographic sampling for a molecular analysis identifies a paraphyletic relationship in Chiroderma trinitatum with respect to Chiroderma doriae. Cis-Andean populations of C. trinitatum are most closely related to the morphologically distinctive and allopatrically distributed C. doriae in the Cerrado and Atlantic Forest of Brazil and Paraguay. The sister taxon to this grouping includes trans-Andean populations of C. trinitatum, which we recommend to elevate to species status as C. gorgasi. This is an example of a cryptic species because C. gorgasi was previously considered morphologically similar to C. trinitatum, but more detailed examination revealed that it lacks a posterolabial accessory cusp on the lower second premolar and has a narrower breadth of the braincase. We provide an amended description of Chiroderma gorgasi.

Next generation sequencing data in the phylogenetic relationships of the genus Molossus (Chiroptera, Molossidae).

The mastiff bat Molossus is a broadly distributed genus within the family Molossidae. Molossus includes groups of species that are either morphologically or genetically very similar, rendering the taxonomy of this genus confusing and unstable. In this paper, we provide inferred phylogenetic relationships of Molossus based on the genotype by sequencing approach from 189 specimens of three species of New World mastiff bats (Molossus, Promops, and Eumops). We also present data on divergent tree topologies produced by alignments using de novo and reference genome approaches and distinct phylogenetic methods (maximum likelihood and coalescent approaches). These data provide the first highly resolved phylogenetic tree for Molossus, not recovered by previous studies using Sanger sequencing. Our dataset brings new insights on relationships among species and show how different approaches might affect phylogenetic resolution and topologies.

Comparative phylogeography of mainland and insular species of Neotropical molossid bats (Molossus).

Historical events, habitat preferences, and geographic barriers might result in distinct genetic patterns in insular versus mainland populations. Comparison between these two biogeographic systems provides an opportunity to investigate the relative role of isolation in phylogeographic patterns and to elucidate the importance of evolution and demographic history in population structure. Herein, we use a genotype-by-sequencing approach (GBS) to explore population structure within three species of mastiff bats (Molossus molossus, M. coibensis, and M. milleri), which represent different ecological histories and geographical distributions in the genus. We tested the hypotheses that oceanic straits serve as barriers to dispersal in Caribbean bats and that isolated island populations are more likely to experience genetic drift and bottlenecks in comparison with highly connected ones, thus leading to different phylogeographic patterns. We show that population structures vary according to general habitat preferences, levels of population isolation, and historical fluctuations in climate. In our dataset, mainland geographic barriers played only a small role in isolation of lineages. However, oceanic straits posed a partial barrier to the dispersal for some populations within some species (M. milleri), but do not seem to disrupt gene flow in others (M. molossus). Lineages on distant islands undergo genetic bottlenecks more frequently than island lineages closer to the mainland, which have a greater exchange of haplotypes.

Molecular data on the CO1 and beta fibrinogen gene in the evolutionary relationships of the mastiff bat (Chiroptera, Molossidae, Molossus).

Molossus is one of the most diverse genera of free-tailed bats in the pantropical family Molossidae and occurs though all the Neotropics. Nevertheless, the taxonomy and phylogeny of this group is poorly understood. Here, we present the data on evolutionary relationships of Molossus based on DNA barcodes of COI gene from 346 specimens of Molossus and its sister genus Promops and another New World molossid Eumops. Of these specimens, 50 are new sequences and 296 were obtained from GenBank. In addition, the nuclear gene beta fibrinogen was sequenced from a subset of 35 specimens. These data provide the basis for further exploration and understanding of the phylogenetic relationships of the genus Molossus (Loureiro et al., 2018) [1].