Diversity Of Subspecies Research Articles

Chromosome-scale genomes provide new insights into subspecies divergence and evolutionary characteristics of the giant panda

Extant giant pandas are divided into Sichuan and Qinling subspecies. The giant panda has many species-specific characteristics, including comparatively small organs for body size, small genitalia of male individuals, and low reproduction. Here, we report the most contiguous, high-quality chromosome-level genomes of two extant giant panda subspecies to date, with the first genome assembly of the Qinling subspecies. Compared with the previously assembled giant panda genomes based on short reads, our two assembled genomes increased contiguity over 200-fold at the contig level. Additional sequencing of 25 individuals dated the divergence of the Sichuan and Qinling subspecies into two distinct clusters from 10,000 to 12,000 years ago. Comparative genomic analyses identified the loss of regulatory elements in the dachshund family transcription factor 2 (DACH2) gene and specific changes in the synaptotagmin 6 (SYT6) gene, which may be responsible for the reduced fertility of the giant panda. Positive selection analysis between the two subspecies indicated that the reproduction-associated IQ motif containing D (IQCD) gene may at least partly explain the different reproduction rates of the two subspecies. Furthermore, several genes in the Hippo pathway exhibited signs of rapid evolution with giant panda-specific variants and divergent regulatory elements, which may contribute to the reduced inner organ sizes of the giant panda.

Phylogeography of subspecies of the sable (Martes zibellina L.) based on mitochondrial genomes: implications for evolutionary history

Phylogeography of the sable is complicated by mass reintroductions that occurred in some areas of Russia from 1940 to 1970. We assessed phylogeny of the sable by sequence analysis of 75 mitogenome haplotypes, including 63 newly sequenced specimens from aboriginal populations. Phylogenetic analysis revealed that the sable exhibited absence of phylogeographical structure across Eurasia with the exception of ‘partially monophyletic lineage’ in the Changbai Mountains (M. z. hamgyenensis) and on Kamchatka Peninsula (M. z. kamtschadalica), and two terminal sharing haplotypes occurred in two long-range subspecies. This may be the result of multiple factors, including ‘secondary contact’, anthropogenic translocation and the opportunity for long-distance movement. At the same time, M. z. hamgyenensis lacked recent demographic expansion and survived with a stable population size during the Last Glacial Maximum (LGM). Combining partially monophyletic lineage, we suggested that the region was a glacial refugia for the species. Considering the geographic distribution and the genetic diversity of sable subspecies, M. z. obscura may be regarded as central and M. z. yeniseensis, M. z. princeps, M. z. linkouensis, M. z. zibellina and M. z. arsenjevi as marginal. M. z. hamgyenensis, M. z. kamtschadalica and M. z. brachyura are categorized as a boundary. The species showed a reduced trend from central to peripheral in genetic diversity. Based on the time to most recent common ancestor (TMRCA) and the Bayesian skyline plots (BSPs) analysis of M. zibellina, the Middle and Late Pleistocene climatic conditions played an important role in phylogenetic differentiation of the sable mitochondrial gene pool. The sable populations experienced a bottleneck during the penultimate glacial maximum (PGM) rather than during the LGM. An expansion of the population continued during the transition from the middle of the Karginian to the Sartanian times, including slow expansion during the LGM. With the rise of postglacial temperatures, a rapid decline of sable populations could be associated with hunting by humans.

Фитопланктон горных озёр Прибайкалья и Забайкалья

A brief description of phytoplankton in the alpine lakes located westward and eastward Lake Baikal within the Baikalian rift zone is given. These lakes are mostly virgin so they could be used as a pure background with almost absence of the anthropogenic activity. In each lake, its peculiar algoflora was formed reflecting climatic, landscape, lake's basin shape, and drainage area conditions. The phytoplankton were mainly consisted of green, chrysophyte, and diatom algae. The species and subspecies diversity was found in a low number of genera not in all one. The baikalian endemic diatoms A. baicalensis (K. Meyer) Sim., Сyclotella baicalensis, C. minuta, Stephanodiscus meyeri, and spore-forming A. islandica (this form of the alga is known to dwell in the lake Khanka and the river Amur, Far East, only), as well as dinoflagellate Gymnodinium baicalense var. minor, have been found there. In most lakes, the relict alga Pliocaenicus costatus, wide-spread in ancient times from Europe to the Kamchatka peninsula, has been observed also. Sets of the dominant species were monodominant. On number and biomass, diatoms were dominant in 59% of the lakes, bluegreen algae and chrysophytes in 15%, green algae in 9%, and dinophytes in 2%. The biomasses varied mainly 50 to 100 mg·m-3, sometimes during the open water period 300 to 600 мгꞏм-3 and 800 мгꞏм-3 in shallows. The phytoplankton exhibited three peaks in their seasonal development and autumn one was maximal. The composition and development of the phytoplankton in the alpine lakes within the Baikalian rift zone testify to their virgin states without acidification.

Emsian (Lower Devonian) conodonts from the Daliantang Formation at Daliantang, southeastern Yunnan, China

The lower/upper Emsian boundary witnessed prominent diversification of biostratigraphically important genera Polygnathus Hinde, Caudicriodus Bultynck, and Latericriodus Müller. In contrast, conodonts previously reported around this boundary from the South China Block were characterized by relatively low species or subspecies diversity, and only a few widely distributed and extensively studied zone-defining taxa were recorded. In this paper, conodonts from the Daliantang Formation at the Daliantang section (Guangnan, southeastern Yunnan), one of the representative sections of the transitional facies between the benthic Xiangzhou facies and the pelagic Nandan facies, are investigated. The conodont fauna shows a remarkably high species or subspecies diversity, with fourteen taxa that are assignable to the notoperbonus Zone and inversus Zone. Five polygnathid species are described: P. gronbergi Klapper and Jonson, P. inversus Klapper and Jonson, Polygnathus sp. B, P. luciae Martínez-Pérez and Valenzuela-Ríos, and P. mashkovae Bardashev. The latter two are reported for the first time from the South China Block. Additionally, three icriodontid taxa are also described, of which L. guangnanensis n. sp. is characterized by distinctly chevron-shaped transverse rows in the anterior part and a well-developed process branching off to the outer side. The new conodont record increases our understanding of the diversification and faunal composition around the lower/upper Emsian boundary in the South China Block. It contributes to the regional and inter-regional biostratigraphical correlation between shallower- and deeper-water facies and also furnishes important South China material for future discussion and deliberation on the position of the lower/upper Emsian boundary.

Subspecies divergence and pronounced phylogenetic incongruence in the East-Asia-endemic shrubMagnolia sieboldii

The biogeographic patterns of the East-Asia-endemic shrub Magnolia sieboldii, in which the range of the subsp. sieboldii is interposed with the disjunct distribution of subsp. japonica, implies a complex evolutionary history, involving rapid speciation and hybridization. Here, we aim to reveal the evolutionary and phylogeographic histories of the species with a particular focus on the time of subspecies divergence, the hypothesis of secondary hybridization and the Pleistocene survival of each subspecies, using a combination of genetic analyses and ecological niche modelling. Genetic variation, genetic structures and phylogenetic relationships were elucidated based on nuclear low-copy genes, chloroplast DNA, and nuclear simple sequence repeats (SSRs). A scenario selection analysis and divergence time estimation were performed using coalescent simulation in DIYABC and *BEAST. Ecological niche modelling and a test of niche differentiation were performed using Maxent and ENMTools. All marker types showed deep, but pronouncedly incongruent, west-east genetic divergences, with the subspecies being delineated only by the nuclear low-copy genes. Phylogenetic tree topologies suggested that ancient hybridization and introgression were likely to have occurred; however, this scenario did not receive significant support in the DIYABC analysis. The subspecies differentiated their niches, but both showed a dependence on high humidity and were predicted to have persisted during the last glacial cycle by maintaining a stable latitudinal distribution via migration to lower altitudes. We found a deep genetic divergence and a pronounced phylogenetic incongruence among the two subspecies of M. sieboldii, which may have been driven by major paleogeographic and paleoclimatic events that have occurred since the Neogene in East Asia, including global cooling, climate oscillations and the formation of land bridges. Both subspecies were, however, considered to persist in situ in stable climatic conditions during the late Pleistocene.

Genetic diversity of native and introduced Phragmites (common reed) in Wisconsin.

Two subspecies of common reed (Phragmites australis; Poaceae) exist in northern North America: the native P. australis subsp. americanus and the introduced P. australis subsp. australis. There are numerous native populations in Wisconsin, in addition to the more recently established populations of the introduced subspecies. We studied populations of both P. australis subspecies across Wisconsin in order to characterize the genetic diversity of both subspecies and to investigate whether any instances of hybridization could be ascertained in Wisconsin. Using eight microsatellite markers, we found minimal overlap in the alleles that could be recovered from native and introduced plants, and we found no evidence to suggest hybridization between subspecies, even in localities where native and introduced plants grow in close proximity. Overall, we found greater genetic variation in plants of the introduced subspecies relative to the native subspecies, and we observed some geographic patterns of allelic diversity.

Genetic architecture of subspecies divergence in trace mineral accumulation and elemental correlations in the rice grain.

Genome differentiation has shaped the divergence in element concentration between rice subspecies and contributed to the correlation among trace minerals in the rice grain. The balance between trace minerals in rice, a staple food for more than half of the world's population, is crucial for human health. However, the genetic basis underlying the correlation between trace minerals has not been fully elucidated. To address this issue, we first quantified the concentrations of 11 trace minerals in the grains of a diversity panel of 575 rice cultivars. We found that eight elements were accumulated at significantly different levels between the indica and japonica subspecies, and we also observed significant correlation patterns among a number of elements. Further, using a genome-wide association study, we identified a total of 96 significant association loci (SALs). The differentiation of the major-effect SALs along with the different number of high-concentration alleles present in the two subspecies shaped the different element performance in indica and japonica varieties. Only a few SALs located in clusters and the majority of SALs showed subspecies/subgroup differentiation, indicating that the correlations between elements in the diversity panel were mainly caused by genome differentiation instead of shared genetic basis. The genetic architecture unveiled in this study will facilitate improvement in breeding for trace mineral content.

Escherichia coli Clonobiome: Assessing the Strain Diversity in Feces and Urine by Deep Amplicon Sequencing.

While microbiome studies have focused on diversity at the species level or higher, bacterial species in microbiomes are represented by different, often multiple, strains. These strains could be clonally and phenotypically very different, making assessment of strain content vital to a full understanding of microbiome function. This is especially important with respect to antibiotic-resistant strains, the clonal spread of which may be dependent on competition between them and susceptible strains from the same species. The pandemic, multidrug-resistant, and highly pathogenic Escherichia coli subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder in healthy people. In order to rapidly assess E. coli clonal diversity, we developed a novel method based on deep sequencing of two loci used for sequence typing, along with an algorithm for analysis of the resulting data. Using this method, we assessed fecal and urinary samples from healthy women carrying H30 and were able to uncover considerable diversity, including strains with frequencies at <1% of the E. coli population. We also found that, even in the absence of antibiotic use, H30 could completely dominate the gut and, especially, urine of healthy carriers. Our study offers a novel tool for assessing a species' clonal diversity (clonobiome) within the microbiome, which could be useful in studying the population structure and dynamics of multidrug-resistant and/or highly pathogenic strains in their natural environments.IMPORTANCE Bacterial species in the microbiome are often represented by multiple genetically and phenotypically different strains, making insight into subspecies diversity critical to a full understanding of the microbiome, especially with respect to opportunistic pathogens. However, methods allowing efficient high-throughput clonal typing are not currently available. This study combines a conventional E. coli typing method with deep amplicon sequencing to allow analysis of many samples concurrently. While our method was developed for E. coli, it may be adapted for other species, allowing microbiome researchers to assess clonal strain diversity in natural samples. Since assessment of subspecies diversity is particularly important for understanding the spread of antibiotic resistance, we applied our method to the study of a pandemic multidrug-resistant E. coli clone. The results we present suggest that this clone could be highly competitive in healthy carriers and that the mechanisms of colonization by such clones need to be studied.

Subspecies discrimination on the basis of acoustic signals: a playback experiment in a Neotropical songbird

When animal mating signals diverge between populations, reproductive isolation and speciation may occur. Variation in animals' responses to these signals may reveal whether differences in perception contribute to behavioural differences between populations. We tested whether signal divergence influences receiver responses to playback in the rufous-capped warbler, Basileuterus rufifrons, a Neotropical resident songbird with a contact zone between two divergent subspecies, B. r. delattrii and B. r. rufifrons, in southern Mexico. Studying nearby populations of birds living in allopatry and sympatry, we presented warblers with playback-simulated territorial male rivals of each subspecies. In sympatry, both delattrii and rufifrons responded more strongly to songs of their own subspecies than to songs of the other subspecies, whereas in allopatry, delattrii responded strongly to songs of both subspecies, suggesting possible reproductive character displacement. Our research demonstrates that sympatric delattrii and rufifrons discriminate between each other's songs, suggesting that song is a premating isolating barrier between these divergent subspecies. This study adds to the growing literature on receiver response to vocal signal divergence in closely related sympatric and allopatric animal populations.

Prevalence of hematologic toxicity from copperhead envenomation: an observational study

Background: Copperhead snakes (Agkistrodon contortrix) are considered as the least toxic of the North American pit vipers. The reported incidence of coagulopathy from copperhead envenomation is variable, possibly secondary to regional variation in subspecies and venom potency. Coagulation studies are often obtained when evaluating for the coagulopathic effects of copperhead venom, but the clinical utility of these indices is unclear. The aim of this study was to determine the prevalence of hematologic toxicity due to copperhead envenomation in hospitalized patients.Methods: This was a multi-center retrospective chart review study using electronic hospital data between January 1, 2006 and December 31, 2016 evaluating prevalence of coagulopathy following copperhead envenomation. Patients presenting to one of three major academic tertiary care centers in Virginia with suspected copperhead envenomation were identified using medical billing codes. The primary outcome was to summarize the prevalence of hematologic toxicity including thrombocytopenia, elevated prothrombin or partial thromboplastin times, or hypofibrinogenemia.Results: There were 244 cases used for final analysis. Hematologic toxicity occurred in 14% (95% CI 10–18%) of patients. Specific indices included thrombocytopenia in 1.2% (95% CI 0.4–3.6%), hypofibrinogenemia in 0.7% (95% CI 0.0–3.8%), elevated PT in 10.0% (95% CI 6.8–14.5%), and aPTT in 3.9% (95% CI 2.1–7.2%) of patients. There was no clinically significant bleeding reported in any case.Conclusions: Subtle hematologic abnormalities due to copperhead envenomation in patients treated in the Commonwealth of Virginia were relatively common, but do not appear to be clinically significant in this study population.

Ancient polymorphisms contribute to genome-wide variation by long-term balancing selection and divergent sorting in Boechera stricta

BackgroundGenomic variation is widespread, and both neutral and selective processes can generate similar patterns in the genome. These processes are not mutually exclusive, so it is difficult to infer the evolutionary mechanisms that govern population and species divergence. Boechera stricta is a perennial relative of Arabidopsis thaliana native to largely undisturbed habitats with two geographic and ecologically divergent subspecies. Here, we delineate the evolutionary processes driving the genetic diversity and population differentiation in this species.ResultsUsing whole-genome re-sequencing data from 517 B. stricta accessions, we identify four genetic groups that diverged around 30–180 thousand years ago, with long-term small effective population sizes and recent population expansion after the Last Glacial Maximum. We find three genomic regions with elevated nucleotide diversity, totaling about 10% of the genome. These three regions of elevated nucleotide diversity show excess of intermediate-frequency alleles, higher absolute divergence (dXY), and lower relative divergence (FST) than genomic background, and significant enrichment in immune-related genes, reflecting long-term balancing selection. Scattered across the genome, we also find regions with both high FST and dXY among the groups, termed FST-islands. Population genetic signatures indicate that FST-islands with elevated divergence, which have experienced directional selection, are derived from divergent sorting of ancient polymorphisms.ConclusionsOur results suggest that long-term balancing selection on disease resistance genes may have maintained ancestral haplotypes across different geographical lineages, and unequal sorting of balanced polymorphisms may have generated genomic regions with elevated divergence. This study highlights the importance of ancestral balanced polymorphisms as crucial components of genome-wide variation.

Integrating phylogenomics, phylogenetics, morphometrics, relative genome size and ecological niche modelling disentangles the diversification of Eurasian Euphorbia seguieriana s. l. (Euphorbiaceae)

Next generation sequencing has revolutionised biology. Restriction-associated DNA sequencing (RADseq) has primarily been used to study infraspecific relationships but has also been applied in multi-species phylogenomic analyses. In this study, we used a combination of phylogenomic (with RADseq data) and phylogenetic (with sequences of the nuclear internal transcribed spacer, ITS) methods to explore relationships within the taxonomically intricate Euphorbia seguieriana s. l., one of the most widespread Euphorbia taxa inhabiting zonal and extrazonal steppes from Iberia to Central Asia. In the inferred phylogenies the southeastern Balkan and Anatolian populations were clearly separated, supporting the distinction of E. niciciana from E. seguieriana at the species level. Within E. seguieriana, the populations from the Caucasus, Iran, and easternmost Anatolia were sister to all other populations based on RADseq, making necessary the description of a new, morphologically divergent subspecies, E. seguieriana subsp. armeniaca. Conversely, additional studies are needed to understand the status of E. seguieriana subsp. hohenackeri, which is sympatric with E. seguieriana subsp. armeniaca. Niche analyses indicated that differences in the climatic niche between E. niciciana and E. seguieriana are relatively small compared with the climatic differences between the regions over which they are distributed. Contrary to previous believes, E. niciciana and E. seguieriana are allopatric and have likely diverged during the Pleistocene in two different glacial refugia as suggested by distribution modelling. Euphorbia niciciana nowadays has a submediterranean distribution, occupying habitats that are slightly warmer, moister, and less seasonal in temperature but more seasonal in precipitation than E. seguieriana, a characteristic species of continental steppes. Using flow cytometry, we demonstrate that the relative genome sizes of E. niciciana and E. seguieriana differ significantly. Additionally, multivariate morphometric analyses of 56 morphological characters indicated clear morphological divergence of the two species. Importantly, we also provide a revised taxonomic treatment including formal nomenclatural changes, an identification key and species descriptions. Our study demonstrates that an integrative approach, combining modern phylogenomic methods with traditional phylogenetic, cytogenetic, environmental and morphological analyses can result in satisfactorily resolved relationships in intricate groups of closely related species. Finally, phylogenetic inference using ITS sequences is still a useful tool for resolving relationships among the taxa at the species level, but the phylogenomic approach based on RADseq data certainly provides better resolution both among and within species.

Biodiversity of Bali Banana (Musaceae) and its Usefulness

Banana (Musa spp.) is one the most important agriculture commodities in Indonesia. Archeological and molecular evidences suggest that speciation of this herb occurred in Indonesia, leading to the high diversity in the archipelago. In Bali Island, banana is not only sought for food but as well as for their symbolic role in religious and cultural ceremonies. However, the high demand for bananas in Bali is not yet supported by the adequate production of local farms. This presented study aimed to investigate the diversity of banana cultivars or sub-species in Bali and its usefulness to determine preferable cultivars to cultivate. We recorded and characterized 43 banana cultivars in 10 villages that represent the 8 regencies and 1 city of Bali province. Out of the 43 cultivars, 7 were highly used and at least one cultivar was discovered in each of the studied village. The presence of these cultivars in the study areas were confirmed by site visit and characterization of the fruits. Among the highly ranked cultivars or species, only biu kayu is unique to Bali as it was not found in the closest provinces of East Java and Madura. Hence, the results suggested that to improve the cultivation and production of these 7 highly used cultivars could be an appropriate solution to meet Bali demand of bananas. Furthermore, cultivating biu kayu would also help conservation effort since this cultivar is also currently listed as a rare genetic resource.

Demographic inference in barn swallows using whole‐genome data shows signal for bottleneck and subspecies differentiation during the Holocene

Accounting for historical demographic features is vital for many types of evolutionary inferences, including the estimation of divergence times between closely related populations. In barn swallow, Hirundo rustica, inferring historical population sizes and subspecies divergence times can shed light on the recent co-evolution of this species with humans. Pairwise sequentially Markovian coalescent uncovered population growth beginning on the order of one million years ago-which may reflect the radiation of the broader Hirundo genus-and a more recent population decline. Additionally, we used approximate Bayesian computation to evaluate hypotheses about recent timescale barn swallow demography, including population growth due to human commensalism, and a potential founder event associated with the onset of nesting on human structures. We found signal for a bottleneck event approximately 7,700years ago, near the time that humans began building substantial structures, although there was considerable uncertainty associated with this estimate. Subspecies differentiation and subsequent growth occurred after the bottleneck in the best-supported model, an order of magnitude more recently than previous estimates in this system. We also compared results obtained from whole-genome sequencing versus reduced representation sequencing, finding many similar results despite substantial allelic dropout in the reduced representation data, which may have affected estimates of some parameters. This study presents the first genetic evidence of a potential barn swallow founder effect and subspecies divergence coinciding with the Holocene, which is an important step in analysing the biogeographical history of a well-known human commensal species.

Quaternary-related genetic differentiation and parallel population dynamics of the Eurasian Jay (Garrulus glandarius) in the circum-Japan Sea region

The circum-Japan Sea region (CJSR) greatly impacted animal diversity in the Eastern Palearctic during the Quaternary. However, its role in avian diversification has been underestimated because of the high dispersal capabilities of birds over the sea. We investigated the phylogeographic and demographic history of the Eurasian Jay (Garrulus glandarius), focusing on CJSR populations. We sequenced a total of 1744 bp of mitochondrial DNA (cytochrome b and control region) from 73 samples. Together with the database sequences, we reconstructed a phylogenetic tree for the Eurasian Jay over the Palearctic. The earliest phylogenetic divergence was inferred to be related to geological vicariance between the Japanese archipelago and Eurasian mainland around the Pliocene–Pleistocene boundary. Several demographic analyses have suggested that there are two divergent subspecies across the CJSR, G. g. brandtii on the mainland, Sakhalin, and Hokkaido and G. g. japonicus in the archipelago. These simultaneously experienced population contractions to independent refugia and subsequent expansions around the last glacial period, i.e., parallel population dynamics. We suggest that the two landmasses of the CJSR were important for generating and preserving the phylogenetic structure of the Eurasian Jay.

Morphological and molecular divergence of Rhipicephalus turanicus tick from Albania and China

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53–4.84, 3.57–4.92, 3.57–4.07, 3.57–4.39 and 3.18–4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.

Population-Genomic Insights into Variation in Prevotella intermedia and Prevotella nigrescens Isolates and Its Association with Periodontal Disease.

High-throughput sequencing has helped to reveal the close relationship between Prevotella and periodontal disease, but the roles of subspecies diversity and genomic variation within this genus in periodontal diseases still need to be investigated. We performed a comparative genome analysis of 48 Prevotella intermedia and Prevotella nigrescens isolates that from the same cohort of subjects to identify the main drivers of their pathogenicity and adaptation to different environments. The comparisons were done between two species and between disease and health based on pooled sequences. The results showed that both P. intermedia and P. nigrescens have highly dynamic genomes and can take up various exogenous factors through horizontal gene transfer. The major differences between disease-derived and health-derived samples of P. intermedia and P. nigrescens were factors related to genome modification and recombination, indicating that the Prevotella isolates from disease sites may be more capable of genomic reconstruction. We also identified genetic elements specific to each sample, and found that disease groups had more unique virulence factors related to capsule and lipopolysaccharide synthesis, secretion systems, proteinases, and toxins, suggesting that strains from disease sites may have more specific virulence, particularly for P. intermedia. The differentially represented pathways between samples from disease and health were related to energy metabolism, carbohydrate and lipid metabolism, and amino acid metabolism, consistent with data from the whole subgingival microbiome in periodontal disease and health. Disease-derived samples had gained or lost several metabolic genes compared to healthy-derived samples, which could be linked with the difference in virulence performance between diseased and healthy sample groups. Our findings suggest that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, which may reflect changes in microbial and environmental dynamics in subgingival microbial ecosystems. This provides insight into the potential of P. intermedia and P. nigrescens as new predictive biomarkers and targets for effective interventions in periodontal disease.

Phospholipase A2 in the venom of three cottonmouth snakes

Two phospholipase A2s (PLA2s), Asp49 (D49) and Lys49 (K49), were purified by one-step reverse-phase high performance liquid chromatography (RP-HPLC) from the venom of each of the three subspecies of cottonmouth snake, Western cottonmouth (Agkistrodon piscivorus leucostoma; Apl), Eastern cottonmouth (Agkistrodon piscivorus piscivorus; App) and Florida cottonmouth (Agkistrodon piscivorus conanti; Apc). Venom protein profiles and PLA2s elution pattern of the three cottonmouth snakes were remarkably similar displaying four similar sharp and two wide peaks; in all cases K49 PLA2 eluted first followed by D49 PLA2. The yields of K49 and D49 PLA2s were, respectively, 13.2 and 17.5 mg/g venom from the Western cottonmouth, 16.8 and 19.2 mg/g from the Eastern cottonmouth, and 17.3 and 22.7 mg/g from the Florida cottonmouth. Biochemical and enzymatic techniques were used to characterize the purified PLA2. The amino acid sequences of all three K49PLA2s were identical; App-D49 and Apc-D49 were also identical but displayed a single amino acid difference with Apl-D49. To our knowledge, this is the first report on the amino acid sequence and molecular mass of Apc-D49 and Apc-K49 PLA2s from Florida cottonmouth venom. As expected, PLA2 enzymatic analysis revealed that D49 PLA2s from all three venoms hydrolyze phospholipids to a similar extent, whereas no phospholipid hydrolysis was detectable by any of the K49 PLA2s purified. Cottonmouth snake venoms contain abundant PLA2 isoforms, thus the identification of PLA2s in these venoms is of interest to facilitate the purification of specific PLA2 from rich sources of subspecies venom for future biological and biomedical research. Results of this study also contribute towards the understanding of venom protein profiles, variation, and evolution in subspecies of venomous snakes.

Associations of anthropometry and lifestyle factors with HDL subspecies according to apolipoprotein C-III

The presence of apoC-III on HDL impairs HDL's inverse association with coronary heart disease (CHD). Little is known about modifiable factors explaining variation in HDL subspecies defined according to apoC-III. The aim was to investigate cross-sectional associations of anthropometry and lifestyle with HDL subspecies in 3,631 participants from the Diet, Cancer, and Health study originally selected for a case-cohort study (36% women; age 50–65 years) who were all free of CHD. Greater adiposity and less activity were associated with higher HDL containing apoC-III and lower HDL lacking apoC-III. Per each 15 cm higher waist circumference, the level of HDL containing apoC-III was 2.8% higher (95% CI: 0.4, 5.3; P = 0.024) and the level of HDL not containing apoC-III was 4.7% lower (95% CI: −6.0, −3.4; P = <0.0001). Associations for physical activity were most robust to multivariable modeling. Each 20 metabolic equivalent task hours per week reported higher physical activity was associated with 0.9% (95% CI: −1.7, −0.1; P = 0.031) lower HDL containing apoC-III and 0.5% higher (95% CI: 0.1, 1.0; P = 0.029) HDL lacking apoC-III. Lower alcohol consumption was associated with lower HDL lacking apoC-III (percent difference per 15 g/day: 1.58 (95% CI: 0.84, 2.32; P = <0.0001). Adiposity and sedentary lifestyle were associated with a less favorable HDL subspecies profile.

Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation

When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal.