Anatomical, phenological and genetic aspects of the host-parasite relationship between Andrena vaga (Hymenoptera) and Stylops ater (Strepsiptera).

Stylops ater is an endoparasite of the mining bee Andrena vaga with extreme sexual dimorphism and hypermetamorphosis. Its population structure, parasitization mode, genetic diversity and impact on host morphology were examined in nesting sites in Germany to better understand this highly specialized host­parasite interaction. The shift in host emergence due to stylopization was proven to be especially strong in A. vaga. Around 10% of bees hosted more than 1 Stylops, with at maximum 4. A trend in Stylops' preference for hosts of their own sex and a sex-specific position of extrusion from the host abdomen was found. Invasion of Andrena eggs by Stylops primary larvae was depicted for the first time. Cephalothoraces of female Stylops were smaller in male and pluristylopized hosts, likely due to lower nutrient supply. The genes H3, 18S and cytochrome c oxidase subunit 1 were highly conserved, revealing near-absence of local variation within Stylops. Ovaries of hosts with male Stylops contained poorly developed eggs while those of hosts with female Stylops were devoid of visible eggs, which might be due to a higher protein demand of female Stylops. Male Stylops, which might have a more energy-consuming development, led to a reduction in head width of their hosts. Host masculinization was present in the leaner shape of the metabasitarsus of stylopized females and is interpreted as a by-product of manipulation of the host's endocrine system to shift its emergence. Stylopization intensified tergal hairiness, most strongly in hosts with female Stylops, near the point of parasite extrusion, hinting towards substance-induced host manipulation.

X chromosomes show relaxed selection and complete somatic dosage compensation across Timema stick insect species.

Sex chromosomes have evolved repeatedly across the tree of life. As they are present in different copy numbers in males and females, they are expected to experience different selection pressures than the autosomes, with consequences including a faster rate of evolution, increased accumulation of sexually antagonistic alleles and the evolution of dosage compensation. Whether these consequences are general or linked to idiosyncrasies of specific taxa is not clear as relatively few taxa have been studied thus far. Here, we use whole-genome sequencing to identify and characterize the evolution of the X chromosome in five species of Timema stick insects with XX:X0 sex determination. The X chromosome had a similar size (approximately 12% of the genome) and gene content across all five species, suggesting that the X chromosome originated prior to the diversification of the genus. Genes on the X showed evidence of relaxed selection (elevated dN/dS) and a slower evolutionary rate (dN + dS) than genes on the autosomes, likely due to sex-biased mutation rates. Genes on the X also showed almost complete dosage compensation in somatic tissues (heads and legs), but dosage compensation was absent in the reproductive tracts. Contrary to prediction, sex-biased genes showed little enrichment on the X, suggesting that the advantage X-linkage provides to the accumulation of sexually antagonistic alleles is weak. Overall, we found the consequences of X-linkage on gene sequences and expression to be similar across Timema species, showing the characteristics of the X chromosome are surprisingly consistent over 30 million years of evolution.

Active mode of excretion across digestive tissues predates the origin of excretory organs.

Most bilaterian animals excrete toxic metabolites through specialized organs, such as nephridia and kidneys, which share morphological and functional correspondences. In contrast, excretion in non-nephrozoans is largely unknown, and therefore the reconstruction of ancestral excretory mechanisms is problematic. Here, we investigated the excretory mode of members of the Xenacoelomorpha, the sister group to Nephrozoa, and Cnidaria, the sister group to Bilateria. By combining gene expression, inhibitor experiments, and exposure to varying environmental ammonia conditions, we show that both Xenacoelomorpha and Cnidaria are able to excrete across digestive-associated tissues. However, although the cnidarian Nematostella vectensis seems to use diffusion as its main excretory mode, the two xenacoelomorphs use both active transport and diffusion mechanisms. Based on these results, we propose that digestive-associated tissues functioned as excretory sites before the evolution of specialized organs in nephrozoans. We conclude that the emergence of a compact, multiple-layered bilaterian body plan necessitated the evolution of active transport mechanisms, which were later recruited into the specialized excretory organs.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects.

Polyneoptera represents one of the major lineages of winged insects, comprising around 40,000 extant species in 10 traditional orders, including grasshoppers, roaches, and stoneflies. Many important aspects of polyneopteran evolution, such as their phylogenetic relationships, changes in their external appearance, their habitat preferences, and social behavior, are unresolved and are a major enigma in entomology. These ambiguities also have direct consequences for our understanding of the evolution of winged insects in general; for example, with respect to the ancestral habitats of adults and juveniles. We addressed these issues with a large-scale phylogenomic analysis and used the reconstructed phylogenetic relationships to trace the evolution of 112 characters associated with the external appearance and the lifestyle of winged insects. Our inferences suggest that the last common ancestors of Polyneoptera and of the winged insects were terrestrial throughout their lives, implying that wings did not evolve in an aquatic environment. The appearance of the first polyneopteran insect was mainly characterized by ancestral traits such as long segmented abdominal appendages and biting mouthparts held below the head capsule. This ancestor lived in association with the ground, which led to various specializations including hardened forewings and unique tarsal attachment structures. However, within Polyneoptera, several groups switched separately to a life on plants. In contrast to a previous hypothesis, we found that social behavior was not part of the polyneopteran ground plan. In other traits, such as the biting mouthparts, Polyneoptera shows a high degree of evolutionary conservatism unique among the major lineages of winged insects.

Why Did a Female Penis Evolve in a Small Group of Cave Insects?

The evolution of a female penis is an extremely rare event and is only known to have occurred in a tribe of small cave insects, Sensitibillini (Psocodea: Trogiomorpha: Prionoglarididae). The female penis, which is protrudable and inserted into the male vagina-like cavity during copulation to receive semen, is thought to have evolved independently twice in this tribe, in the Brazilian Neotrogla and the African Afrotrogla. These findings strongly suggest that there are some factors unique to Sensitibillini that have facilitated female penis evolution. Here, several hypothetical factors are presented that may have enabled the evolution of the female penis in Sensitibillini. The female-female competition for nutritious semen, the oligotrophic environment, and the twin insemination slots with switching valve are considered to be the driving factors for female penis evolution. Additionally, the following factors are considered responsible for relaxing the constraint against female penis evolution: preexistence of the female-above mating position, the elongated duct connecting the female pre-penis with the sperm storage organ, and the small male genital cavity accepting the female genital tubercle bearing the opening of this duct. Understanding the factors enabling female penis evolution may also shed light on the evolution of the male penis.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Genome-wide signatures of local adaptation among seven stoneflies species along a nationwide latitudinal gradient in Japan.

BACKGROUND: Environmental heterogeneity continuously produces a selective pressure that results in genomic variation among organisms; understanding this relationship remains a challenge in evolutionary biology. Here, we evaluated the degree of genome-environmental association of seven stonefly species across a wide geographic area in Japan and additionally identified putative environmental drivers and their effect on co-existing multiple stonefly species. Double-digest restriction-associated DNA (ddRAD) libraries were independently sequenced for 219 individuals from 23 sites across four geographical regions along a nationwide latitudinal gradient in Japan. RESULTS: A total of 4251 candidate single nucleotide polymorphisms (SNPs) strongly associated with local adaptation were discovered using Latent mixed models; of these, 294 SNPs showed strong correlation with environmental variables, specifically precipitation and altitude, using distance-based redundancy analysis. Genome-genome comparison among the seven species revealed a high sequence similarity of candidate SNPs within a geographical region, suggesting the occurrence of a parallel evolution process. CONCLUSIONS: Our results revealed genomic signatures of local adaptation and their influence on multiple, co-occurring species. These results can be potentially applied for future studies on river management and climatic stressor impacts.

The role of structural genomic variants in population differentiation and ecotype formation in Timema cristinae walking sticks.

Theory predicts that structural genomic variants such as inversions can promote adaptive diversification and speciation. Despite increasing empirical evidence that adaptive divergence can be triggered by one or a few large inversions, the degree to which widespread genomic regions under divergent selection are associated with structural variants remains unclear. Here we test for an association between structural variants and genomic regions that underlie parallel host-plant-associated ecotype formation in Timema cristinae stick insects. Using mate-pair resequencing of 20 new whole genomes we find that moderately sized structural variants such as inversions, deletions and duplications are widespread across the genome, being retained as standing variation within and among populations. Using 160 previously published, standard-orientation whole genome sequences we find little to no evidence that the DNA sequences within inversions exhibit accentuated differentiation between ecotypes. In contrast, a formerly described large region of reduced recombination that harbours genes controlling colour-pattern exhibits evidence for accentuated differentiation between ecotypes, which is consistent with differences in the frequency of colour-pattern morphs between host-associated ecotypes. Our results suggest that some types of structural variants (e.g., large inversions) are more likely to underlie adaptive divergence than others, and that structural variants are not required for subtle yet genome-wide genetic differentiation with gene flow.

Ecological gradients drive insect wing loss and speciation: The role of the alpine treeline.

Alpine ecosystems are frequently characterized by an abundance of wing-reduced insect species, but the drivers of this biodiversity remain poorly understood. Insect wing reduction in these environments has variously been attributed to altitude, temperature, isolation, habitat stability or decreased habitat size. We used fine-scale ecotypic and genomic analyses, along with broad-scale distributional analyses of ecotypes, to unravel the ecological drivers of wing reduction in the wing-dimorphic stonefly Zelandoperla fenestrata complex. Altitudinal transects within populations revealed dramatic wing reduction over very fine spatial scales, tightly linked to the alpine treeline. Broad biogeographical analyses confirm that the treeline has a much stronger effect on these ecotype distributions than altitude per se. Molecular analyses revealed parallel genomic divergence between vestigial-winged (high altitude) and full-winged (low altitude) ecotypes across distinct streams. These data thus highlight the role of the alpine treeline as a key driver of rapid speciation, providing a new model for ecological diversification along exposure gradients.

Parallel evolution of an alpine type ecomorph in a scorpionfly: Independent adaptation to high-altitude environments in multiple mountain locations.

Elucidation of the diversification process of organisms is one of the important tasks of biology. From the viewpoint of species diversity, insects are the most successful group among the diverse organisms on earth and evolutionary adaptation is one of the important factors driving this pattern. Evolutionary adaptation is one of the important factors in the diversification of insects. One of the representative examples of environmental adaptation in insects is the shortening and loss of wings in subalpine and alpine zones. In this study, we focused on the Japanese scorpionfly, Panorpodes paradoxus. In this species, individuals that inhabit mountainous regions and subalpine zones have long wings (the "general type"), and individuals that inhabit higher altitudinal ranges have short wings (the "alpine type"). We collected samples of all Japanese Panorpodes species and one Korean Panorpodes species, and conducted molecular phylogenetic analyses of the mtDNA COI (610 bp), COII (688 bp), and 16S rRNA (888 bp) and nuDNA EF1-α (658 bp) and 28S rRNA (524 bp) regions in order to reveal the evolutionary history of the alpine type of P. paradoxus. As a result of molecular phylogenetic analyses, it was revealed that the alpine type of P. paradoxus was polyphyletic, and had evolved to become the alpine type at least twice independently at separate mountain locations. In addition, the result of divergence time estimation suggested that the alpine type is an "ecomorph", having recently adapted to low temperature habitats following mountain uplift within the Japanese Archipelago and subsequent glacial-interglacial cycles.

Population biology of a selfish sex ratio distorting element in a booklouse (Psocodea: Liposcelis).

Arthropods harbour a variety of selfish genetic elements that manipulate reproduction to be preferentially transmitted to future generations. A major ongoing question is to understand how these elements persist in nature. In this study, we examine the population dynamics of an unusual selfish sex ratio distorter in a recently discovered species of booklouse, Liposcelis sp. (Psocodea: Liposcelididae) to gain a better understanding of some of the factors that may affect the persistence of this element. Females that carry the selfish genetic element only ever produce daughters, although they are obligately sexual. These females also only transmit the maternal half of their genome. We performed a replicated population cage experiment, varying the initial frequency of females that harbour the selfish element, and following female frequencies for 20 months. The selfish genetic element persisted in all cages, often reaching very high (and thus severely female-biased) frequencies. Surprisingly, we also found that females that carry the selfish genetic element had much lower fitness than their nondistorter counterparts, with lower lifetime fecundity, slower development and a shorter egg-laying period. We suggest that differential fitness plays a role in the maintenance of the selfish genetic element in this species. We believe that the genetic system in this species, paternal genome elimination, which allows maternal control of offspring sex ratio, may also be important in the persistence of the selfish genetic element, highlighting the need to consider species with diverse ecologies and genetic systems when investigating the effects of sex ratio manipulators on host populations.

Assembling large genomes: analysis of the stick insect (Clitarchus hookeri) genome reveals a high repeat content and sex-biased genes associated with reproduction.

BACKGROUND: Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects. RESULTS: The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified. CONCLUSIONS: The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects.

The chromosome-level genome assembly of the giant dobsonfly Acanthacorydalis orientalis (McLachlan, 1899).

Acanthacorydalis orientalis (McLachlan, 1899) (Megaloptera: Corydalidae) is an important freshwater-benthic invertebrate species that serves as an indicator for water-quality biomonitoring and is valuable for conservation from East Asia. Here, a high-quality reference genome for A. orientalis was constructed using Oxford Nanopore sequencing and High throughput Chromosome Conformation Capture (Hi-C) technology. The final genome size is 547.98 Mb, with the N50 values of contig and scaffold being 7.77 Mb and 50.53 Mb, respectively. The longest contig and scaffold are 20.57 Mb and 62.26 Mb in length, respectively. There are 99.75% contigs anchored onto 13 pseudo-chromosomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed that the completeness of the genome assembly is 99.01%. There are 10,977 protein-coding genes identified, of which 84.00% are functionally annotated. The genome contains 44.86% repeat sequences. This high-quality genome provides substantial data for future studies on population genetics, aquatic adaptation, and evolution of Megaloptera and other related insect groups.

Morphology of the pterothoracic musculature in Paraneoptera and its phylogenetic implication (Insecta: Neoptera).

Although the monophyly of Paraneoptera (=hemipteroid orders or Acercaria, composed of Psocodea, Thysanoptera and Hemiptera) has been widely accepted morphologically, the results from molecular phylogenetic and phylogenomic analyses contradict this hypothesis. In particular, phylogenomic analyses provide strong bootstrap support for the sister group relationship between Psocodea and Holometabola, that is, paraphyly of Paraneoptera. Here, we examined the pterothoracic musculature of Paraneoptera, as well as a wide range of other neopterous insect orders, and analysed its phylogenetic implication. By using the synchrotron microcomputed tomography (µCT) and parsimony-based ancestral state reconstruction, several apomorphic conditions suggesting the monophyly of Paraneoptera, such as the absence of the II/IIItpm7, IIscm3, IIIspm2 and IIIscm3 muscles, were identified. In contrast, no characters supporting Psocodea + Holometabola were recovered from the thoracic muscles. These results provide additional support for the monophyly of Paraneoptera, together with the previously detected morphological apomorphies of the head, wing base, and abdomen.

An integrative approach to the study of Kempnyia Klapálek, 1914 (Plecoptera: Perlidae) from Brazil: Support for the description of four new species and a basis for future studies.

Kempnyia (Plecoptera: Perlidae) is an endemic genus of Brazilian stoneflies that has 36 valid species and is distributed primarily in the Atlantic Forest and the mountainous areas of Central Brazil, particularly in Goiás and Tocantins states. Despite being the Brazilian genus with the most DNA sequences available on GenBank, integrative studies on the genus began only recently, in 2014. In this context, herein we studied the morphology and molecular data of Kempnyia specimens deposited in the Aquatic Biology Laboratory (UNESP, Assis) and the Entomology Museum of the Federal University of Viçosa (UFVB, Viçosa) collections. For the integrative approach adopted, in addition to studying the specimens morphologically, we used sequences of the COI mitochondrial gene combined with the following species delimitation methods: Automatic Barcode Gap Discovery (ABGD), both primary (ABGDp) and recursive (ABGDr) partitions; Assemble Species by Automatic Partitioning (ASAP); Poisson Tree Processes (PTP) and the Bayesian implementation of the Poisson Tree Processes (bPTP). As a result, we provided 28 new COI sequences of 21 species and support the description of four new species, namely, K. guarani sp. nov., K. tupiniquim sp. nov., K. una sp. nov., and K. zwickii sp. nov., consequently increasing the known diversity of the genus to 40 species. We also discuss the morphological variations observed in other species of the genus and provide several new geographic records. Therefore, our study brings new insights into the values of intra- and interspecific molecular divergence within Kempnyia, serving as a basis for new studies.

Morphological description of a new species of Capnia (Plecoptera: Capniidae) with DNA barcoding of genus members from the Russian Far East.

Capnia yavorskayae, a new species of the stonefly family Capniidae, is described from the Low Amur River Basin, Khabarovskiy Kray of the Russian Far East, on the basis of female morphological features. Confirmation of the uniqueness of the new species was also molecularly compared to other Capnia, including a few Far Eastern species, C. aligera Zapekina-Dulkeit, C. bargusinica Zapekina-Dulkeit, C. khingana Teslenko, C. kurnakovi Zhiltzova, C. nearctica Banks, C. nigra (Pictet), and C. rara Zapekina-Dulkeit for which DNA barcodes were obtained. We support the distinctiveness of the new species with mitochondrial DNA sequences, comparing it to Capnia from the eastern Palaearctic and Nearctic realms and one Zwicknia species. The new species forms a common clade with C. khingana, C. kurnakovi from the Russian Far East, and an undetermined Capnia species from Honshu, Japan. Each species from the Russian Far East has high interspecific distances from other Capnia species except C. nearctica which was close to C. atra Morton.

Convergent consequences of parthenogenesis on stick insect genomes.

The shift from sexual reproduction to parthenogenesis has occurred repeatedly in animals, but how the loss of sex affects genome evolution remains poorly understood. We generated reference genomes for five independently evolved parthenogenetic species in the stick insect genus Timema and their closest sexual relatives. Using these references and population genomic data, we show that parthenogenesis results in an extreme reduction of heterozygosity and often leads to genetically uniform populations. We also find evidence for less effective positive selection in parthenogenetic species, suggesting that sex is ubiquitous in natural populations because it facilitates fast rates of adaptation. Parthenogenetic species did not show increased transposable element (TE) accumulation, likely because there is little TE activity in the genus. By using replicated sexual-parthenogenetic comparisons, our study reveals how the absence of sex affects genome evolution in natural populations, providing empirical support for the negative consequences of parthenogenesis as predicted by theory.

The phylogenic position of aschiphasmatidae in euphasmatodea based on mitochondrial genomic evidence.

The mitochondrial genome (mitogenome) has been regarded as significant source of data to better understand the phylogenetic relationships within the Euphasmatodea, but no mitogenome in Aschiphasmatoidea has been sequenced to date. In this study, two mitogenomes of Orthomeria smaragdinum and Nanhuaphasma hamicercum of Aschiphasmatidae were sequenced and annotated for the first time. The same mitochondrial gene rearrangement structure was present in the two mitogenomes sequenced, showing as the translocation of tRNA-Arg and tRNA-Asn, which conformed to the tandem duplication-random loss and could be used as a possible synapomorphy for Aschiphasmatidae. The phylogenetic results based on the maximum likelihood (ML) and bayesian inference (BI) methods both showed that Aschiphasmatidae and Neophasmatodea in Euphasmatodea are sister taxa. Although the monophyly of Oriophasmata, Occidophasmata, Diapheromeridae, Phasmatidae, Lonchodidae and Bacilloidea has not been solved, the monophyly of Neophasmatodea and Phyllioidea was well supported.

Calculating Orthologous Protein-Coding Sequence Set Probability Using the Poisson Process.

We extend the popular Jukes-Cantor evolution model and calculate the probability of an orthologous nucleotide sequence set [a reference sequence (B1) stays with the other sequences (B-1)], where the sequence evolution [from a last common ancestral sequence (ɑ)] follows the (prospective) Poisson process with the overall event rate λ prorated among mutation types (nucleotide/codon substitution, insertion, and deletion) and sites along each sequence. The corresponding retrospective process (reversing the prospective process) facilitates developing algorithms to calculate the marginal probability [Pr(B1)] (Monte Carlo integration) and sample ɑ (given B1). We calculate probability Pr(B-1|ɑ) based on the identified events (during "ɑ→B-1") from pairwise sequence alignment to implement Pr(B-1|B1) calculation (Monte Carlo integration). Event queue sampling and probability magnifiers are used to improve the computational efficiency when the number of events is large. We finally test our procedure on both simulated and recently studied hexapod transcriptome data (Brandt et al.), where each asexual lineage pairs with its closest related sexual lineage. Rate estimates (for Phasmatodea and Zygentoma) and model comparison indicate that the asexual lineages likely mutate several times faster than their sexual relatives.