Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

An efficient Agrobacterium‐mediated site‐specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.     

Comparative phylogeography of two sympatric beeches in subtropical China: Species‐specific geographic mosaic of lineages

In subtropical China, large‐scale phylogeographic comparisons among multiple sympatric plants with similar ecological preferences are scarce, making generalizations about common response to historical events necessarily tentative. A phylogeographic comparison of two sympatric Chinese beeches (Fagus lucida and F. longipetiolata, 21 and 28 populations, respectively) was conducted to test whether they have responded to historical events in a concerted fashion and to determine whether their phylogeographic structure is exclusively due to Quaternary events or it is also associated with pre‐Quaternary events. Twenty‐three haplotypes were recovered for F. lucida and F. longipetiolata (14 each one and five shared). Both species exhibited a species‐specific mosaic distribution of haplotypes, with many of them being range‐restricted and even private to populations. The two beeches had comparable total haplotype diversity but F. lucida had much higher within‐population diversity than F. longipetiolata. Molecular dating showed that the time to most recent common ancestor of all haplotypes was 6.36 Ma, with most haplotypes differentiating during the Quaternary. [Correction added on 14 October 2013, after first online publication: the time unit has been corrected to ‘6.36’.] Our results support a late Miocene origin and southwards colonization of Chinese beeches when the aridity in Central Asia intensified and the monsoon climate began to dominate the East Asia. During the Quaternary, long‐term isolation in subtropical mountains of China coupled with limited gene flow would have lead to the current species‐specific mosaic distribution of lineages.     

Deep divergence among mitochondrial lineages in African jackals

Recently, molecular analyses revealed that African and Eurasian golden jackals are distinct species. This finding suggests re‐investigation of the phylogenetic relationships and taxonomy of other African members of the Canidae. Here, we provide a study on the phylogenetic relationship between populations of African jackals Lupulella mesomelas and L. adusta inferred from 962 bp of the mitochondrial cytochrome b (cytb) gene. As expected from its disjunct distribution, with one population in eastern Africa and the other one in southern Africa, we found two mitochondrial lineages within L. mesomelas, which diverged about 2.5 million years ago (Ma). In contrast, in L. adusta with its more continuous distribution in sub‐Saharan Africa, we found only a shallower genetic diversification, with the exception of the West African population, which diverged around 1.4 Ma from the Central and East African populations. Both divergence ages are older than, for example the 1.1–0.9 million years between the grey wolf Canis lupus and the African golden wolf C. lupaster. One taxonomic implication of our findings might be that the two L. mesomelas populations warrant species status. However, genome‐wide data with adequate geographical sampling are needed to substantiate our results.     

Application of multigene phylogenetics and site‐stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta)

Previous molecular assessments of the red algal order Rhodymeniales have confirmed its monophyly and distinguished the six currently recognized families (viz. Champiaceae, Faucheaceae, Fryeellaceae, Hymenocladiaceae, Lomentariaceae, and Rhodymeniaceae); however, relationships among most of these families have remained unresolved possibly as a result of substitution saturation at deeper phylogenetic nodes. The objective of the current study was to improve rhodymenialean systematics by increasing taxonomic representation and using a more robust multigene dataset of mitochondrial (COB, COI/COI‐5P), nuclear (LSU, EF2) and plastid markers (psbA, rbcL). Additionally, we aimed to prevent phylogenetic inference problems associated with substitution saturation (particularly at the interfamilial nodes) by removing fast‐evolving sites and analyzing a series of progressively more conservative alignments. The Rhodymeniales was resolved as two major lineages: (i) the Fryeellaceae as sister to the Faucheaceae and Lomentariaceae; and (ii) the Rhodymeniaceae allied to the Champiaceae and Hymenocladiaceae. Support at the interfamilial nodes was highest when 20% of variable sites were removed. Inclusion of Binghamiopsis, Chamaebotrys, and Minium, which were absent in previous phylogenetic investigations, established their phylogenetic affinities while assessment of two genera consistently polyphyletic in phylogenetic analyses, Erythrymenia and Lomentaria, resulted in the proposition of the novel genera Perbella and Fushitsunagia. The taxonomic position of Drouetia was reinvestigated with re‐examination of holotype material of D. coalescens to clarify tetrasporangial development in this genus. In addition, we added three novel Australian species to Drouetia as a result of ongoing DNA barcoding assessments—D. aggregata sp. nov., D. scutellata sp. nov., and D. viridescens sp. nov.     

Molecular phylogenetics of slit‐faced bats (Chiroptera: Nycteridae) reveal deeply divergent African lineages

The bat family Nycteridae contains only the genus Nycteris, which comprises 13 currently recognized species from Africa and the Arabian Peninsula, one species from Madagascar, and two species restricted to Malaysia and Indonesia in South‐East Asia. We investigated genetic variation, clade membership, and phylogenetic relationships in Nycteridae with broad sampling across Africa for most clades. We sequenced mitochondrial cytochrome b (cytb) and four independent nuclear introns (2,166 bp) from 253 individuals. Although our samples did not include all recognized species, we recovered at least 16 deeply divergent monophyletic lineages using independent mitochondrial and multilocus nuclear datasets in both gene tree and species tree analyses. Mean pairwise uncorrected genetic distances among species‐ranked Nycteris clades (17% for cytb and 4% for concatenated introns) suggest high levels of phylogenetic diversity in Nycteridae. We found a large number of designated clades whose members are distributed wholly or partly in East Africa (10 of 16 clades), indicating that Nycteris diversity has been historically underestimated and raising the possibility that additional unsampled and/or undescribed Nycteris species occur in more poorly sampled Central and West Africa. Well‐resolved mitochondrial, concatenated nuclear, and species trees strongly supported African ancestry for SE Asian species. Species tree analyses strongly support two deeply diverged subclades that have not previously been recognized, and these clades may warrant recognition as subgenera. Our analyses also strongly support four traditionally recognized species groups of Nycteris. Mitonuclear discordance regarding geographic population structure in Nycteris thebaica appears to result from male‐biased dispersal in this species. Our analyses, almost wholly based on museum voucher specimens, serve to identify species‐rank clades that can be tested with independent datasets, such as morphology, vocalizations, distributions, and ectoparasites. Our analyses highlight the need for a comprehensive revision of Nycteridae.     

Extensive hybridization and past introgression between divergent lineages in a quasi‐clonal hermaphroditic fish: Ramifications for species concepts and taxonomy

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self‐fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a ‘Central clade’ closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self‐fertilization for 1–3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.     

Between a rock and a hard place: adaptive sensing and site‐specific dispersal

Environmental variability can lead to dispersal: why stay put if it is better elsewhere? Without clues about local conditions, the optimal strategy is often to disperse a set fraction of offspring. Many habitats contain environmentally differing sub‐habitats. Is it adaptive for individuals to sense in which sub‐habitat they find themselves, using environmental clues, and respond plastically by altering the dispersal rates? This appears to be done by some plants which produce dimorphic seeds with differential dispersal properties in response to ambient temperature. Here we develop a mathematical model to show, that in highly variable environments, not only does sensing promote plasticity of dispersal morph ratio, individuals who can sense their sub‐habitat and respond in this way have an adaptive advantage over those who cannot. With a rise in environmental variability due to climate change, our understanding of how natural populations persist and respond to changes has become crucially important.     

Experimental evolution reveals habitat‐specific fitness dynamics among Wolbachia clades in Drosophila melanogaster

The diversity and infection dynamics of the endosymbiont Wolbachia can be influenced by many factors, such as transmission rate, cytoplasmic incompatibility, environment, selection and genetic drift. The interplay of these factors in natural populations can result in heterogeneous infection patterns with substantial differences between populations and strains. The causes of these heterogeneities are not yet understood, partly due to the complexity of natural environments. We present experimental evolution as a new approach to study Wolbachia infection dynamics in replicate populations exposed to a controlled environment. A natural Drosophila melanogaster population infected with strains of Wolbachia belonging to different clades evolved in two laboratory environments (hot and cold) for 1.5 years. In both treatments, the rate of Wolbachia infection increased until fixation. In the hot environment, the relative frequency of different Wolbachia clades remained stable over 37 generations. In the cold environment, however, we observed marked changes in the composition of the Wolbachia population: within 15 generations, one Wolbachia clade increased more than 50% in frequency, whereas the other two clades decreased in frequency, resulting in the loss of one clade. The frequency change was highly reproducible not only among replicates, but also when flies that evolved for 42 generations in the hot environment were transferred to the cold environment. These results document how environmental factors can affect the composition of Wolbachia in D. melanogaster. The high reproducibility of the pattern suggests that experimental evolution studies can efficiently determine the functional basis of habitat‐specific fitness among Wolbachia strains.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Long‐term endemism of two highly divergent lineages of the amphibian‐killing fungus in the Atlantic Forest of Brazil

The recent global spread of the amphibian‐killing fungus [Batrachochytrium dendrobatidis (Bd)] has been closely tied to anthropogenic activities; however, regional patterns of spread are not completely understood. Using historical samples, we can test whether Bd was a spreading or endemic pathogen in a region within a particular time frame, because those two disease states provide different predictions for the regional demographic dynamics and population genetics of Bd. Testing historical patterns of pathogen prevalence and population genetics under these predictions is key to understanding the evolution and origin of Bd. Focusing on the Atlantic Forest (AF) of Brazil, we used qPCR assays to determine the presence or absence of Bd on 2799 preserved postmetamorphic anurans collected between 1894 and 2010 and used semi‐nested PCRs to determine the frequency of rRNA ITS1 haplotypes from 52 samples. Our earliest date of detection was 1894. A mean prevalence of 23.9% over time and spatiotemporal patterns of Bd clusters indicate that Bd has been enzootic in the Brazilian AF with no evidence of regional spread within the last 116 years. ITS1 haplotypes confirm the long‐term presence of two divergent strains of Bd (BdGPL and Bd‐Brazil) and three spatiotemporally broad genetic demes within BdGPL, indicating that Bd was not introduced into southeast Brazil by the bullfrog trade. Our data show that the evolutionary history and pathogen dynamics of Bd in Brazil is better explained by the endemic pathogen hypothesis.     

Parting ways: parasite release in nature leads to sex‐specific evolution of defence

We evaluated the extent to which males and females evolve along similar or different trajectories in response to the same environmental shift. Specifically, we used replicate experimental introductions in nature to consider how release from a key parasite (Gyrodactylus) generates similar or different defence evolution in male vs. female guppies (Poecilia reticulata). After 4–8 generations of evolution, guppies were collected from the ancestral (parasite still present) and derived (parasite now absent) populations and bred for two generations in the laboratory to control for nongenetic effects. These F2 guppies were then individually infected with Gyrodactylus, and infection dynamics were monitored on each fish. We found that parasite release in nature led to sex‐specific evolutionary responses: males did not show much evolution of resistance, whereas females showed the evolution of increased resistance. Given that male guppies in the ancestral population had greater resistance to Gyrodactylus than did females, evolution in the derived populations led to reduction of sexual dimorphism in resistance. We argue that previous selection for high resistance in males constrained (relative to females) further evolution of the trait. We advocate more experiments considering sex‐specific evolutionary responses to environmental change.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

WNT‐3A and WNT‐5A counteract lipopolysaccharide‐induced pro‐inflammatory changes in mouse primary microglia

Surveying microglia, the resident macrophage‐like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro‐ and anti‐inflammatory manner. We have previously shown that the lipoglycoproteins WNT‐3A and WNT‐5A drive pro‐inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT‐3A and WNT‐5A on lipopolysaccharide (LPS)‐activated mouse primary microglia to investigate the putative anti‐inflammatory modulation of microglia by WNTs. While both WNT‐3A and WNT‐5A alone induce an up‐regulation of cyclooxygenase 2 (COX2), a generic pro‐inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose‐dependent decrease in LPS‐induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context‐dependent effect on microglia acting in a homeostatic pro‐ and anti‐inflammatory manner.     

Anti‐aging drugs reduce hypothalamic inflammation in a sex‐specific manner

Aging leads to hypothalamic inflammation, but does so more slowly in mice whose lifespan has been extended by mutations that affect GH/IGF‐1 signals. Early‐life exposure to GH by injection, or to nutrient restriction in the first 3 weeks of life, also modulate both lifespan and the pace of hypothalamic inflammation. Three drugs extend lifespan of UM‐HET3 mice in a sex‐specific way: acarbose (ACA), 17‐α‐estradiol (17αE2), and nordihydroguaiaretic acid (NDGA), with more dramatic longevity increases in males in each case. In this study, we examined the effect of these anti‐aging drugs on neuro‐inflammation in hypothalamus and hippocampus. We found that age‐associated hypothalamic inflammation is reduced in males but not in females at 12 months of age by ACA and 17αE2 and at 22 months of age in NDGA‐treated mice. The three drugs blocked indices of hypothalamic reactive gliosis associated with aging, such as Iba‐1‐positive microglia and GFAP‐positive astrocytes, as well as age‐associated overproduction of TNF‐α. This effect was not observed in drug‐treated female mice or in the hippocampus of the drug‐treated animals. On the other hand, caloric restriction (CR; an intervention that extends the lifespan in both sexes) significantly reduced hypothalamic microglia and TNF‐α in both sexes at 12 months of age. Together, these results suggest that the extent of drug‐induced changes in hypothalamic inflammatory processes is sexually dimorphic in a pattern that parallels the effects of these agents on mouse longevity and that mimics the changes seen, in both sexes, of long‐lived nutrient restricted or mutant mice.