Chromosomal-level reference genome assembly of the North American wolverine (Gulo gulo luscus): a resource for conservation genomics.

We report a chromosomal-level genome assembly of a male North American wolverine (Gulo gulo luscus) from the Kugluktuk region of Nunavut, Canada. The genome was assembled directly from long-reads, comprising: 758 contigs with a contig N50 of 36.6 Mb; contig L50 of 20; base count of 2.39 Gb; and a near complete representation (99.98%) of the BUSCO 5.2.2 set of 9,226 genes. A presumptive chromosomal-level assembly was generated by scaffolding against two chromosomal-level Mustelidae reference genomes, the ermine and the Eurasian river otter, to derive a final scaffold N50 of 144.0 Mb and a scaffold L50 of 7. We annotated a comprehensive set of genes that have been associated with models of aggressive behavior, a trait which the wolverine is purported to have in the popular literature. To support an integrated, genomics-based wildlife management strategy at a time of environmental disruption from climate change, we annotated the principal genes of the innate immune system to provide a resource to study the wolverine's susceptibility to new infectious and parasitic diseases. As a resource, we annotated genes involved in the modality of infection by the coronaviruses, an important class of viral pathogens of growing concern as shown by the recent spillover infections by severe acute respiratory syndrome coronavirus-2 to naïve wildlife. Tabulation of heterozygous single nucleotide variants in our specimen revealed a heterozygosity level of 0.065%, indicating a relatively diverse genetic pool that would serve as a baseline for the genomics-based conservation of the wolverine, a rare cold-adapted carnivore now under threat.

Effect of vitamin D source and dietary cation-anion difference in peripartum dairy cows on calcium homeostasis and milk production.

The objective of this experiment was to determine the effects of dietary vitamin D source on serum calcium (Ca), urinary Ca excretion, and milk production when fed in combination with a prepartum acidogenic negative dietary cation-anion difference (DCAD) diet. Nonlactating, pregnant multiparous cows (n = 15), balanced for breed (Holstein n = 9 and Jersey n = 6), and previous mature equivalent milk production, were assigned to one of three treatments (five cows/treatment), consisting of a control (PCH; positive DCAD, 8.9 mEq/100 g DM) and two negative DCAD diets (-15.4 mEq/100 g DM), one with vitamin D3 (cholecalciferol; NCH) and one with 25-hydroxyvitamin D3 (calcidiol; NCA; DSM nutritional products). The treatments were formulated to provide 1.95 mg/d of vitamin D and were fed 28 d prior to expected calving date. Delivery of vitamin D sources was accomplished by manufacture of a pellet and 2 kg of these pellets were individually fed simultaneously each day along with 2 kg of ground corn daily at 0800 hours. Negative DCAD treatments were formulated to provide 0.46 kg/d of Animate (Phibro Animal Health) and, if needed, additional Animate was top-dressed at each feeding to achieve a urine pH between 5.5 and 6.0 based on the previous day's urine pH. Close-up cows had ad libitum access to chopped bermudagrass (Cynodon dactylon L.) hay and hay intake was measured using SmartFeed Pro systems (C-Lock Inc.; Rapid City, SD). Prepartum urine and serum samples were collected weekly and serum was collected 36, 48, and 72 h post-calving. Prepartum dry matter intake (DMI) as a percent of body weight was not (P = 0.66) affected by treatments. Cows fed NCH and NCA had greater (P = 0.02) prepartum serum Ca than PCH and tended to have greater urinary Ca excretions (P = 0.10). Average postpartum serum Ca (mg/dL) was greater (P = 0.05) for cows fed NCH (8.8) compared with PCH (7.8), whereas NCA (8.4) was numerically intermediate and not (P > 0.05) different from either of the other treatments. Postpartum DMI was not affected by treatment (P = 0.39). Daily milk yield (MY) (kg/d) was greatest (P < 0.01) for NCA (37.5) compared with the other treatments and NCH (34.1) was intermediate and greater than PCH (29.9). These results suggest that an acidogenic prepartum diet in combination with vitamin D was effective in maintaining peripartum serum Ca and the 25-hydroxy form of vitamin D improved MY compared with NCH in early lactation.

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.

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.

Feeding supplemental 25-hydroxyvitamin D3 increases serum mineral concentrations and alters mammary immunity of lactating dairy cows.

Objectives were to determine the effects of feeding supplemental 25-hydroxyvitamin D3 [25(OH)D3] on concentrations of vitamin D metabolites and minerals in serum, mammary immune status, and responses to intramammary bacterial infection in dairy cows. Sixty multiparous, pregnant lactating Holstein cows with somatic cell count <200,000/mL were blocked by days in milk and milk yield and randomly assigned to receive a daily top-dressed dietary supplement containing 1 or 3 mg of vitamin D3 (1mgD or 3mgD), or 1 or 3 mg 25(OH)D3 (1mg25D or 3mg25D) for 28 d (n = 15/treatment). Cows were kept in a freestall barn and fed a total mixed ration in individual feeding gates. Individual dry matter intake (DMI) and milk yield were recorded daily, and milk and blood samples were collected at 0, 7, 14, and 21 d relative to the start of treatment. At 21 d, cows fed 1mgD and 3mg25D received an intramammary challenge with Streptococcus uberis. Cows were observed for severity of mastitis, and blood and milk samples were collected every 12 h to measure inflammation. The 1mg25D and 3mg25D cows had greater serum 25(OH)D3 concentrations at 21 d compared with 1mgD and 3mgD cows (62 ± 7, 66 ± 8, 135 ± 15, and 232 ± 26 ng/mL for 1mgD, 3mgD, 1mg25D, and 3mg25D, respectively). The 3mg25D cows had greater concentrations of Ca and P in serum at 21 d compared with other treatments (Ca = 2.38, 2.4, 2.37, and 2.48 ± 0.02 mM, 1.87, 1.88, and 2.10 ± 0.08 mM for 1mgD, 3mgD, 1mg25D, and 3mg25D, respectively). Yields of milk and milk components, DMI, body weight, and concentrations of 1,25-dihydroxyvitamin D and Mg in serum did not differ among treatments. Abundance of mRNA transcripts for interleukin-1ß (IL1B) and inducible nitric oxide synthase (iNOS) in milk somatic cells before S. uberis challenge were increased in cows fed 25(OH)D3 compared with cows fed vitamin D3. Furthermore, IL1B, iNOS, ß-defensin 7, and ß-defensin 10 in milk somatic cells increased as concentrations of 25(OH)D3 increased in serum. Cows fed 3mg25D had less severe mastitis at 60 and 72 h after challenge with S. uberis compared with cows fed 1mgD. Concentrations of bacteria, somatic cells, and serum albumin in milk after challenge did not differ between treatments; however, an interaction between treatment and day was detected for lactate dehydrogenase in milk. Expression of adhesion protein CD11b on milk neutrophils after the S. uberis challenge was greater among 3mg25D cows compared with 1mgD cows. Transcripts of CYP24A1 and iNOS in milk somatic cells during mastitis also were greater in 3mg25D cows compared with 1mgD cows. Feeding 25(OH)D3 increased serum 25(OH)D3 more effectively than supplemental vitamin D3, resulting in increased serum mineral concentrations, increased expression of vitamin D-responsive genes, and altered immune responses to intramammary bacterial challenge.

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.

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].

Evolutionary relationships and climatic niche evolution in the genus Handleyomys (Sigmodontinae: Oryzomyini).

Mesoamerica is considered a biodiversity hot spot with levels of endemism and species diversity likely underestimated. Unfortunately, the region continues to experience some of the highest deforestation rates in the world. For mammals, the evolutionary relationships of many endemic taxa are controversial, as it is the case for members of the genus Handleyomys. Estimation of a time-calibrated hypothesis for the evolution of these six genera (Euryoryzomys, Handleyomys, Hylaeamys, Nephelomys, Oecomys and Transandinomys) supported a monophyletic Handleyomys sensu lato. Based on their distinctive morphology and the amount of inter-generic genetic divergence, Handleyomys sensu stricto, H. alfaroi, the H. chapmani, and the H. melanotis species groups warrant recognition as separate genera. In addition, species delimitation documents the existence of cryptic species-level lineages within H. alfaroi and H. rostratus. Cryptic lineages within H. rostratus exhibited significant niche differentiation, but this was not the pattern among species-level clades within H. alfaroi. Similarly, age-range correlations revealed that niche evolution within Handleyomys is not correlated with evolutionary time, instead, ancestral climate tolerance reconstructions show niche disparities at specific diversification events within the chapmani and melanotis species groups, while the climatic niche of the rest of species of Handleyomys tended to be conservative.

Safety evaluation of dietary levels of 25-hydroxyvitamin D3 in growing calves.

The objective of this study was to evaluate the safety of 25-OH-D3 addition to the diet of bovine calves. The investigation was conducted as a feeding experiment for 90 days. 40 calves were allotted to four groups: T1 - control group which received 30 IU vitamin D3/kg feed, and three experimental groups, which received 25-OH-D3 in increasing dosages: T2 - 25-OH-D3 at 1.7 µg/kg, T3 - 25-OH-D3 at 5.1 µg/kg and T4 - 25-OH-D3 at 8.5 µg/kg. All calves in the four groups gained weight continually: no growth depression was observed. No adverse effects of 25- OH-D3 were observed for any of the hematology and serum chemistry parameters measured or during the routine clinical examinations. Plasma 25-OH-D3 concentration was higher (p<0.05) in groups T2, T3 and T4 compared to that observed in group T1. Tissues content of 25-OH-D3 was significantly higher in in groups T2, T3 and T4 than in group T1. In the post-mortem evaluation, no adverse effects of the different 25-OH-D3 doses were observed, neither during the gross pathology nor in the histological examination. The results of this study show explicitly that there were no adverse effects of 25-OH-D3 compared to the control group, supplemented with vitamin D3.

Nuclear and mtDNA phylogenetic analyses clarify the evolutionary history of two species of native Hawaiian bats and the taxonomy of Lasiurini (Mammalia: Chiroptera).

Previous studies on genetics of hoary bats produced differing conclusions on the timing of their colonization of the Hawaiian Islands and whether or not North American (Aeorestes cinereus) and Hawaiian (A. semotus) hoary bats are distinct species. One study, using mtDNA COI and nuclear Rag2 and CMA1, concluded that hoary bats colonized the Hawaiian Islands no more than 10,000 years ago based on indications of population expansion at that time using Extended Bayesian Skyline Plots. The other study, using 3 mtDNA and 1 Y-chromosome locus, concluded that the Hawaiian Islands were colonized about 1 million years ago. To address the marked inconsistencies between those studies, we examined DNA sequences from 4 mitochondrial and 2 nuclear loci in lasiurine bats to investigate the timing of colonization of the Hawaiian Islands by hoary bats, test the hypothesis that Hawaiian and North American hoary bats belong to different species, and further investigate the generic level taxonomy within the tribe. Phylogenetic analysis and dating of the nodes of mtDNA haplotypes and of nuclear CMA1 alleles show that A. semotus invaded the Hawaiian Islands approximately 1.35 Ma and that multiple arrivals of A. cinereus occurred much more recently. Extended Bayesian Skyline plots show population expansion at about 20,000 years ago in the Hawaiian Islands, which we conclude does not represent the timing of colonization of the Hawaiian Islands given the high degree of genetic differentiation among A. cinereus and A. semotus (4.2% divergence at mtDNA Cytb) and the high degree of genetic diversity within A. semotus. Rather, population expansion 20,000 years ago could have resulted from colonization of additional islands, expansion after a bottleneck, or other factors. New genetic data also support the recognition of A. semotus and A. cinereus as distinct species, a finding consistent with previous morphological and behavioral studies. The phylogenetic analysis of CMA1 alleles shows the presence of 2 clades that are primarily associated with A. semotus mtDNA haplotypes, and are unique to the Hawaiian Islands. There is evidence for low levels of hybridization between A. semotus and A. cinereus on the Hawaiian Islands, but it is not extensive (<15% of individuals are of hybrid origin), and clearly each species is able to maintain its own genetic distinctiveness. Both mtDNA and nuclear DNA sequences show deep divergence between the 3 groups (genera) of lasiurine bats that correspond to the previously recognized morphological differences between them. We show that the Tribe Lasiurini contains the genera Aeorestes (hoary bats), Lasiurus (red bats), and Dasypterus (yellow bats).

De Novo Genome and Transcriptome Assembly of the Canadian Beaver (Castor canadensis).

The Canadian beaver (Castor canadensis) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon-gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology.

Stereotypic vocalizations in harvest mice (Reithrodontomys): Harmonic structure contains prominent and distinctive audible, ultrasonic, and non-linear elements.

Reports of audible vocalizations are rare in adult muroid rodents, animals generally very small in body size and under strong predation pressure. By contrast, communication using high, often ultrasonic vocal frequencies is relatively common. There are anecdotal reports of audible vocalizations for some harvest mice (genus Reithrodontomys), however none have been recorded or analyzed. Several species of harvest mice are studied, representing the subgenera Reithrodontomys (R. fulvescens, R. sumichrasti) and Aporodon (R. creper, R. mexicanus, R. spp.), as part of a larger phylogenetic analysis of stereotypical vocal communication. Only R. mexicanus produced stereotyped vocalizations on a regular basis. Acoustic signals of R. mexicanus contain prominent harmonics bridging both the audible and ultrasonic range, with evidence of non-linear distribution of energy within and between notes of an individual call. Harmonic emphasis varies, making the carrier frequency difficult to locate. These Reithrodontomys vocalizations are compared with members of their phylogenetic sister group Onychomys+Peromyscus, genera whose stereotypic calls exclusively occupy the audible or ultrasonic spectrum, respectively. It is hypothesized that the stereotypic signals of harvest mice represent announcement calls. Given the number of anecdotal reports of stereotyped 1, 2, and 3-note calls among Reithrodontomys species, further interspecific comparisons are warranted.

TOWARD A MOLECULAR PHYLOGENY FOR PEROMYSCUS: EVIDENCE FROM MITOCHONDRIAL CYTOCHROME-b SEQUENCES.

One hundred DNA sequences from the mitochondrial cytochrome-b gene of 44 species of deer mice (Peromyscus (sensu stricto), 1 of Habromys, 1 of Isthmomys, 2 of Megadontomys, and the monotypic genera Neotomodon, Osgoodomys, and Podomys were used to develop a molecular phylogeny for Peromyscus. Phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) were conducted to evaluate alternative hypotheses concerning taxonomic arrangements (sensu stricto versus sensu lato) of the genus. In all analyses, monophyletic clades were obtained that corresponded to species groups proposed by previous authors; however, relationships among species groups generally were poorly resolved. The concept of the genus Peromyscus based on molecular data differed significantly from the most current taxonomic arrangement. Maximum-likelihood and Bayesian trees depicted strong support for a clade placing Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys within Peromyscus. If Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys are regarded as genera, then several species groups within Peromyscus (sensu stricto) should be elevated to generic rank. Isthmomys was associated with the genus Reithrodontomys; in turn this clade was sister to Baiomys, indicating a distant relationship of Isthmomys to Peromyscus. A formal taxonomic revision awaits synthesis of additional sequence data from nuclear markers together with inclusion of available allozymic and karyotypic data.