Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction

Several genetic factors are assumed to influence developmental instability (DI). One is the level of heterozygosity, with higher levels often being associated with decreased DI; another is genetic incompatibility in hybrids, which in several cases has been shown to increase DI. The genus Bacillus includes species which have both amphigonic heterozygous reproducing populations and homozygous parthenogenetic reproducing populations (B. rossius rossius and B. r. redtenbacheri). Furthermore, Bacillus includes hybrid parthenogenetic species, which have very high levels of almost fixed heterozygosities (B. atticus, B. whitei, B. lynceorum). We investigated the phenotypic variance (σ²p) and the impact of hybridization and level of heterozygosity on DI in females from these populations and species of Bacillus. DI was estimated as fluctuating asymmetry (FA) for three bilateral traits: the labial palpus, the maxillary palpus and the antenna. For the labial palpus and maxillary palpus we found, in general, a lower level of DI in the amphigonic females compared with parthenogenetic counterparts from the same species and with parthenogenetic females from the three hybrid species. A higher DI of the antenna was found in the hybrid species when compared with both parthenogenetic and amphigonic populations of the nonhybrid species, suggesting that the genes controlling antenna development are located on the sex chromosomes. The development of the investigated bilateral characters in the hybrid species seemed to be affected more by factors relating to genetic incompatibilities as a consequence of hybridization than by the stabilizing force of increased heterozygosity. Only few differences in σ²p were observed, supporting the possibility that the observed differences in DI are related mainly to internal genetic factors. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 249–259.     

An enigmatic new stick insect from the Philippine Islands (Insecta: Phasmatodea)

A new genus and species of stick insect is described and figured from Mount Halcon, on the Philippine island of Mindoro. Conlephasma enigma gen. et sp. n. is a stout, flightless, and apparently ground-dwelling species with vivid integumental colors. When disturbed, specimens spray a defensive secretion from the prothoracic exocrine glands. The systematic position of Conlephasma within Euphasmatodea is unclear. The elongated galealobulus and the trichome area located laterally in the galea, represent unusual apomorphic characters of the maxilla that could indicate affinities with Necrosciinae or Pseudophasmatinae. All tibiae exhibit the anareolate condition. Euplantulae are of two types: those of tarsomeres I–IV feature a nubby microstructure, whilst the one on the ventral side of the pretarsus is smooth. Males are characterized by the presence of a well-developed vomer on the tenth abdominal segment. A distinctive and apomorphic trait of female terminalia is represented by the elongated tenth abdominal tergum. Conlephasma can represent an interesting taxon for studies on the evolution of the stick and leaf insects.     

Interpopulation differences in egg sculpturing of the stick insect,Clonopsis gallica (Charp.) (Phasmatodea: Bacillidae)

Eggs of 6 Clonopsis gallica (Phasmatodea: Bacillidae) populations have been investigated by means of scanning electron microscopy (SEM) to detect intraspecific differences. Two main chorionic patterns have been observed: type 1 is common to 5 populations, while type 2 is found in the Motril (South Spain) sample. Type-2 sculpturing differs from type-1 in the absence of prominences on the operculum; and hemispheric, isolated mamelons replace the smooth and diffuse prominences of the common pattern on the egg capsule. This suggests that a considerable degree of difference exists in the South Spanish C. gallica. In the 5 type-1 populations, only minor and partially overlapping differences in the opercular pattern have been detected, so that the existence of subspecific taxa is very unlikely among them. Therefore, the Calabrian subspecies C. gallica affinis does not appear to be tenable.This is the 1st report on egg interpopulation differences in the Phasmatodea. These findings suggest further field search, along with karyological and gene-enzyme system analysis, within the C. gallica complex.     

Morphology of the terminalia of the stick insect Dajaca napolovi from Vietnam (Insecta: Phasmatodea)

Aschiphasmatinae is a small group of stick insects from the Oriental region whose genital morphology has been rarely described in detail. The subfamily is of particular interest, as phylogenetic studies have shown Aschiphasmatinae to be the sister group to the remaining Euphasmatodea. In this paper, the male and female terminalia are described for the first time in Dajaca napolovi Brock, a little known aschiphasmatine species from Vietnam. In the male, the transversally undivided abdominal sternum IX and gently incurved cerci with a conspicuous apical tooth represent apomorphies of Aschiphasmatinae. Thorn pads on the hind margin of abdominal tergum X consist of only a single row of 6–7 ventrally oriented teeth. The simple thorn pad structure of Aschiphasmatinae can represent an ancestral condition for Euphasmatodea. The vomer on venter X is well‐developed and features two unusually large basal apodemes and a strongly developed apical spine showing a specialized streaked surface micropattern. Female terminalia are characterized by an unkeeled abdominal sternum VIII covering the reduced primary ovipositor. Gonapophysis VIII does not extend beyond the tip of gonapophysis IX. An asymmetry concerning the size of the paired gonapophyses is reported for the first time in Phasmatodea.     

Phylogeography and species delineation of the genus Phoxinus Rafinesque, 1820 (Actinopterygii: Leuciscidae) in the Iberian Peninsula

The genus Phoxinus is comprised of at least 15 currently recognized species inhabiting Eurasia. Morphological traits have been traditionally used to delineate species in Phoxinus; however, the high level of phenotypic plasticity observed in the genus has confounded this process. Molecular genetic analyses have revealed a higher than expected genetic structure within Phoxinus. Here, we analyzed both nuclear and mitochondrial molecular genetic markers to infer the phylogeography and divergence times of Phoxinus in the Iberian Peninsula. Our results show that the Iberian lineages of Phoxinus were polyphyletic. They also support the co‐existence of three species in the Iberian Peninsula, two corresponding to two previously recognized species (Phoxinus bigerri and Phoxinus septimaniae) and a third undescribed species (Phoxinus sp.). Phoxinus bigerri is structured into western Cantabrian, eastern Cantabrian, and Artibai basins. We hypothesize that this structure is a consequence of glaciation–deglaciation cycles during the Pleistocene. While the presence of P. septimaniae in the Iberian Peninsula is possibly the result of human translocation, that of Phoxinus sp. in lower Ebro rivers may be attributed to past fluvial captures. Our study represents the first report to show a relationship among Phoxinus populations from central Pyrenean rivers of Spain and France. Furthermore, we found genetic hybridization between Phoxinus sp. and P. septimaniae in the shared localities, a likely consequence of anthropogenic activities. Overall, our findings provide insight into the genetic structure of Iberian Phoxinus populations, including the presence of an undescribed species and the putative introduction of some species that may have implications for conservation.     

Endogenous cellulase enzymes in the stick insect (Phasmatodea) gut

High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of the walking stick (Phasmatodea) Eurycantha calcarata. The enzyme was isolated and analyzed via mass spectrometry. RT-PCR revealed two endoglucanase genes, EcEG1 and EcEG2. Mascot analysis of the purified enzyme confirms it to be the product of gene EcEG1. Homologous cDNAs were also isolated from a distantly related species, Entoria okinawaensis, suggesting a general distribution of cellulase genes in phasmids. Phasmid cellulases showed high homology to endogenously-produced glycoside hydrolase family 9 (GH9) endoglucanases from insects, especially to those of termites, cockroaches, and crickets. The purified E. calcarata enzyme showed clear antigency against an anti-serum for termite GH9 cellulase, which, together with the sequence homology, further suggests an endogenous origin of the enzyme. This discovery suggests a possible nutritive value for cellulose in the leaf-feeding phasmids, unlike in herbivorous Lepidoptera.     

Chromosomal and NOR patterns in the polyclonal stick insect Bacillus atticus atticus (Insecta; Phasmatodea).

Bacillus atticus atticus is a complex of thelytokous parthenogens, related to the bisexual Bacillus grandii, that ranges from Sardinia to Near Eastern countries. Karyotypic and cytogenetic differentiation of the B. atticus atticus diploid unisexual "isolates" is really higher than expected. Its standard karyotype has 2n = 34 chromosomes, but several instances of repatterned or even aneuploid complements have been found. The number and location of silver-stained NORs are particularly intriguing, since in addition to homozygous NOR patterns, simple or double hemizygous strains are found spread over specific and wide regions. The odd patterns are not due to Ag-NOR staining technique artifacts, since the FISH method, using rDNA probes, apparently labels the same ribosomal clusters. Transpositions and translocations have been suggested to account for some NOR patterns, but hybridizations between different NOR-bearing races are also a possible cause. This chromosomal survey clearly contributes to a better understanding of B. atticus phylogeny.     

A genomic evaluation of taxonomic trends through time in coast horned lizards (genus Phrynosoma)

Determining the boundaries between species and deciding when to describe new species are challenging practices that are particularly difficult in groups with high levels of geographic variation. The coast horned lizards (Phrynosoma blainvillii, Phrynosoma cerroense and P. coronatum) have an extensive geographic distribution spanning many distinctive ecological regions ranging from northern California to the Cape Region of Baja California, Mexico, and populations differ substantially with respect to external morphology across much of this range. The number of taxa recognized in the group has been reevaluated by herpetologists over 20 times during the last 180 years, and typically without the aid of explicit species delimitation methods, resulting in a turbulent taxonomy containing anywhere from one to seven taxa. In this study, we evaluate taxonomic trends through time by ranking 15 of these species delimitation models (SDMs) using coalescent analyses of nuclear loci and SNPs in a Bayesian model comparison framework. Species delimitation models containing more species were generally favoured by Bayesian model selection; however, several three‐species models outperformed some four‐ and five‐species SDMs, and the top‐ranked model, which contained five species, outperformed all SDMs containing six species. Model performance peaked in the 1950s based on marginal likelihoods estimated from nuclear loci and SNPs. Not surprisingly, SDMs based on genetic data outperformed morphological taxonomies when using genetic data alone to evaluate models. The de novo estimation of population structure favours a three‐population model that matches the currently recognized integrative taxonomy containing three species. We discuss why Bayesian model selection might favour models containing more species, and why recognizing more than three species might be warranted.     

The follicle cell-oocyte interaction in ovarian follicles of the stick insect Bacillus rossius (Rossi): (Insecta: Phasmatodea)

Developing ovarian follicles of Bacillus rossius have been examined ultrastructurally in an attempt to understand how inception of vitel-logenesis is controlled. Early vitellogenic follicles are characterized by a thick cuboidal epithelium that is highly interlocked with the oocyte plasma membrane. Gap junctional contacts are present both at the follicle cell/oocyte interface and in between adjacent follicle cells. In addition, microvilli of follicle cells protrude deeply into the cortical ooplasm of these early vitellogenic oocytes. With the onset of vitellogenesis, wide intercellular spaces appear in the follicle cell epithelium and at the follicle cell/oocyte interface. Gap junctions become progressively reduced both on the follicle cell surface and on the oocyte plasma membrane. Microvilli from the two cell types no longer interlock. From a theoretical standpoint each of the two structural differentiations present at the follicle cell/oocyte interface—gap junctions and follicle cell microvilli—could potentially trigger inception of vitellogenesis. Gap junctions might permit the passage of a regulatory molecule, transferring from follicle cells to oocyte, which would control the assembly of coated pits on the oocyte plasma membrane. Alternatively cell interaction via microvilli might induce the appearance of coated pits, thus creating a membrane focus for vitellogenin receptors. Both possibilities are discussed in relation to current literature.     

An early winged crown group stick insect from the Early Eocene amber of France (Insecta,Phasmatodea)

The new stick insect family Gallophasmatidae, based on Gallophasma longipalpis gen. et sp.n. , from the Earliest Eocene French amber has a pattern of tegmina venation typical of Archaeorthoptera, also present in at least some Mesozoic ‘Phasmatodea’. On the other hand, Gallophasma displays in its body anatomy some apomorphies of the extant Euphasmatodea, e.g. fusion of metatergum and abdominal tergum 1, correlated with the reduction of abdominal sternum 1 to lateral triangular sclerites. A unique autapomorphy of Gallophasma is the presence of annulated and apparently multi‐segmented or pseudo‐segmented cerci; all other Phasmatodea have one‐segmented cerci. The venation of the tegmina of Gallophasma differs from that of extant winged Phasmatodea in the plesiomorphic absence of a knob‐like dorsal eversion. This and other differences in the wing venation between extant and extinct Phasmatodea might have been caused by the loss of wings at some point in the evolutionary history of the order and their secondary gain in a subclade of the extant phasmids.     

Influence of aminergic and peptidergic substances on heart beat frequency in the stick insect Carausius morosus (Insecta,Phasmatodea)

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi‐exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo‐HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo‐HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo‐HrTH‐II, thereby rendering the HrTH peptide “not active.” In ligated stick insects, 6.7 × 10^?8 M Carmo‐HrTH‐II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose‐dependent manner; inhibition was achieved with 10^?4 M of octopamine.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Loss and gain of sexual reproduction in the same stick insect

The outcome of competition between different reproductive strategies within a single species can be used to infer selective advantage of the winning strategy. Where multiple populations have independently lost or gained sexual reproduction it is possible to investigate whether the advantage is contingent on local conditions. In the New Zealand stick insect Clitarchus hookeri, three populations are distinguished by recent change in reproductive strategy and we determine their likely origins. One parthenogenetic population has established in the United Kingdom and we provide evidence that sexual reproduction has been lost in this population. We identify the sexual population from which the parthenogenetic population was derived, but show that the UK females have a post‐mating barrier to fertilisation. We also demonstrate that two sexual populations have recently arisen in New Zealand within the natural range of the mtDNA lineage that otherwise characterizes parthenogenesis in this species. We infer independent origins of males at these two locations using microsatellite genotypes. In one population, a mixture of local and nonlocal alleles suggested males were the result of invasion. Males in another population were most probably the result of loss of an X chromosome that produced a male phenotype in situ. Two successful switches in reproductive strategy suggest local competitive advantage for outcrossing over parthenogenetic reproduction. Clitarchus hookeri provides remarkable evidence of repeated and rapid changes in reproductive strategy, with competitive outcomes dependent on local conditions.     

Phylogeography and ecological niche modelling of the New Zealand stick insect Clitarchus hookeri (White) support survival in multiple coastal refugia

Aim Increasing our understanding of the effects of the Last Glacial Maximum (LGM) and determining the location of refugia requires studies on widely distributed species with dense sampling of populations. We have reconstructed the biogeographic history of Clitarchus hookeri (White), a widespread species of New Zealand stick insect that exhibits geographic parthenogenesis, using phylogeographic analysis and ecological niche modelling. Location New Zealand. Methods We used DNA sequence data from the mitochondrial cytochrome c oxidase subunit I gene to reconstruct phylogenetic relationships among haplotypes from C. hookeri and two undescribed Clitarchus species. We also used distribution data from our own field surveys and museum records to reconstruct the geographic distribution of C. hookeri during the present and the LGM, using ecological niche modelling. Results The ecological niche models showed that the geographic distribution of C. hookeri has expanded dramatically since the LGM. Our model predicted large areas of suitable LGM habitat in upper North Island, and small patches along the east coast of South Island. The phylogeographic analysis shows that populations in the northern half of North Island contain much higher levels of genetic variation than those from southern North Island and South Island, and is congruent with the ecological niche model. The distribution of bisexual populations is also non-random, with males completely absent from South Island and very rare in southern North Island. Main conclusions During the LGM C. hookeri was most likely restricted to several refugia in upper North Island and one or more smaller refugia along the east coast of South Island. The unisexual populations predominate in post-glacial landscapes and are clearly favoured in the recolonization of such areas. Our study exemplifies the utility of integrating ecological niche modelling and phylogeographic analysis.     

External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations

External morphological features of adult males are described in the stick insect Hermarchus leytensis from the Philippines, a species belonging to the little-known euphasmatodean lineage Stephanacridini. Mouthparts are characterized by some likely specialized features: (1) a dentate dorsal cutting edge on the mandibles; (2) distinct differences in size and shape between the galeae; (3) absence of an apical field of trichomes on the galeae; and (4) lacinial setae not protruding from the mesal margin, which features three bearing-like protuberances. The latter character state represents a very unusual condition, not known in other phasmatodeans or even in polyneopteran insects. A distinctive characteristic of attachment devices is that each euplantula is divided into two separated pads with a smooth microstructure, as it also occurs in some members of the clade Schizodecema. Male terminalia exhibit character states previously unknown in Stephanacridini, including (1) a symmetrical type of vomer and (2) claspers equipped with ∼70 very minute ventral teeth on each thorn pad. Potential implications for the systematic placement of H. leytensis are discussed. The results also underline the importance of microanatomical investigations as source of substantial characters for future analyses on phasmatodean systematics.     

Speciation and genetic divergence of three species of charr from ancient Lake El'gygytgyn (Chukotka) and their phylogenetic relationships with other representatives of the genus Salvelinus

The diversity of phenotypically different and often reproductively isolated lacustrine forms of charrs of the genus Salvelinus represents a substantial problem for taxonomists and evolutionary biologists. Based on the analysis of variability of ten microsatellite loci and two fragments of mitochondrial DNA (control region and cyt‐b gene), the evolutionary history of three charr species from Lake El'gygytgyn was reconstructed, and phylogenetic relationships between the main representatives of the genus were revealed. Three species from Lake El'gygytgyn were strongly reproductively isolated. Long‐finned charr described previously as Salvethymus svetovidovi, an ancient endemic form in the lake, originated 3.5 Mya (95% Bayesian credible intervals: 1.7, 6.1). Placement of this species in the phylogenetic tree of Salvelinus was not determined strictly, but it should be located in the basal part of the clade Salvelinus alpinus – S. malma species complex. The origin of small‐mouth charr S. elgyticus and Boganida charr S. boganidae in Lake El'gygytgyn was related to allopatric speciation. Their ancestors were represented by two glacial lineages of Taranets charr S. alpinus taranetzi from Asia. In Lake El'gygytgyn, these lineages entered into secondary contact postglacially. A revision of the main phylogenetic groups within the Salvelinus alpinus – S. malma complex is conducted. The Boganida charrs from Lakes El'gygytgyn and Lama (Taimyr) belong to different phylogenetic groups of Arctic charr and should not be regarded as a single species S. boganidae. Using the charrs from Lakes El'gygytgyn and Lama as a case study, we show that a model of sympatric speciation, which seemed more probable based on previous empirical evidence, was rejected by other data.     

Positive selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes across 22 hare species (genus Lepus)

Amino acid changes in mitochondrial (mt) oxidative phosphorylation (OXPHOS) genes have been suggested as a key adaptation to environmental variation. Here, we analyzed 416 sequences of ATPase synthase 6 (MT‐ATP6) and NADH dehydrogenase 2 (MT‐ND2) in 22 different hare (Lepus) species from across a wide range of habitats and climates. We used site‐ and branch‐based methods to test for positive selection on specific codons and lineages. We found four codons in MT‐ATP6 and five in MT‐ND2 under positive selection, affecting several species lineages. We investigated the association of protein variants at each locus with climate zone, using multinomial generalized linear models (glm), including species, regions, historical introgression events, and the co‐occurring protein variant at the other locus as additional explanatory variables. A significant climate effect as based on the “Köppen climate classification” was observed for MT‐ND2 protein variants as translated from our nucleotide sequences. Moreover, MT‐ND2 protein variants were significantly affected by the co‐occurring MT‐ATP6 protein variant in the same mtDNA molecule. Contrary to the expectation for non‐recombining mitochondrial DNA molecules, the presence of an evolutionarily relatively ancestral protein variant at one locus was associated with a relatively derived protein at the other locus in the same mitochondrial molecule, respectively. The relative evolutionary status of a protein variant was evaluated according to its positions relative to the respective out‐group protein variant in a network analysis of nucleotide sequences. All our results suggest a complex effect of various climatic parameters acting on multiple mtOXPHOS genes in a co‐adaptive way, favoring combinations of ancestral and derived variants.     

Mitochondrial introgressive hybridization following a demographic expansion in the tomato frogs of Madagascar,genus Dyscophus

Madagascar is a biodiversity hotspot with a unique fauna and flora largely endemic at the species level and highly threatened by habitat destruction. The processes underlying population‐level differentiation in Madagascar's biota are poorly understood and have been proposed to be related to Pleistocene climatic cycles, yet the levels of genetic divergence observed are often suggestive of ancient events. We combined molecular markers of different variability to assess the phylogeography of Madagascar's emblematic tomato frogs (Dyscophus guineti and D. antongilii) and interpret the observed pattern as resulting from ancient and recent processes. Our results suggest that the initial divergence between these taxa is probably old as reflected by protein‐coding nuclear genes and by a strong mitochondrial differentiation of the southernmost population. Dramatic changes in their demography appear to have been triggered by the end of the last glacial period and possibly by the short return of glacial conditions known as the 8K event. This dramatic change resulted in an approximately 50‐fold reduction of the effective population size in various populations of both species. We hypothesize these species' current mitochondrial DNA diversity distribution reflects a swamping of the mitochondrial genetic diversity of D. guineti by that of D. antongilii previous to the populations' bottlenecks during the Holocene, and probably as a consequence of D. antongilii demographic expansion approximately 1 million years ago. Our data support the continued recognition of D. antongilii and D. guineti as separate species and flag D. guineti as the more vulnerable species to past and probably also future environmental changes.