A common goal of population genomics and molecular ecology is to reconstruct the demographic history of a species of interest. A pair of powerful tools based on the sequentially Markovian coalescent have been developed to infer past population sizes using genome sequences. These methods are most useful when sequences are available for only a limited number of genomes and when the aim is to study ancient demographic events. The results of these analyses can be difficult to interpret accurately, because doing so requires some understanding of their theoretical basis and of their sensitivity to confounding factors. In this practical review, we explain some of the key concepts underpinning the pairwise and multiple sequentially Markovian coalescent methods (PSMC and MSMC, respectively). We relate these concepts to the use and interpretation of these methods, and we explain how the choice of different parameter values by the user can affect the accuracy and precision of the inferences. Based on our survey of 100 PSMC studies and 30 MSMC studies, we describe how the two methods are used in practice. Readers of this article will become familiar with the principles, practice, and interpretation of the sequentially Markovian coalescent for inferring demographic history.

The arrival of the ectoparasitic mite Varroa destructor on the western honeybee Apis mellifera saw a change in the diversity and prevalence of honeybee RNA viruses. One virus in particular, deformed wing virus (DWV) has become closely associated with V. destructor , leading many to conclude that V. destructor has affected viral virulence by changing the mode of transmission. While DWV is normally transmitted via feeding and faeces, V. destructor transmits viruses by direct injection. This change could have resulted in higher viral prevalence causing increased damage to the bees. Here we test the effect of a change in the mode of transmission on the composition and levels of honeybee RNA viruses in the absence of V. destructor . We find a rapid increase in levels of two viruses, sacbrood virus (SBV) and black queen cell virus (BQCV) after direct injection of viral extracts into honeybee pupae. In pupae injected with high levels of DWV extracted from symptomatic adult bees, DWV levels rapidly decline in the presence of SBV and BQCV. Further, we observe high mortality in honeybee pupae when injected with SBV and BQCV, whereas injecting pupae with high levels of DWV results in near 100% survival. Our results suggest a different explanation for the observed association between V. destructor and DWV. Instead of V. destructor causing an increase in DWV virulence, we hypothesize that direct virus inoculation, such as that mediated by a vector, quickly eliminates the most virulent honeybee viruses resulting in an association with less virulent viruses such as DWV.

Summary Homotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [ 1 , 2 , 3 , 4 ]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [ 5 ]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [ 6 , 7 , 8 ]. However, mitochondrial phylogenies can be misled by hybridization [ 9 ], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [ 10 ], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [ 10 ]. To examine the evolutionary history of Homotherium , we generated a ∼7x nuclear genome and a ∼38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (∼22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [ 5 ]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [ 3 , 4 , 11 , 12 , 13 , 14 ]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage.

The historical signal in nucleotide sequences becomes eroded over time by substitutions occurring repeatedly at the same sites. This phenomenon, known as substitution saturation, is recognized as one of the primary obstacles to deep-time phylogenetic inference using genome-scale data sets. We present a new test of substitution saturation and demonstrate its performance in simulated and empirical data. For some of the 36 empirical phylogenomic data sets that we examined, we detect substitution saturation in around 50% of loci. We found that saturation tends to be flagged as problematic in loci with highly discordant phylogenetic signals across sites. Within each data set, the loci with smaller numbers of informative sites are more likely to be flagged as containing problematic levels of saturation. The entropy saturation test proposed here is sensitive to high evolutionary rates relative to the evolutionary timeframe, while also being sensitive to several factors known to mislead phylogenetic inference, including short internal branches relative to external branches, short nucleotide sequences, and tree imbalance. Our study demonstrates that excluding loci with substitution saturation can be an effective means of mitigating the negative impact of multiple substitutions on phylogenetic inferences.

Examination of the Streptococcus gordonii chromosomal region, which lies immediately upstream of the glucosyltransferase positive regulatory determinant rgg, revealed two open reading frames. Based on nucleotide sequences, these genes were similar to the Listeria monocytogenes lemA gene, which is involved in antigen presentation, and the Escherichia coli htpX heat shock gene, which has an unknown function. Northern hybridization analysis indicated that S. gordonii lemA and htpX genes were associated with a ca. 1.7-kb polycistronic transcript. Although levels of the lemA/htpX transcript did not increase in response to heat to levels seen with dnaK controls, insertional inactivation of htpX resulted in changes in adhesiveness, cellular morphology and detergent-extractable surface antigens in cells grown at 41 degrees C, implying that htpX may be involved in surface protein expression. Insertional inactivation of lemA and htpX indicated that, despite their proximity to rgg and the structural gene, gtfG, these upstream genes do not affect S. gordonii glucosyltransferase activity.

The historical signal in nucleotide sequences becomes eroded over time by substitutions occurring repeatedly at the same sites. This phenomenon, known as substitution saturation, is recognized as one of the primary obstacles to deep-time phylogenetic inference using genome-scale data sets. We present a new test of substitution saturation and demonstrate its performance in simulated and empirical data. For some of the 36 empirical phylogenomic data sets that we examined, we detect substitution saturation in around 50% of loci. We found that saturation tends to be flagged as problematic in loci with highly discordant phylogenetic signals across sites. Within each data set, the loci with smaller numbers of informative sites are more likely to be flagged as containing problematic levels of saturation. The entropy saturation test proposed here is sensitive to high evolutionary rates relative to the evolutionary timeframe, while also being sensitive to several factors known to mislead phylogenetic inference, including short internal branches relative to external branches, short nucleotide sequences, and tree imbalance. Our study demonstrates that excluding loci with substitution saturation can be an effective means of mitigating the negative impact of multiple substitutions on phylogenetic inferences.

The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by the upstream determinant rgg. In the present study, two ORFs, transcribed on the opposite DNA strand, were identified immediately downstream of gtfG. The first, designated dsg, shares a convergent putative transcriptional terminator with gtfG, and encodes a predicted 46 kDa transmembrane protein similar to the Yersinia enterocolitica TrsA involved in polysaccharide biosynthesis. Insertional inactivation of dsg resulted in only " " 60 % of the parental level of glucosyltransferase activity. The 870 bp gene 5' to dsg is similar to the gtfG regulatory determinant. Designated rggD, this rgglike determinant downstream of gtfG encodes a putative 336 kDa cytoplasmic protein. Despite their sequence similarity, the functions of rgg and rggD appear specific. Strains in which rggD was insertionally inactivated and strains containing plasmid-borne rggD had parental levels of glucosyltransferase activity. Northern blot hybridization analyses showed " 13 kb dsg-specific and " 10 kb rggD-specific mRNA transcripts associated with this region ; no polycistronic transcript was observed. Although rgg-like gene products have been demonstrated to function as positive transcriptional regulators of adjacent genes in several streptococcal species, Northern blot analysis suggested that rggD did not influence the transcription of dsg or the divergent downstream ylbN-like determinant under the conditions in the present study. Comparison of this S. gordonii chromosome region to other streptococcal genomes, which do not contain the rgg/rggD-flanked region involved in glucan synthesis, raised intriguing possibilities about the origins of this chromosomal region, and also suggested that rggD might regulate a distally located gene.

A B S T R A C TThe illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/ optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action.