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Simple Summary: The phylogenetic relationship and origin time of Phasmatodea have been discussed by many researchers. In this study, five mitogenomes of Phasmatodea were newly sequenced and the disputes about their phylogenetic relationships were explored. Additionally, five reliable fossil calibration points were used to explore the origin and diversification time of Phasmatodea. Stick and leaf insects are widely distributed in tropical and subtropical regions, with only a few species distributed at high latitudes. We collected a Phraortes liaoningensis (Phasmatodea: Lonchodidae) from Anshan City, Liaoning Province, China, to analyze whether its mitochondrial genes were under selective pressure to adapt to a low-temperature environment. Stick and leaf insects are a group among the Insecta that are famous for their extraordinary mimicry ability. Since the establishment of the Phasmatodea, their internal classification has been constantly revised. Mitochondrial genes as molecular markers have been widely used for species classification, but the phylogenetic relationships within the Phasmatodea remain to be thoroughly discussed. In the present study, five mitogenomes of Phasmatodea ranging from 15,746 bp to 16,747 bp in length were sequenced. Bayesian inference (BI) and maximum likelihood (ML) analyses were carried out based on a 13 PCGs data matrix (nt123) and a combined matrix of 13 PCGs and two rRNA genes (nt123_rRNA). The present study supports the conclusion that Phylliidae was the basal group of Neophasmatodea and confirms the monophyly of Lonchodinae and Necrosciinae, but it shows that Lonchodidae was polyphyletic. A sister group of Bacillidae and Pseudophasmatidae was also recovered. The phylogenetic tree based on the nt_123 dataset showed higher node support values. The construction of a divergent time tree in this study supported the conclusion that extant Phasmatodea originated in the Jurassic (170 Mya) and most lineages diverged after the Cretaceous–Paleogene extinction event. To explore whether the mitochondrial genes of Phraortes liaoningensis collected from high latitudes where low temperatures occur for eight months of the year are under selection pressure, this study used the branch-site model and the branch model to analyze the selection pressure on the 13 mitochondria protein-coding genes (PCGs). We found that both ND2 and ND4L of Ph. liaoningensis exhibited positive selection sites using the branch-site model. This study shows that a low-temperature environment causes mitochondrial genes to be selected to meet the energy requirements for survival. [ABSTRACT FROM AUTHOR]

The genus Presbistus and the family Aschiphasmatidae are recorded from Thailand for the first time. A new species of Presbistus Kirby, 1896, Presbistus muka sp. nov., is described from the Mahidol University Campus in Kanchanaburi, West Thailand based on both sexes and eggs. The new species is diagnosed and differentiated from its congenerics by its distinct colouration and by the notably short wings. Illustrations of adults, specimens in situ, host plants, records on biology and a distribution map are provided. A second Presbistus species, Presbistus vitivorus Bresseel & Constant, 2022 is also recorded from West Thailand, its distribution and morphology are briefly discussed. [ABSTRACT FROM AUTHOR]

Two new species of Neooxyartes Ho, 2018 are described from Central Vietnam: N. minimus sp. nov. from Kon Ka Kinh National Park and N. neohiraseoides sp. nov. from Chu Yang Sin National Park. A new genus Mycovartes gen. nov. is described to accommodate Oxyartes vietnamensis Ho, 2018 resulting into the new combination Mycovartes vietnamensis (Ho, 2018) gen. et comb. nov. Two new species are described in the new genus: M. khoii gen. et sp. nov. from Chu Yang Sin and Ta Dung National Parks and M. montanus gen. et sp. nov. from Chu Yang Sin and Bidoup-Nui Ba National Parks. For all species, adults of both sexes, nymphs and eggs of wild and captive bred origin are described and figured. Pictures of living specimens, and distribution maps of the species of both genera are provided. The male vomer was dissected for all treated species and is described and figured. The relationship of the herein treated genera to closely related taxa is discussed and supported with recent phylogenetic studies.

During hybrid speciation, homoeologues combine in a single genome. Homoeologue expression bias (HEB) occurs when one homoeologue has higher gene expression than another. HEB has been well characterized in plants but rarely investigated in animals, especially invertebrates. Consequently, we have little idea as to the role that HEB plays in allopolyploid invertebrate genomes. If HEB is constrained by features of the parental genomes, then we predict repeated evolution of similar HEB patterns among hybrid genomes formed from the same parental lineages. To address this, we reconstructed the history of hybridization between the New Zealand stick insect genera Acanthoxyla and Clitarchus using a high‐quality genome assembly from Clitarchus hookeri to call variants and phase alleles. These analyses revealed the formation of three independent diploid and triploid hybrid lineages between these genera. RNA sequencing revealed a similar magnitude and direction of HEB among these hybrid lineages, and we observed that many enriched functions and pathways were also shared among lineages, consistent with repeated evolution due to parental genome constraints. In most hybrid lineages, a slight majority of the genes involved in mitochondrial function showed HEB towards the maternal homoeologues, consistent with only weak effects of mitonuclear incompatibility. We also observed a proteasome functional enrichment in most lineages and hypothesize this may result from the need to maintain proteostasis in hybrid genomes. Reference bias was a pervasive problem, and we caution against relying on HEB estimates from a single parental reference genome. [ABSTRACT FROM AUTHOR]

Stick insects (Phasmatodea) have many different oviposition strategies, reflecting a range of adaptive behaviours and morphologies to best place and secure eggs in their environments. Oviposition strategies in Aotearoa New Zealand phasmids are not well documented, but the literature so far suggests that they drop individual eggs to the ground from their position in the foliage. Here, we present evidence for an oviposition strategy unique among the Rō stick insects of Aotearoa New Zealand. Individual female Spinotectarchus acornutus were observed inserting their eggs in a range of substrates, in particular the bark of trees and in spaces within the textured surfaces of tree fern trunks. We also highlight how the specific morphology of their eggs may be an adaptation to assist in substrate attachment, while their elongated secondary ovipositor could aid in egg insertion into substrates. [ABSTRACT FROM AUTHOR]

Reproduction is a key feature of all organisms, yet the way in which it is achieved varies greatly across the tree of life. One striking example of this variation is the stick insect genus Bacillus , in which five different reproductive modes have been described: sex, facultative and obligate parthenogenesis, and two highly unusual reproductive modes: hybridogenesis and androgenesis. Under hybridogenesis, the entire genome from the paternal species is eliminated and replaced each generation by mating with the corresponding species. Under androgenesis, an egg is fertilized, but the developing diploid offspring bear two paternal genomes and no maternal genome, as a consequence of unknown mechanisms. Here, we reevaluate the previous descriptions of Bacillus lineages and the proposed F1 hybrid ancestries of the hybridogenetic and obligately parthenogenetic lineages (based on allozymes and karyotypes) from Sicily, where all these reproductive modes are found. We generate a chromosome-level genome assembly for a facultative parthenogenetic species (B. rossius) and combine extensive field sampling with RADseq and mtDNA data. We identify and genetically corroborate all previously described species and confirm the ancestry of hybrid lineages. All hybrid lineages have fully retained their F1 hybrid constitution throughout the genome, indicating that the elimination of the paternal genome in hybridogens is always complete and that obligate parthenogenesis in Bacillus hybrid species is not associated with an erosion of heterozygosity as known in other hybrid asexuals. Our results provide a stepping stone toward understanding the transitions between reproductive modes and the proximate mechanisms of genome elimination. [ABSTRACT FROM AUTHOR]

Stick and leaf insects are a group among the Insecta that are famous for their extraordinary mimicry ability. Since the establishment of the Phasmatodea, their internal classification has been constantly revised. Mitochondrial genes as molecular markers have been widely used for species classification, but the phylogenetic relationships within the Phasmatodea remain to be thoroughly discussed. In the present study, five mitogenomes of Phasmatodea ranging from 15,746 bp to 16,747 bp in length were sequenced. Bayesian inference (BI) and maximum likelihood (ML) analyses were carried out based on a 13 PCGs data matrix (nt123) and a combined matrix of 13 PCGs and two rRNA genes (nt123_rRNA). The present study supports the conclusion that Phylliidae was the basal group of Neophasmatodea and confirms the monophyly of Lonchodinae and Necrosciinae, but it shows that Lonchodidae was polyphyletic. A sister group of Bacillidae and Pseudophasmatidae was also recovered. The phylogenetic tree based on the nt_123 dataset showed higher node support values. The construction of a divergent time tree in this study supported the conclusion that extant Phasmatodea originated in the Jurassic (170 Mya) and most lineages diverged after the Cretaceous–Paleogene extinction event. To explore whether the mitochondrial genes of Phraortes liaoningensis collected from high latitudes where low temperatures occur for eight months of the year are under selection pressure, this study used the branch-site model and the branch model to analyze the selection pressure on the 13 mitochondria protein-coding genes (PCGs). We found that both ND2 and ND4L of Ph. liaoningensis exhibited positive selection sites using the branch-site model. This study shows that a low-temperature environment causes mitochondrial genes to be selected to meet the energy requirements for survival.

Attachment to the substrate is an important phenomenon that determines the survival of many organisms. Most insects utilize wet adhesion to support attachment, which is characterized by fluids that are secreted into the interface between the tarsus and the substrates. Previous research has investigated the composition and function of tarsal secretions of different insect groups, showing that the secretions are likely viscous emulsions that contribute to attachment by generating capillary and viscous adhesion, leveling surface roughness and providing self-cleaning of the adhesive systems. Details of the structural organization of these secretions are, however, largely unknown. Here, we analyzed footprints originating from the arolium and euplantulae of the stick insect Medauroidea extradentata using cryo-scanning electron microscopy (cryo-SEM) and white light interferometry (WLI). The secretion was investigated with cryo-SEM, revealing four morphologically distinguishable components. The 3D WLI measurements of the droplet shapes and volumes over time revealed distinctly different evaporation rates for different types of droplets. Our results indicate that the subfunctionalization of the tarsal secretion is facilitated by morphologically distinct components, which are likely a result of different proportions of components within the emulsion. Understanding these components and their functions may aid in gaining insights for developing adaptive and multifunctional biomimetic adhesive systems. [ABSTRACT FROM AUTHOR]

Herbivorous insects require an active lignocellulolytic microbiome to process their diet. Stick insects (phasmids) are common in the tropics and display a cosmopolitan host plant feeding preference. The microbiomes of social insects are vertically transmitted to offspring, while for solitary species, such as phasmids, it has been assumed that microbiomes are acquired from their diet. This study reports the characterization of the gut microbiome for the Gray’s Malayan stick insect, Lonchodes brevipes, reared on native and introduced species of host plants and compared to the microbiome of the host plant and surrounding soil to gain insight into possible sources of recruitment. Clear differences in the gut microbiome occurred between insects fed on native and exotic plant diets, and the native diet displayed a more species-rich fungal microbiome. While the findings suggest that phasmids may be capable of adapting their gut microbiome to changing diets, it is uncertain whether this may lead to any change in dietary efficiency or organismal fitness. Further insight in this regard may assist conservation and management decision-making. [ABSTRACT FROM AUTHOR]

A vast majority of insects can fly, but some cannot. Flight generally increases how far an individual can travel to access mates, enables the exploitation of additional food resources, and aids in predator avoidance. Despite its functional significance, much remains unknown about the factors that influence the evolution of flight. Here, I use phylogenetic comparative methods to investigate whether average annual temperature or wind speed, two components of the flying environment, is correlated with the evolution of flight using data from 107 species of stick and leaf insects (Insecta: Phasmatodea). I find no association between wind speed and flying ability in this clade. However, I find that colder temperatures are associated with the lack of flying ability. This pattern may be explained by the additional metabolic costs required for insects to fly when it is cold. This finding contradicts previous patterns observed in other insect groups and supports the hypothesis that cold temperatures can influence the evolution of flight. [ABSTRACT FROM AUTHOR]

Simple Summary: Stick and leaf insects are herbivorous species widely distributed in tropical and subtropical areas, disguising themselves as leaves, twigs, or moss through morphology and behavior to avoid visually hunting predators. Currently, Phasmatodea present difficulties in taxonomy, and their phylogeny is unresolved. Mitochondria, as maternally inherited organelles, also contain evolutionary information. Compared to nuclear genes, mitogenomes have become a powerful marker for inferring phylogenetic relationships due to advantages including fast evolution rates, conserved structure, and easy amplification. With rapid advances in sequencing technology and assembly algorithms, mitogenomes can be sequenced in a very cost-effective way. As of March 2023, there are thirty-seven complete or nearly complete Phasmatodea mitogenomes listed in the NCBI. Considering the richness of Phasmatodea, additional study is warranted. In the present study, nine new mitogenomes were sequenced to examine gene rearrangements and phylogenetic relationships within the Phasmatodea. The classification of stick and leaf insects (Order Phasmatodea) is flawed at various taxonomic ranks due to a lack of robust phylogenetic relationships and convergent morphological characteristics. In this study, we sequenced nine new mitogenomes that ranged from 15,011 bp to 17,761 bp in length. In the mitogenome of Carausis sp., we found a translocation of trnR and trnA, which can be explained by the tandem duplication/random loss (TDRL) model. In the Stheneboea repudiosa Brunner von Wattenwyl, 1907, a novel mitochondrial structure of 12S rRNA-CR1-trnI-CR2-trnQ-trnM was found for the first time in Phasmatodea. Due to the low homology of CR1 and CR2, we hypothesized that trnI was inverted through recombination and then translocated into the middle of the control region. Control region repeats were frequently detected in the newly sequenced mitogenomes. To explore phylogenetic relationships in Phasmatodea, mtPCGs from 56 Phasmatodean species (composed of 9 stick insects from this study, 31 GenBank data, and 16 data derived from transcriptome splicing) were used for Bayesian inference (BI), and maximum likelihood (ML) analyses. Both analyses supported the monophyly of Lonchodinae and Necrosciinae, but Lonchodidae was polyphyletic. Phasmatidae was monophyletic, and Clitumninae was paraphyletic. Phyllidae was located at the base of Neophasmatodea and formed a sister group with the remaining Neophasmatodea. Bacillidae and Pseudophasmatidae were recovered as a sister group. Heteroptergidae was monophyletic, and the Heteropteryginae sister to the clade (Obriminae + Dataminae) was supported by BI analysis and ML analysis. [ABSTRACT FROM AUTHOR]

Abstract A vast majority of insects can fly, but some cannot. Flight generally increases how far an individual can travel to access mates, enables the exploitation of additional food resources, and aids in predator avoidance. Despite its functional significance, much remains unknown about the factors that influence the evolution of flight. Here, I use phylogenetic comparative methods to investigate whether average annual temperature or wind speed, two components of the flying environment, is correlated with the evolution of flight using data from 107 species of stick and leaf insects (Insecta: Phasmatodea). I find no association between wind speed and flying ability in this clade. However, I find that colder temperatures are associated with the lack of flying ability. This pattern may be explained by the additional metabolic costs required for insects to fly when it is cold. This finding contradicts previous patterns observed in other insect groups and supports the hypothesis that cold temperatures can influence the evolution of flight.

The south Asian genus Cladobethylus Kieffer, 1922, is reviewed, with a key to the species. Five new species are described, one from the island of Borneo (Cl. darlingi), two from Thailand (Cl. densepunctatus and Cl. thailandicus), one from Korea (Cl. koreensis), and one from Sumatra (acehensis).

Insect attachment devices and capabilities have been subject to research efforts for decades, and even though during that time considerable progress has been made, numerous questions remain. Different types of attachment devices are known, alongside most of their working principles, however, some details have yet to be understood. For instance, it is not clear why insects for the most part developed pairs of claws, instead of either three or a single one. In this paper, we investigated the gripping forces generated by the stick insect Sungaya inexpectata, in dependence on the number of available claws. The gripping force experiments were carried out on multiple, standardized substrates of known roughness, and conducted in directions both perpendicular and parallel to the substrate. This was repeated two times: first with a single claw being amputated from each of the animals' legs, then with both claws removed, prior to the measurement. The adhesive pads (arolia) and frictional pads (euplantulae) remained intact. It was discovered that the removal of claws had a detractive effect on the gripping forces in both directions, and on all substrates. Notably, this also included the control of smooth surfaces on which the claws were unable to find any asperities to grip on. The results show that there is a direct connection between the adhesive performance of the distal adhesive pad (arolium) and the presence of intact claws. These observations show collective effects between different attachment devices that work in concert during locomotion, and grant insight into why most insects possess two claws. [ABSTRACT FROM AUTHOR]

Abstract Background In most arthropods, adult females are larger than males, and male competition is a race to quickly locate and mate with scattered females (scramble competition polygyny). Variation in body size among males may confer advantages that depend on context. Smaller males may be favored due to more efficient locomotion leading to higher mobility during mate searching. Alternatively, larger males may benefit from increased speed and higher survivorship. While the relationship between male body size and mobility has been investigated in several systems, how different aspects of male body morphology specifically affect their locomotor performance in different contexts is often unclear. Results Using a combination of empirical measures of flight performance and modelling of body aerodynamics, we show that large body size impairs flight performance in male leaf insects (Phyllium philippinicum), a species where relatively small and skinny males fly through the canopy in search of large sedentary females. Smaller males were more agile in the air and ascended more rapidly during flight. Our models further predicted that variation in body shape would affect body lift and drag but suggested that flight costs may not explain the evolution of strong sexual dimorphism in body shape in this species. Finally, empirical measurements of substrate adhesion and subsequent modelling of landing impact forces suggested that smaller males had a lower risk of detaching from the substrates on which they walk and land. Conclusions By showing that male body size impairs their flight and substrate adhesion performance, we provide support to the hypothesis that smaller scrambling males benefit from an increased locomotor performance and shed light on the evolution of sexual dimorphism in scramble competition mating systems.

Simple Summary: The order Phasmatodea includes the longest Brazilian insects, known by their remarkable morphological and behavioural adaptations for camouflage such as sticks, moss, and leaves; they are predominantly nocturnal and phytophagous insects. Cladomorphus phyllinus Gray, 1835 is one of the most common and best-known stick insect species in Brazil. It feeds mainly on guava leaves, angico, and powder-puff, and reproduces sexually and asexually. Cladomorphus phyllinus presents marked sexual dimorphism in the adult: winged males are significantly smaller in size than females and can reach up to 13 cm, while females can reach 23 cm in length and are apterous. A female specimen collected in the Atlantic Forest in Petrópolis, Rio de Janeiro, Brazil, was compared with a C. phyllinus specimen, identified according to published literature. The differences between the two specimens related to the general size and several morphological characters were observed. In order to add evidence that the recently collected specimen belonged to a species distinct from C. phyllinus, part of the cytochrome oxidase I (COI) gene was sequenced and analysed. The comparative analysis of the COI sequences from the two specimens revealed significant differences that, together with the morphological characters and recorded sympatry of the two specimens, support the existence of a new species, which is described here as Cladomorphus petropolisensis.Cladomorphus petropolisensis sp. nov., a new species of stick insect from Petrópolis, Rio de Janeiro, Brazil, is herein described and compared to the other sympatric species, C. phyllinus Gray, 1835 (Phasmatidae, Cladomorphinae). The description of the new species is supported by morphological and molecular evidence. Kimura-2-parameter (K2P) intraspecific COI divergences among the holotype of C. petropolisensis sp. nov. and C. phyllinus individuals ranged from 2.9% to 4.4%, which are suggestive of distinct species, especially when considering that all Cladomorphus individuals studied were collected in the Petrópolis municipality. The new species can be distinguished from C. phyllinus Gray, 1835 by several characteristics: smaller size, the presence of two spines on the hind femora, the relative longer length of the ovipositor, and spiny tegument, especially in the mesonotum, sculpturing of the operculum of the egg. [ABSTRACT FROM AUTHOR]

Simple Summary: Cytochrome P450s are a group of detoxification enzymes found in all animals. They are highly diverse, with multiple copies of different families of these genes in individual organisms, especially those that need to digest chemically-defended foods, such as toxic plants. The stick insects are a group of leaf-feeding herbivores whose diets can include toxic species, such as Eucalyptus, so this study sought to identify what groups of cytochrome P450s are expressed in the digestive tracts of six species of stick insects and how they evolved. The results show a complement of detoxification genes comparable to or slightly more limited than those of other insects, except for multiple enzymes per species in the subfamily CYP15A1, while most insects will only have one enzyme in this group. The functions of these expanded enzyme groups should be investigated further. Cytochrome P450s (CYPs) are xenobiotic detoxification genes found in most eukaryotes, and linked in insects to the tolerance of plant secondary chemicals and insecticide resistance. The number and diversity of CYP clans, families, and subfamilies that an organism produces could correlate with its dietary breadth or specialization. This study examined the CYP diversity expressed in the midguts of six species of folivorous stick insects (Phasmatodea), to identify their CYP complement and see if any CYPs correlate with diet toxicity or specialization, and see what factors influenced their evolution in this insect order. CYP genes were mined from six published Phasmatodea transcriptomes and analyzed phylogenetically. The Phasmatodea CYP complement resembles that of other insects, though with relatively low numbers, and with significant expansions in the CYP clades 6J1, 6A13/14, 4C1, and 15A1. The CYP6 group is known to be the dominant CYP family in insects, but most insects have no more than one CYP15 gene, so the function of the multiple CYP15A1 genes in Phasmatodea is unknown, with neofunctionalization following gene duplication hypothesized. No correlation was found between CYPs and diet specialization or toxicity, with some CYP clades expanding within the Phasmatodea and others likely inherited from a common ancestor. [ABSTRACT FROM AUTHOR]

Attachment to the substrate is an important phenomenon that determines the survival of many organisms. Most insects utilize wet adhesion to support attachment, which is characterized by fluids that are secreted into the interface between the tarsus and the substrates. Previous research has investigated the composition and function of tarsal secretions of different insect groups, showing that the secretions are likely viscous emulsions that contribute to attachment by generating capillary and viscous adhesion, leveling surface roughness and providing self-cleaning of the adhesive systems. Details of the structural organization of these secretions are, however, largely unknown. Here, we analyzed footprints originating from the arolium and euplantulae of the stick insect Medauroidea extradentata using cryo-scanning electron microscopy (cryo-SEM) and white light interferometry (WLI). The secretion was investigated with cryo-SEM, revealing four morphologically distinguishable components. The 3D WLI measurements of the droplet shapes and volumes over time revealed distinctly different evaporation rates for different types of droplets. Our results indicate that the subfunctionalization of the tarsal secretion is facilitated by morphologically distinct components, which are likely a result of different proportions of components within the emulsion. Understanding these components and their functions may aid in gaining insights for developing adaptive and multifunctional biomimetic adhesive systems.

Recently, the species of the insect order Phasmatodea, have attracted the interest of more and more enthusiasts. Here, we obtained the complete mitochondrial genome of Ramulus irregulatiter dentatus (R. irregulatiter dentatus), which belongs to the subfamily of Phasmatidae, detected by Illumina next-generation sequencing. The entire mitochondrial genome is 16,060 bp in length and contains a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a putative A + T-rich region. The base composition and codon usage were typical of Phasmatodea species. The mitochondrial gene organization (37 genes) was consistent with that of other Phasmatidae. A phylogenetic tree was built from the sequence information of the 13 protein-coding genes by Bayesian analyses. The newly sequenced R. irregulatiter dentatus was most closely related to the family Phasmatidae. The complete mitochondrial genome of R. irregulatiter dentatus also provides valuable molecular information for future studies on Phasmatidae insect taxonomy and a framework to unveil more of their cryptic and unknown diversity, so that it can be used to control forest pests and protect crops. [ABSTRACT FROM AUTHOR]

In facultatively parthenogenetic populations, the prevalence of sexual reproduction depends on whether females mate and therefore produce sons and daughters or avoid mating and produce daughters only.The relative advantage of mating in such species may depend on a female's own reproductive origin (i.e. development from a fertilised or unfertilised egg) if parthenogenesis reduces heterozygosity similar to sexual inbreeding, or if it inhibits mating, sperm storage or fertilisation. But effects of reproductive origin on development and performance are poorly understood.Using the facultatively parthenogenetic stick insect, Extatosoma tiaratum, we quantified morphology, mating probability, and reproductive success in mated versus unmated females of sexual versus automictic (parthenogenetic) origin.We found strong evidence that increased homozygosity negatively impacted some traits in parthenogenetically produced females: compared to sexually produced females, parthenogenetically produced females were smaller and more prone to deformities in vestigial wings, but not more prone to fluctuating asymmetry in their legs.Parthenogenetically produced females received fewer mating attempts and avoided mating more often than sexually produced females. Yet, contrary to the expectation that sex should rescue parthenogenetic lineages from the detrimental effects of increased homozygosity, parthenogenetically produced females gained no net reproductive benefit from mating, suggesting that physiological constraints limit fitness returns of sexual reproduction for these females.Our findings indicate that advantages of mating in this species depend on female reproductive origin. These results could help to explain spatial distributions of sex in facultatively parthenogenetic animals and evolutionary transitions to obligate asexuality. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

A new species of Presbistus Kirby, 1896, Presbistus vitivorus sp. nov., is described from Cambodia based on both sexes, nymphs, and eggs. Male genitalia and vomer are described and figured. Illustrations of adults, nymphs, specimens in situ, host plants, a distribution map and records on biology and breeding in captivity are provided. The host plants of the species belong to the family Vitaceae. The genus Presbistus and the family Aschiphasmatidae are recorded from Cambodia for the first time. The species diversity and the distribution of the genus are discussed, and it is shown that the genus is restricted to Sundaland. A nomenclature for the morphology of the dissected vomer is proposed and tries to homologize the previously used terms. [ABSTRACT FROM AUTHOR]

The classification of stick and leaf insects (Order Phasmatodea) is flawed at various taxonomic ranks due to a lack of robust phylogenetic relationships and convergent morphological characteristics. In this study, we sequenced nine new mitogenomes that ranged from 15,011 bp to 17,761 bp in length. In the mitogenome of Carausis sp., we found a translocation of trnR and trnA, which can be explained by the tandem duplication/random loss (TDRL) model. In the Stheneboea repudiosa Brunner von Wattenwyl, 1907, a novel mitochondrial structure of 12S rRNA-CR1-trnI-CR2-trnQ-trnM was found for the first time in Phasmatodea. Due to the low homology of CR1 and CR2, we hypothesized that trnI was inverted through recombination and then translocated into the middle of the control region. Control region repeats were frequently detected in the newly sequenced mitogenomes. To explore phylogenetic relationships in Phasmatodea, mtPCGs from 56 Phasmatodean species (composed of 9 stick insects from this study, 31 GenBank data, and 16 data derived from transcriptome splicing) were used for Bayesian inference (BI), and maximum likelihood (ML) analyses. Both analyses supported the monophyly of Lonchodinae and Necrosciinae, but Lonchodidae was polyphyletic. Phasmatidae was monophyletic, and Clitumninae was paraphyletic. Phyllidae was located at the base of Neophasmatodea and formed a sister group with the remaining Neophasmatodea. Bacillidae and Pseudophasmatidae were recovered as a sister group. Heteroptergidae was monophyletic, and the Heteropteryginae sister to the clade (Obriminae + Dataminae) was supported by BI analysis and ML analysis.

The diversity of insect eggs is astounding but still largely unexplained. Here, we apply phylogenetic analyses to 208 species of stick and leaf insects, coupled with physiological measurements of metabolic rate and water loss on five species, to evaluate classes of factors that may drive egg morphological diversification: life history constraints, material costs, mechanical constraints, and ecological circumstances. We show support for all three classes, but egg size is primarily influenced by female body size and strongly trades off with egg number. Females that lay relatively fewer but larger eggs, which develop more slowly because of disproportionately low metabolic rates, also tend to bury or glue them in specific locations instead of simply dropping them from the foliage (ancestral state). This form of parental care then directly favors relatively elongated eggs, which may facilitate their placement and allow easier passage through the oviducts in slender species. In addition, flightless females display a higher reproductive output and consequently lay relatively more and larger eggs compared with flight-capable females. Surprisingly, local climatic conditions had only weak effects on egg traits. Overall, our results suggest that morphological diversification of stick insect eggs is driven by a complex web of causal relationships among traits, with dominant effects of resource allocation and oviposition strategies, and of mechanical constraints. [Display omitted] • We analyzed patterns of evolution of egg traits from over 200 stick insect species • Egg traits have diversified within a complex network of causal relationships • Variation in size is mainly explained by adult size and a trade-off with egg number • Egg shape evolution is mainly driven by oviposition strategy and female body shape Boisseau and Woods investigate factors influencing the evolution of egg morphology in stick insects. Relative egg size appears to be influenced primarily by a trade-off with egg number, while egg shape reflects mechanical constraints arising from the diameter of the female oviduct and oviposition strategy. [ABSTRACT FROM AUTHOR]

The new Hesperophasmatini–genus Sigaruphasma gen. nov. from Hispaniola is described and illustrated. It is remarkable for violating several of the previously stated diagnostic features of the tribe Hesperophasmatini Bradley and Galil, 1977. It is well characterized by the cylindrical, cigar–like body of females, proportionally very short legs, strongly broadened profemora of females, lacking sensory–areas on the probasisternum and profurcasternum as well as the smooth egg–capsule. Two new species are described from the Dominican Republic, both from the males, females and eggs. S. bouladoui sp. nov. occurs in the eastern portion of Hispaniola (Monseñor Nouel, San Cristóbal, San José de Ocoa and Samaná provinces) and has fully winged males. S. armatum sp. nov. occurs in the southern and southeastern Dominican Republic (Barahona, La Vega, San José de Ocoa, Elías Piña, Santiago provinces) and has apterous males with a strong body spination. Both species show a remarkable range of intraspecific variability and polymorphism, which is described and illustrated.

Although morphological adaptations leading to crypsis or mimicry have been studied extensively, their interaction with particular behaviors to avoid detection or recognition is understudied. Yet animal behaviors interact with morphology to reduce detection risk, and the level of protection conferred likely changes according to the surrounding environment. Apart from providing a locational cue for predators, prey motion can also serve as concealing behavior in a dynamic environment to prevent detection by potential predators or prey. Phasmids are conventionally known to rely on their adaptive resemblance to plant parts for protection, and this resemblance may vary across life stages and species. However, little is known about how their behaviors interact with their appearance and their environment. We investigated two species of phasmids with varying morphology and color patterns at different ontogenetic stages and examined their behavioral responses to a wind stimulus as a proxy for a dynamic environment. While adult behaviors were mostly species-specific, behavioral responses of nymphs varied with appearance and environmental condition. Display of different behaviors classified as revealing was positively correlated, while the display of concealing behaviors, except for swaying, was mostly negatively correlated with other behaviors. Exhibition of specific behaviors varied with appearance and environmental condition, suggesting that these behavioral responses could help reduce detection or recognition cues. We discuss the differences in behavioral responses in the context of how the behaviors could reveal or conceal the phasmids from potential predators. Our results provide a novel investigation into adaptive resemblance strategies of phasmids through the interaction of behavior and morphology, and highlight the importance of considering the effects of dynamic environments on sending and receiving cues.

We present the complete mitochondrial genome of Carausius morosus from Salinas, CA. The mitochondrial genome of C. morosus is circular, AT rich (78.1%), and 16,671 bp in length. It consists of 13 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes and is identical in gene content to Carausius sp., Competing Interests: The authors declare no conflict of interest.

Cladomorphus petropolisensis sp. nov., a new species of stick insect from Petrópolis, Rio de Janeiro, Brazil, is herein described and compared to the other sympatric species, C. phyllinus Gray, 1835 (Phasmatidae, Cladomorphinae). The description of the new species is supported by morphological and molecular evidence. Kimura-2-parameter (K2P) intraspecific COI divergences among the holotype of C. petropolisensis sp. nov. and C. phyllinus individuals ranged from 2.9% to 4.4%, which are suggestive of distinct species, especially when considering that all Cladomorphus individuals studied were collected in the Petrópolis municipality. The new species can be distinguished from C. phyllinus Gray, 1835 by several characteristics: smaller size, the presence of two spines on the hind femora, the relative longer length of the ovipositor, and spiny tegument, especially in the mesonotum, sculpturing of the operculum of the egg.

Senescence is a decline in reproduction and survival rate with advancing age resulting from deterioration of somatic tissues and systems throughout the body. Age-related somatic changes (somatic ageing) have been studied extensively in vertebrates but are less well known in other animals, including insects. Since adult insects have very limited ability to repair their exoskeleton, somatic ageing could involve deterioration and discolouration of the cuticle. We investigated age-related changes in wing pigmentation and abdominal cuticle necrosis in females of the Australian leaf insect Phyllium monteithi. Adult females varied markedly in the extent and pattern of pigmentation on their bodies, and we found that pigment spots on the forewings increased in size with age in most individuals. As females aged, most individuals also exhibited increasing levels of abdominal cuticle necrosis, resulting in the loss of abdominal cuticle along the margin of the abdomen. Neither the extent of pigmentation nor cuticle loss were clearly associated with reduced fecundity or longevity in the protected laboratory environment, but it remains unknown whether these age-related changes have functional implications in the wild. Our results show that the P. monteithi exoskeleton undergoes complex changes with age, with potential implications for functional traits and fitness. Australian leaf insects have leaf-shaped bodies that enable them to blend in to the tree canopy and confer protection against predators. We investigated whether the female leaf insect's exoskeleton – the tough outer-layer that supports and protects the insect's body – changes and deteriorates with age, and discovered that the wings develop enlarged brown patches and the margins of the abdomen become necrotic as females age. Our findings reveal novel manifestations of ageing in the insect exoskeleton. Photo by R. Bonduriansky. [ABSTRACT FROM AUTHOR]

The stick insects or phasmids (Phamsatodea) have a series of pyriform ampulles with long, thin filaments on the posterior end of their midgut referred to as the "appendices of the midgut." Found only in phasmids, their function had never been determined until now. Their similarity to the Malpighian tubules, which are ubiquitous insect organs of excretion, suggested a similar function. To differentiate between the appendices and the Malpighian tubules and compare functional differences between the two tissue types, vital staining (the injection of histological stains into living organisms) was done in conjunction with light and scanning electron microscopy in multiple phasmid species. The results showed that the appendices originated in the basal phasmids (Timematidae) and grew more numerous in derived species. The appendices stain selectively, notably failing to pick up the indicators of the two known systems of invertebrate excretory function, indigo carmine and ammonium carmine. Appendices sequester stains in the ampule portion before eliminating the compounds into the midgut. We conclude by confirming that the appendices do have an excretory function, but one unlike any other known in invertebrates. Their function is likely cation excretion, playing a role in calcium regulation and/or organic alkaloid sequestration. The appendices must thus be considered distinct organs from the Malpighian tubules.

Cytochrome P450s (CYPs) are xenobiotic detoxification genes found in most eukaryotes, and linked in insects to the tolerance of plant secondary chemicals and insecticide resistance. The number and diversity of CYP clans, families, and subfamilies that an organism produces could correlate with its dietary breadth or specialization. This study examined the CYP diversity expressed in the midguts of six species of folivorous stick insects (Phasmatodea), to identify their CYP complement and see if any CYPs correlate with diet toxicity or specialization, and see what factors influenced their evolution in this insect order. CYP genes were mined from six published Phasmatodea transcriptomes and analyzed phylogenetically. The Phasmatodea CYP complement resembles that of other insects, though with relatively low numbers, and with significant expansions in the CYP clades 6J1, 6A13/14, 4C1, and 15A1. The CYP6 group is known to be the dominant CYP family in insects, but most insects have no more than one CYP15 gene, so the function of the multiple CYP15A1 genes in Phasmatodea is unknown, with neofunctionalization following gene duplication hypothesized. No correlation was found between CYPs and diet specialization or toxicity, with some CYP clades expanding within the Phasmatodea and others likely inherited from a common ancestor.

Some animal groups, such as stick insects (Phasmatodea), have repeatedly evolved alternative reproductive strategies, including parthenogenesis. Genomic studies have found modification of the genes underlying meiosis exists in some of these animals. Here we examine the evolution of copy number, evolutionary rate, and gene expression in candidate meiotic genes of the New Zealand geographic parthenogenetic stick insect Clitarchus hookeri. We characterized 101 genes from a de novo transcriptome assembly from female and male gonads that have homology with meiotic genes from other arthropods. For each gene we determined copy number, the pattern of gene duplication relative to other arthropod orthologs, and the potential for meiosis-specific expression. There are five genes duplicated in C. hookeri , including one also duplicated in the stick insect Timema cristinae , that are not or are uncommonly duplicated in other arthropods. These included two sister chromatid cohesion associated genes (SA2 and SCC2), a recombination gene (HOP1), an RNA-silencing gene (AGO2) and a cell-cycle regulation gene (WEE1). Interestingly, WEE1 and SA2 are also duplicated in the cyclical parthenogenetic aphid Acyrthosiphon pisum and Daphnia duplex , respectively, indicating possible roles in the evolution of reproductive mode. Three of these genes (SA2 , SCC2 , and WEE1) have one copy displaying gonad-specific expression. All genes, with the exception of WEE1 , have significantly different nonsynonymous/synonymous ratios between the gene duplicates, indicative of a shift in evolutionary constraints following duplication. These results suggest that stick insects may have evolved genes with novel functions in gamete production by gene duplication. [ABSTRACT FROM AUTHOR]

Neuropeptides are among the structurally most diverse signaling molecules and participate in intercellular information transfer from neurotransmission to intrinsic or extrinsic neuromodulation. Many of the peptidergic systems have a very ancient origin that can be traced back to the early evolution of the Metazoa. In recent years, new insights into the evolution of these peptidergic systems resulted from the increasing availability of genome and transcriptome data which facilitated the investigation of the complete neuropeptide precursor sequences. Here we used a comprehensive transcriptome dataset of about 200 species from the 1KITE initiative to study the evolution of single-copy neuropeptide precursors in Polyneoptera. This group comprises well-known orders such as cockroaches, termites, locusts, and stick insects. Due to their phylogenetic position within the insects and the large number of old lineages, these insects are ideal candidates for studying the evolution of insect neuropeptides and their precursors. Our analyses include the orthologs of 21 single-copy neuropeptide precursors, namely ACP, allatotropin, AST-CC, AST-CCC, CCAP, CCHamide-1 and 2, CNMamide, corazonin, CRF-DH, CT-DH, elevenin, HanSolin, NPF-1 and 2, MS, proctolin, RFLamide, SIFamide, sNPF, and trissin. Based on the sequences obtained, the degree of sequence conservation between and within the different polyneopteran lineages is discussed. Furthermore, the data are used to postulate the individual neuropeptide sequences that were present at the time of the insect emergence more than 400 million years ago. The data confirm that the extent of sequence conservation across Polyneoptera is remarkably different between the different neuropeptides. Furthermore, the average evolutionary distance for the single-copy neuropeptides differs significantly between the polyneopteran orders. Nonetheless, the single-copy neuropeptide precursors of the Polyneoptera show a relatively high degree of sequence conservation. Basic features of these precursors in this very heterogeneous insect group are explained here in detail for the first time. [ABSTRACT FROM AUTHOR]

Short report about breeding Lamachodes sp."Bokor", a lately disvorvered, yet undescribed and rather colourful Phasmatodea species. [ABSTRACT FROM AUTHOR]

Abstract 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 rarity of facultative asexuality in animals is an evolutionary puzzle. It has been hypothesized that male factors that influence female performance could be key to this paradox because parthenogens that fail to obtain fitness-increasing stimulation from males at certain life stages may reproduce poorly via parthenogenesis. However, given that certain male factors can reduce female fitness, an alternative hypothesis is that exposure to male factors could exert a sexually antagonistic suppressive effect on females' capacity for parthenogenesis. To test the contrasting predictions of these two hypotheses, we used the spiny leaf stick insect, Extatosoma tiaratum , a species capable of both sexual and asexual reproduction, to investigate developmental age-dependent effects of nonmating exposure to males on female behaviour and parthenogenetic performance, with exposure to other females as a control. We found that females reared with immature males were more likely than controls to show resistance-like behaviours as juveniles. Moreover, as adults, females reared with immature males produced asexual eggs with greatly depressed hatching success, resulting in a two-fold reduction in asexual performance compared to controls. By contrast, nonmating exposure to adult males at maturity had little effect on female behaviour or performance. However, females maintained exclusively with other females had slightly reduced fecundity, perhaps due to deprivation of fecundity-increasing male stimulation. Our results suggest that interactions with juvenile males can suppress the development of females' parthenogenetic capacity, an effect that appears to be sexually antagonistic. Such effects could help to explain the rarity of facultative asexuality in animal systems. • We investigated developmental age-dependent effects of male factors on asexuality. • Juvenile females exposed to juvenile males were more defensive than controls. • As adults, these females produced 50% fewer offspring asexually than controls. • Males appear to suppress females' capacity for asexuality during development. • Juvenile females may avoid costs of male exposure through behavioural resistance. [ABSTRACT FROM AUTHOR]

Four new species of Lobolibethra Hennemann & Conle, 2007 (Diapheromerinae: Diapheromerini: “Clonistria-group”) are described and illustrated. Lobolibethra carbonelli sp. nov. and L. pozuzoae sp. nov. from eastern Peru are described from both sexes and the eggs, L. tricarinata sp. nov. from southeast Ecuador is described from the female and male, and L. verruculosa sp. nov. from eastern Peru is described from the male. The previously unknown males of the Peruvian L. mainerii (Giglio-Tos, 1910) and L. mutica Hennemann & Conle, 2007 are described and illustrated. Dyme iconnicoffi Caudell, 1918 is shown to represent the previously unknown male of L. mainerii (Giglio-Tos, 1910) and is synonymised (syn. nov.). An updated key is presented for the eleven known species of Lobolibethra.

The South American genus Phantasca Redtenbacher, 1906 (Phasmatodea: Diapheromeridae: Diapheromaerinae) is re-diagnosed and revised at the species level. The precedingly unknown eggs are described for the first time. The genus Pterolibethra Günther, 1940 (type species: P. heteronemia Günther, 1940) is re-synonymised, with Phantasca (syn. nov.) and consequently the two species originally contained, P. heteronemia Günther, 1940 and P. poeciloptera Günther, 1940, are transferred to Phantasca (comb. rev.). P. laeta Conle, Hennemann & Gutierréz, 2011 is not congeneric and is transferred to the genus Jeremiodes Hennemann & Conle, 2007 (Cladomorphinae: Cladomorphini; comb. nov.). Two species are removed from Bacteria Berthold, 1827 and transferred to Phantasca; these are B. quadrilobata Chopard, 1911 and B. montana Redtenbacher, 1906 (comb. nov.). Six new species are described: P. adiposa sp. nov., P. amabile sp. nov., P. femorata sp. nov., P. guianensis sp. nov., P. nigrolineata sp. nov. and P. ruboligata sp. nov. The male and egg of P. quadrilobata (Chopard, 1911) are described and illustrated for the first time. The genus now contains 13 species that are distributed throughout the northern half of South America. A key as well as detailed descriptions and illustrations are presented for all known species.

Environmental factors that regulate egg diapause in the walking-stick insect, Ramulus irregulariterdentatus, were studied. Insects collected in fields at Okayama, Japan soon after hatching in spring were reared under constant laboratory conditions. After adult emergence, their eggs were placed outdoors or incubated under constant laboratory conditions. Most eggs kept at 15 or 20°C hatched more than 100 days after oviposition. At 25°C, however, many eggs remained in diapause throughout the experimental period although a small number hatched slightly earlier than those kept at the lower temperature. The maternal photoperiod affected the egg period at 20 and 25°C; eggs from females reared under short day conditions hatched significantly earlier. A low temperature of 10°C shortens diapause development, whereas 5°C did not have a clear effect on time to hatching. When eggs of females reared under long day conditions were placed outdoors from June to early July, most hatched the next spring. On the other hand, eggs of females reared under short day conditions hatched before winter if they were laid before mid-July. The short day eggs laid in August and September hatched successfully the following April, and there was a positive correlation between the date of oviposition and time of hatching. It is concluded that low temperatures in mid-winter do not terminate diapause development. The results revealed that both maternal regulation of diapause intensity and the rate of diapause development play an important role in maintaining a monovoltine life cycle in Ramulus irregulariterdentatus.

A new species of stick insect, Diapheromera arena n.sp., from western Texas and New Mexico is the first new species of Diapheromera described from the United States in several decades. It lacks armature on the head of both sexes and has a unique apomorphy of a distinctly swollen third antennal segment that easily differentiates it from other species in that genus. Despite being broadly sympatric with Diapheromera velii Walsh, the occurrence of this new species only in areas of windblown sand and sand dunes appears to function as habitat separation between these two related stick insects at the local level. Despite the new species' relatively large geographic range, it is a rare taxon and much less common than Diapheromera velii in the same region. This new species is part of the specialized sandy environment ecosystem including endemic plants, other insects, and vertebrates in western Texas and New Mexico [ABSTRACT FROM AUTHOR]

The new species Acrophylla bhaskarai sp. n. from Larat Island, Indonesia, is described from both sexes and the egg. It is the second species of the genus Acrophylla GRAY, 1835 known from outside Australia and the first record of the genus from Wallacea. The discovery raises the number of genera of the tribe Phasmatini known to occur within the boundaries of Wallacea to five. [ABSTRACT FROM AUTHOR]