Drivers of sociality in Gobiodon fishes: An assessment of phylogeny, ecology and life-history.

What drives the evolution of sociality in animals? Many robust studies in terrestrial organisms have pointed toward various kinship-based, ecological and life-history traits or phylogenetic constraint which have played a role in the evolution of sociality. These traits are not mutually exclusive and the exact combination of traits is likely taxon-specific. Phylogenetic comparative analyses have been instrumental in identifying social lineages and comparing various traits with non-social lineages to give broad evolutionary perspectives on the development of sociality. Few studies have attempted this approach in marine vertebrate systems. Social marine fishes are particularly interesting because many have a pelagic larval phase and non-conventional life-history strategies (e.g. bi-directional sex-change) not often observed in terrestrial animals. Such strategies provide novel insights into terrestrially-derived theories of social evolution. Here, we assess the strength of the phylogenetic signal of sociality in the Gobiodon genus with Pagel's lambda and Blomberg's K parameters. We found some evidence of a phylogenetic signal of sociality, but factors other than phylogenetic constraint also have a strong influence on the extant social state of each species. We then use phylogenetic generalized least squares analyses to examine several ecological and life-history traits that may have influenced the evolution of sociality in the genus. We found an interaction of habitat size and fish length was the strongest predictor of sociality. Sociality in larger species was more dependent on coral size than in smaller species, but smaller species were more social overall, regardless of coral size. Finally, we comment on findings regarding the validity of the species G. spilophthalmus which arose during the course of our research. These findings in a group of marine fishes add a unique perspective on the evolution of sociality to the excellent terrestrial work conducted in this field.

Coexistence of minicircular and a highly rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome organization.

Recombination has been proposed as a possible mechanism to explain mitochondrial (mt) gene rearrangements, although the issue of whether mtDNA recombination occurs in animals has been controversial. In this study, we sequenced the entire mt genome of the megaspilid wasp Conostigmus sp., which possessed a highly rearranged mt genome. The sequence of the A+T-rich region contained a number of different types of repeats, similar to those reported previously in the nematode Meloidogyne javanica, in which recombination was discovered. In Conostigmus, we detected the end products of recombination: a range of minicircles. However, using isolated (cloned) fragments of the A+T-rich region, we established that some of these minicircles were found to be polymerase chain reaction (PCR) artifacts. It appears that regions with repeats are prone to PCR template switching or PCR jumping. Nevertheless, there is strong evidence that one minicircle is real, as amplification primers that straddle the putative breakpoint junction produce a single strong amplicon from genomic DNA but not from the cloned A+T-rich region. The results provide support for the direct link between recombination and mt gene rearrangement. Furthermore, we developed a model of recombination which is important for our understanding of mtDNA evolution.

Higher-level phylogeny of the Hymenoptera inferred from mitochondrial genomes.

Higher-level hymenopteran relationships remain unresolved in both morphological and molecular analyses. In this study, we present the most comprehensive analyses of hymenopteran relationships based on 48 mitochondrial (mt) genomes. One complete and two nearly complete mt genomes representing three hymenopteran superfamilies were newly sequenced. We assessed the influence of inclusion/exclusion of 3rd codon positions, alignment approaches, partition schemes and phylogenetic approaches on topology and nodal support within the Hymenoptera. The results showed that the topologies were sensitive to the variation of dataset and analytical approach. However, some robust and highly supported relationships were recovered: the Ichneumonomorpha was monophyletic; the Trigonalyoidea+Megalyroidea and the Diaprioidea+Chalcidoidea were consistently recovered; the Cynipoidea was generally recovered as the sister group to the Diaprioidea+Chalcidoidea. In addition, the monophyletic Aculeata and Proctotrupomorpha were recovered in some analyses. Several gene rearrangements were detected in each of the three newly sequenced mt genomes. Specifically, the Ibalia leucospoides mt genome harbors a large inversion of a gene block from trnE to trnS2. Inverted, duplicated A+T rich regions were detected in the Ibalia leucospoides mt genome, which probably played an important role during the formation of the large gene block inversion via recombination.

R270C polymorphism leads to loss of function of the canine P2X7 receptor.

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

Complete mitochondrial genomes of Ceratobaeus sp. and Idris sp. (Hymenoptera: Scelionidae): shared gene rearrangements as potential phylogenetic markers at the tribal level.

We sequenced the complete mitochondrial genomes of two sceliond taxa (Ceratobaeus sp. and Idris sp.). An atypical tRNA-Arg which lacks a D-stem was identified in both taxa, and represents a potentially derived character of sceliond wasps. A number of tRNA genes have rearranged in the two mitochondrial genomes compared with the ancestral organization. Some of these derived genome organizations are shared, and thus have much potential as phylogenetic markers at the tribal level in the subfamily Scelioninae. We test the influence of third codon inclusion/exclusion, alignment methods and partition schemes on the reconstruction of phylogenetic relationships. The results show that inclusion of third codon positions does not appear to be problematic when investigating the phylogeny of closely related taxa. Muscle and PartitionFinder schemes significantly improve the likelihood scores.

Utility of COI, CAD and morphological data for resolving relationships within the genus Sarcophaga (sensu lato) (Diptera: Sarcophagidae): a preliminary study.

Currently there are ≈ 3000 known species of Sarcophagidae (Diptera), which are classified into 173 genera in three subfamilies. Almost 25% of sarcophagids belong to the genus Sarcophaga (sensu lato) however little is known about the validity of, and relationships between the ≈ 150 (or more) subgenera of Sarcophaga s.l. In this preliminary study, we evaluated the usefulness of three sources of data for resolving relationships between 35 species from 14 Sarcophaga s.l. subgenera: the mitochondrial COI barcode region, ≈ 800 bp of the nuclear gene CAD, and 110 morphological characters. Bayesian, maximum likelihood (ML) and maximum parsimony (MP) analyses were performed on the combined dataset. Much of the tree was only supported by the Bayesian and ML analyses, with the MP tree poorly resolved. The genus Sarcophaga s.l. was resolved as monophyletic in both the Bayesian and ML analyses and strong support was obtained at the species-level. Notably, the only subgenus consistently resolved as monophyletic was Liopygia. The monophyly of and relationships between the remaining Sarcophaga s.l. subgenera sampled remain questionable. We suggest that future phylogenetic studies on the genus Sarcophaga s.l. use combined datasets for analyses. We also advocate the use of additional data and a range of inference strategies to assist with resolving relationships within Sarcophaga s.l.

Delayed recovery and host specialization may spell disaster for coral-fish mutualism.

Mutualisms are prevalent in many ecosystems, yet little is known about how symbioses are affected by ecological pressures. Here, we show delayed recovery for 13 coral-dwelling goby fishes (genus Gobiodon) compared with their host Acropora corals following four consecutive cyclones and heatwaves. While corals became twice as abundant in 3 years postdisturbances, gobies were only half as abundant relative to predisturbances and half of the goby species disappeared. Although gobies primarily occupied one coral species in greater abundance predisturbances, surviving goby species shifted hosts to newly abundant coral species when their previously occupied hosts became rare postdisturbances. As host specialization is key for goby fitness, shifting hosts may have negative fitness consequences for gobies and corals alike and affect their survival in response to environmental changes. Our study is an early sign that mutualistic partners may not recover similarly from multiple disturbances, and that goby host plasticity, while potentially detrimental, may be the only possibility for early recovery.

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

The first mitochondrial genome for the wasp superfamily Platygastroidea: the egg parasitoid Trissolcus basalis.

The nearly complete mitochondrial (mt) genome of an egg parasitoid, Trissolcus basalis (Wollaston), was sequenced using both 454 and Illumina next-generation sequencing technologies. A portion of the noncoding region remained unsequenced, possibly owing to the presence of repeats. The sequenced portion of the genome is 15,768 bp and has a high A+T content (84.2%), as is typical for hymenopteran mt genomes. A total of 36 of the 37 genes normally present in animal mt genomes were located. The one exception was trnR; a truncated version of this gene is present between trnS(1) and nd5, but it is unclear whether this gene fragment could code for the entire trnR gene. The mt gene arrangement of T. basalis is different from other Proctotrupomorpha mt genomes, with a number of trn genes in different positions. However, no shared derived gene rearrangements were identified in the present study. Bayesian analyses of mt genomes from 29 hymenopteran taxa and seven other orders of holometabolous insects support some uncontroversial evolutionary relationships, but indicate that much higher levels of taxonomic sampling are necessary for the resolution of family and superfamily relationships.

A Comparison of Three Molecular Markers for the Identification of Populations of Globodera pallida.

Potato cyst nematodes cost the potato industry substantial financial losses annually. Through the use of molecular markers, the distribution and infestation routes of these nematodes can be better elucidated, permitting the development of more effective preventative methods. Here we assess the ability of three molecular markers to resolve multiple representatives of five Globodera pallida populations as monophyletic groups. Molecular markers included a region of the rbp-1 gene (an effector), a non-coding nuclear DNA region (the ITS region), and a novel marker for G. pallida, a ∼3.4 kb non-coding mitochondrial DNA (mtDNA) region. Multiple phylogenetic analysis methods were performed on the three DNA regions separately, and on a data set of these three regions combined. The analyses of the combined data set were similar to that of the sole mtDNA marker; resolving more populations as monophyletic groups, relative to that of the ITS region and rbp-1 gene region. This suggests that individual markers may be inadequate for distinguishing populations of G. pallida. The use of this new non-coding mtDNA marker may provide further insights into the historical distribution of G. pallida, as well as enable the development of more sensitive diagnostic methods.

Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artefact of amplification.

We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10(-5) indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the 'biological + artefactual' rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.

Paternal leakage of mitochondrial DNA in experimental crosses of populations of the potato cyst nematode Globodera pallida.

Animal mtDNA is typically assumed to be maternally inherited. Paternal mtDNA has been shown to be excluded from entering the egg or eliminated post-fertilization in several animals. However, in the contact zones of hybridizing species and populations, the reproductive barriers between hybridizing organisms may not be as efficient at preventing paternal mtDNA inheritance, resulting in paternal leakage. We assessed paternal mtDNA leakage in experimental crosses of populations of a cyst-forming nematode, Globodera pallida. A UK population, Lindley, was crossed with two South American populations, P5A and P4A. Hybridization of these populations was supported by evidence of nuclear DNA from both the maternal and paternal populations in the progeny. To assess paternal mtDNA leakage, a ~3.4 kb non-coding mtDNA region was analyzed in the parental populations and in the progeny. Paternal mtDNA was evident in the progeny of both crosses involving populations P5A and P4A. Further, paternal mtDNA replaced the maternal mtDNA in 22 and 40 % of the hybrid cysts from these crosses, respectively. These results indicate that under appropriate conditions, paternal leakage occurs in the mtDNA of parasitic nematodes, and supports the hypothesis that hybrid zones facilitate paternal leakage. Thus, assumptions of strictly maternal mtDNA inheritance may be frequently violated, particularly when divergent populations interbreed.

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines.

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.

DNA-based identification of forensically important Australian Sarcophagidae (Diptera).

The utility of the forensically important Sarcophagidae (Diptera) for time since death estimates has been severely limited, as morphological identification is difficult and thermobiological histories are inadequately documented. A molecular identification method involving the sequencing of a 658-bp 'barcode' fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene from 85 specimens, representing 16 Australian species from varying populations, was evaluated. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining phylogenetic tree generated. All species were resolved as reciprocally monophyletic, except Sarcophaga dux. Intraspecific and interspecific variation ranged from 0.000% to 1.499% (SE = 0.044%) and 6.658% to 8.983% (SE = 0.653%), respectively. The COI 'barcode' sequence was found to be suitable for the molecular identification of the studied Australian Sarcophagidae: 96.5% of the examined specimens were assigned to the correct species. Given that the sarcophagid fauna is poorly described, it is feasible that the few incorrectly assigned specimens represent cryptic species. The results of this research will be instrumental for implementation of the Australian Sarcophagidae in forensic entomology.

Genome-Wide SNPs Detect Hybridisation of Marsupial Gliders (Petaurus breviceps breviceps × Petaurus norfolcensis) in the Wild.

Petaurus breviceps and Petaurus norfolcensis have produced hybrids in captivity, however there are no reported cases of Petaurus hybridisation in the wild. This study uses morphological data, mitochondrial DNA, and nuclear genome-wide SNP markers to confirm P. breviceps breviceps × P. norfolcensis hybridisation within their natural range on the central coast of New South Wales, Australia. Morphological data identified a potential hybrid that was confirmed with next-generation sequencing technology and 10,111 genome-wide SNPs. Both STRUCTURE and NewHybrid analyses identified the hybrid as a P. norfolcensis backcross, which suggests an initial F1 hybrid was fertile. The mitochondrial DNA matched that of a P. b. breviceps, indicating that a P. b. breviceps female initially mated with a P. norfolcensis male to produce a fertile female offspring. Our study is an important example of how genome-wide SNPs can be used to identify hybrids where the distribution of congeners overlaps. Hybridisation between congeners is likely to become more frequent as climate changes and habitats fragment, resulting in increased interactions and competition for resources and mates.

Uneven declines between corals and cryptobenthic fish symbionts from multiple disturbances.

With the onset and increasing frequency of multiple disturbances, the recovery potential of critical ecosystem-building species and their mutual symbionts is threatened. Similar effects to both hosts and their symbionts following disturbances have been assumed. However, we report unequal declines between hosts and symbionts throughout multiple climate-driven disturbances in reef-building Acropora corals and cryptobenthic coral-dwelling Gobiodon gobies. Communities were surveyed before and after consecutive cyclones (2014, 2015) and heatwaves (2016, 2017). After cyclones, coral diameter and goby group size (i.e., the number of gobies within each coral) decreased similarly by 28-30%. After heatwave-induced bleaching, coral diameter decreased substantially (47%) and gobies mostly inhabited corals singly. Despite several coral species persisting after bleaching, all goby species declined, leaving 78% of corals uninhabited. These findings suggest that gobies, which are important mutual symbionts for corals, are unable to cope with consecutive disturbances. This disproportionate decline could lead to ecosystem-level disruptions through loss of key symbiont services to corals.

Characterization of 67 mitochondrial tRNA gene rearrangements in the Hymenoptera suggests that mitochondrial tRNA gene position is selectively neutral.

We present entire sequences of two hymenopteran mitochondrial genomes and the major portion of three others. We combined these data with nine previously sequenced hymenopteran mitochondrial genomes. This allowed us to infer and analyze the evolution of the 67 mitochondrial gene rearrangements so far found in this order. All of these involve tRNA genes, whereas four also involve larger (protein-coding or ribosomal RNA) genes. We find that the vast majority of mitochondrial gene rearrangements are independently derived. A maximum of four of these rearrangements represent shared, derived organizations, whereas three are convergently derived. The remaining mitochondrial gene rearrangements represent new mitochondrial genome organizations. These data are consistent with the proposal that there are an enormous number of alternative mitochondrial genome organizations possible and that mitochondrial genome organization is, for the most part, selectively neutral. Nevertheless, some mitochondrial genes appear less mobile than others. Genes close to the noncoding region are generally more mobile but only marginally so. Some mitochondrial genes rearrange in a pattern consistent with the duplication/random loss model, but more mitochondrial genes move in a pattern inconsistent with this model. An increased rate of mitochondrial gene rearrangement is not tightly associated with the evolution of parasitism. Although parasitic lineages tend to have more mitochondrial gene rearrangements than nonparasitic lineages, there are exceptions (e.g., Orussus and Schlettererius). It is likely that only a small proportion of the total number of mitochondrial gene rearrangements that have occurred during the evolution of the Hymenoptera have been sampled in the present study.

Latitudinal Biogeographic Structuring in the Globally Distributed Moss Ceratodon purpureus.

Biogeographic patterns of globally widespread species are expected to reflect regional structure, as well as connectivity caused by occasional long-distance dispersal. We assessed the level and drivers of population structure, connectivity, and timescales of population isolation in one of the most widespread and ruderal plants in the world - the common moss Ceratodon purpureus. We applied phylogenetic, population genetic, and molecular dating analyses to a global (n = 147) sampling data set, using three chloroplast loci and one nuclear locus. The plastid data revealed several distinct and geographically structured lineages, with connectivity patterns associated with worldwide, latitudinal "bands." These imply that connectivity is strongly influenced by global atmospheric circulation patterns, with dispersal and establishment beyond these latitudinal bands less common. Biogeographic patterns were less clear within the nuclear marker, with gene duplication likely hindering the detection of these. Divergence time analyses indicated that the current matrilineal population structure in C. purpureus has developed over the past six million years, with lineages diverging during the late Miocene, Pliocene, and Quaternary. Several colonization events in the Antarctic were apparent, as well as one old and distinct Antarctic clade, possibly isolated on the continent since the Pliocene. As C. purpureus is considered a model organism, the matrilineal biogeographic structure identified here provides a useful framework for future genetic and developmental studies on bryophytes. Our general findings may also be relevant to understanding global environmental influences on the biogeography of other organisms with microscopic propagules (e.g., spores) dispersed by wind.

Pharmacological and genetic characterisation of the canine P2X4 receptor.

BACKGROUND AND PURPOSE: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. EXPERIMENTAL APPROACH: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. KEY RESULTS: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. CONCLUSION AND IMPLICATIONS: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.