Comparative morphology,biology and phylogeny of terminal‐instar larvae of the European species of Toryminae (Hym., Chalcidoidea,Torymidae) parasitoids of gall wasps (Hym. Cynipidae)

We present a phylogenetic and taxonomic study of the morphology and biology of the terminal‐instar larval stage of 19 species representing all the genera of Torymidae parasitoids of gall wasps in Europe, with the single exception of Megastigmus. The genera studied include Adontomerus Nikol'skaya, Idiomacromerus Crawford, Chalcimerus Steffan & Andriescu, Glyphomerus Förster, Pseudotorymus Masi and Torymus Dalman. We primarily used chaetotaxy and some head structures. The terminal‐instar larvae of all studied species are thoroughly described for the first time and illustrated with SEM images. We provide diagnostic characters for the family and the genera studied, and keys to genera and species for the identification of torymid larvae associated with cynipid galls. The majority of the torymid larvae studied are solitary monophagous parasitoids. Finally, to assess the potential use of larval characters in systematic studies of the family, a phylogenetic analysis of the studied taxa based on 42 larval morphological characters is proposed and compared with the current taxonomy of Torymidae. Our results suggest that body chaetotaxy, and characters of the head and mouthparts could be used for genera and species discrimination. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 676–721.     

Phylogenetics links monster larva to deep‐sea shrimp

Mid‐water plankton collections commonly include bizarre and mysterious developmental stages that differ conspicuously from their adult counterparts in morphology and habitat. Unaware of the existence of planktonic larval stages, early zoologists often misidentified these unique morphologies as independent adult lineages. Many such mistakes have since been corrected by collecting larvae, raising them in the lab, and identifying the adult forms. However, challenges arise when the larva is remarkably rare in nature and relatively inaccessible due to its changing habitats over the course of ontogeny. The mid‐water marine species Cerataspis monstrosa (Gray 1828) is an armored crustacean larva whose adult identity has remained a mystery for over 180 years. Our phylogenetic analyses, based in part on recent collections from the Gulf of Mexico, provide definitive evidence that the rare, yet broadly distributed larva, C. monstrosa, is an early developmental stage of the globally distributed deepwater aristeid shrimp, Plesiopenaeus armatus. Divergence estimates and phylogenetic relationships across five genes confirm the larva and adult are the same species. Our work demonstrates the diagnostic power of molecular systematics in instances where larval rearing seldom succeeds and morphology and habitat are not indicative of identity. Larval–adult linkages not only aid in our understanding of biodiversity, they provide insights into the life history, distribution, and ecology of an organism.     

DNA Taxonomy and the identification of immature insect stages: the true larva of Tauriphila argo (Hagen 1869) (Odonata: Anisoptera: Libellulidae)

For many insect taxa, larval morphology plays a decisive role in various fields like taxonomy, phylogeny or ecology. However, species identification is usually based on imaginal characters and the identification of larvae depends upon an established link to unequivocally identified imagines. This taxonomic correspondence of larvae and imagines is far from being established in many odonate species. We have employed a molecular approach to link larval and adult specimens in Tauriphila argo (Hagen, 1869). The sequenced mt SSU gene fragments of the reared female, supposedly a T. argo female, and a clearly identified male specimen of the species were identical. However, the larva of the reared female clearly differed from the described T. argo larva, previously matched to the species. From this observation, we conclude that the previously described larva of T. argo does not belong to this species because of too many phenotypic differences that far exceed the generally observed intraspecific variation.

It can be foreseen that the molecular approach will prove to be effective in identifying unknown larvae in many insect species. Additionally, the discrimination of sibling species or the linkage of allotypes and holotypes will become feasible with this approach.     

Larval morphology and phylogenetic position of Horelophopsis hanseni Satô et Yoshitomi (Coleoptera,Hydrophilidae, Horelophopsinae)

The subfamily Horelophopsinae was originally proposed as one of the earliest diverging clades of Hydrophilidae (s.s.), but its phylogenetic placement has never been tested. We describe the larva of Horelophopsis hanseni Satô et Yoshitomi, 2004 of the Horelophopsinae. Larval data are based on larval specimens collected together with adults, and unambiguously associated with them by means of DNA barcoding. We perform an analysis testing the phylogenetic position of H. hanseni based on larval and adult morphological characters. Horelophopsis hanseni is unambiguously placed within the hydrophilid subfamily Hydrophilinae and its close relationships to the genus Agraphydrus Régimbart, 1903 (Hydrophilinae, Acidocerini) is recognized. The results suggest that the subfamily Horelophopsinae is unlikely to be a basal taxon of Hydrophilidae, as originally suggested.     

The larval head structures of Podagricomela shirahatai (Chûjô) (Chrysomelidae,Galerucinae, Alticini) and morphological effects of leaf mining

Head structures of the leaf mining larva of the chrysomelid species Podagricomela shirahatai are described and illustrated. Internal and external structures were reconstructed three dimensionally based on image stacks obtained with microcomputed tomography. The larval head is characterized by prognathism, a dorsoventrally compressed shape, a flattened maxillolabial complex, a completely reduced coronal suture, and the presence of a deep, V‐shaped posterior emargination of the head capsule. Internal structures are not distinctly affected by leaf mining. The cephalic features are compared with conditions found in surface feeding and root feeding alticine larvae and also with characters of chrysomeline larvae of Chrysomela populi Linnaeus. Possible correlations between modifications of the larval head and different feeding behaviors are discussed. Characters are also discussed with respect to possible phylogenetic implications. Some derived features are apparently due to phylogenetic constraints. Apomorphies characterizing alticine larvae with distinctly different life habits are the loss of M. frontoepharyngalis (M. 9), the origin of M. tentoriostipitalis (M. 18) from the head capsule, two insertions of M. tentoriopraementalis inferior (M. 29) and the reduction of stemmata. The study underlines that the anatomical study of chrysomeloid larvae is not only highly desirable in a phylogenetic context, but also crucial for understanding the evolution of different life strategies in this extremely successful group of Coleoptera. J. Morphol. 276:446–457, 2015. © 2014 Wiley Periodicals, Inc.     

Phylogeny of the death's head hawkmoths, Acherontia[Laspeyres], and related genera (Lepidoptera: Sphingidae: Sphinginae: Acherontiini)

Abstract. Adult death's head hawkmoths (Acherontia species) have a unique feeding biology as cleptoparasites of honeybees, stealing honey from the combs, rather than imbibing nectar from flowers. The moths have a range of features, both morphological and behavioural, that enable them to successfully enter, feed and escape from the colonies. These adaptations may vary among the three Acherontia species and allow them each to target different species of honeybee. A cladistic analysis is presented of the hawkmoths of tribe Acherontiini. The study aims to resolve the relationships of the genera and species of Acherontiini, with a particular focus on the three species of Acherontia. The dataset comprises sixty‐five characters derived from adult, larval and pupal morphology, and larva host‐plant biology. These data are analysed using equal weighting and implied weighting. Acherontiini and each constituent genus are recovered as monophyletic. However, within Coelonia, there is ambiguity in that the sister‐species relationships C. brevis+C. fulvinotata and C. fulvinotata + C. solani are equally parsimonious under both weighting schemes. Furthermore, under equal weighting Agrius is placed equally parsimoniously as the sister group of either Acherontia + Coelonia or Callosphingia. Under implied weighting, however, only the latter relationship is most parsimonious (fit). Within Acherontia, A. atropos and A. styx are always recovered as sister species to the exclusion of A. lachesis. The results of the phylogenetic analysis provide an objective basis for future studies of the unique cleptoparasitic association of these moths.     

Phylogenetic analysis of the Pinnotheridae (Crustacea, Brachyura) based on larval morphology, with emphasis on the Dissodactylus species complex

Comparative larval morphology was used to elucidate phylogenetic relationships within the Pinnotheridae and the Dissodactylus species complex. Within the family, seven zoeal and six megalopal characters suggested two equally parsimonious phylogenetic hypotheses for pinnotherid larvae, both with Ostracotheres tridacnae representing the sister group for the Dissodactylus complex. Results indicated that the genus Pinnotheres is a polyphyletic taxon, and that the traditional subfamilial arrangement comprises paraphyletic taxa within the subfamilies Pinnotherinae and Pinnothereliinae. Certain evidence has suggested that Fabia and Juxtafubia should be excluded from the Pinnotherinae and placed into the Pinnothereliinae. Larval and adult morphology suggested that Pinnotheres politus should be included within Tumidotheres. The phylogenetic analysis within the Dissodactylus complex involved one zoeal and 16 megalopal characters. Results suggested a single phylogenetic hypothesis based on larval morphology. Combining adult morphology with larval evidence resulted in two equally parsimonious phylogenetic hypotheses, one of which agreed with a previously suggested hypothesis based only on adult characters.     

Coding characters from different life stages for phylogenetic reconstruction: a case study on dragonfly adults and larvae,including a description of the larval head anatomy of Epiophlebia superstes (Odonata: Epiophlebiidae)

The exclusive use of characters coding for specific life stages may bias tree reconstruction. If characters from several life stages are coded, the type of coding becomes important. Here, we simulate the influence on tree reconstruction of morphological characters of Odonata larvae incorporated into a data matrix based on the adult body under different coding schemes. For testing purposes, our analysis is focused on a well‐supported hypothesis: the relationships of the suborders Zygoptera, ‘Anisozygoptera’, and Anisoptera. We studied the cephalic morphology of Epiophlebia, a key taxon among Odonata, and compared it with representatives of Zygoptera and Anisoptera in order to complement the data matrix. Odonate larvae are characterized by a peculiar morphology, such as the specific head form, mouthpart configuration, ridge configuration, cephalic musculature, and leg and gill morphology. Four coding strategies were used to incorporate the larval data: artificial coding (AC), treating larvae as independent terminal taxa; non‐multistate coding (NMC), preferring the adult life stage; multistate coding (MC); and coding larval and adult characters separately (SC) within the same taxon. As expected, larvae are ‘monophyletic’ in the AC strategy, but with anisopteran and zygopteran larvae as sister groups. Excluding larvae in the NMC approach leads to strong support for both monophyletic Odonata and Epiprocta, whereas MC erodes phylogenetic signal completely. This is an obvious result of the larval morphology leading to many multistate characters. SC results in the strongest support for Odonata, and Epiprocta receives the same support as with NMC. Our results show the deleterious effects of larval morphology on tree reconstruction when multistate coding is applied. Coding larval characters separately is still the best approach in a phylogenetic framework. © 2015 The Linnean Society of London     

The larvae of the burrowing mayfly genus Tortopus (Ephemeroptera: Polymitarcyidae)

The larval stage of Tortopus is redescribed based on three species: T. puella from North America, the only species of the genus previously known from larva, and the larvae of T. obscuripennis and T. sarae from South America described here. Generic characters of the larva include: relatively large finger-like gill near base of maxilla, inner margin of mandibular tusks with a subdistal tubercle, straight or weakly convex frontal ridge present between antennae, reduced unilamellated gill on abdominal segment I. Additionally the male imagines of both Neotropical species are described for the first time, and T. obscuripennis is recorded from Bolivia. Diagnoses, SEM photographs, and illustrations are given for the new stages described and for the identification of the three Tortopus species known as larvae.     

The species‐level phylogeny of archostematan beetles—where do Micromalthus debilis and Crowsoniella relicta belong?

A species‐level phylogenetic analysis comprising 37 of the 45 known extant species of archostematan beetles and a total of 110 morphological characters from adults and larvae is presented. For the first time, characters of the male genitalia are included in a phylogenetic analysis of Archostemata. The dataset is analysed with parsimony as well as with Bayesian algorithms. Analyses with differently arranged datasets, with larval characters included or excluded, and including or excluding Micromalthus debilis and Crowsoniella relicta are conducted. The resulting hypothesis of the species‐level phylogeny of Archostemata confirms Cupedidae and Ommatidae as monophyletic taxa. Within Cupedidae, the South American Paracupes and the North American Priacma together are the sister group to all remaining Cupedidae. Among the latter, the identification of a clade comprising Rhipsideigma, Cupes capitatus and Tenomerga leucophaea renders Tenomerga polyphyletic. Ascioplaga scalena comb. nov . is transferred from Adinolepis. Characters of the male genitalia support phylogenetic affinities of Micromalthus debilis with Ommatidae and of Crowsoniella relicta with Cupedidae.     

Development in Berthella californica (Gastropoda: Opisthobranchia) with comparative observations on phylogenetically relevant larval characters among nudipleuran opisthobranchs

Abstract. Morphological criteria defining the Nudipleura and sister group relationships among the three nudipleuran subgroups (pleurobranchoideans, anthobranch nudibranchs, and cladobranch nudibranchs) have been controversial. Analysis of larval stages may help resolve these uncertainties by identifying additional phylogenetically informative characters, but existing information on pleurobranchoidean larvae is meager. We studied larval development and metamorphosis of the pleurobranchoidean Berthella californica using histological sections, scanning and transmission electron microscopy, and immunolabeling of neurons within the larval apical ganglion. We also provide comparative data on other nudipleuran larvae that may be useful for phylogenetic reconstruction. Berthella californica fills a previously unoccupied place within an evolutionary scenario that derives nudibranchs from pleurobranchoideans, two groups in which the larval mantle fold forms the post‐metamorphic notum (dorsal epidermis). In B. californica, reflection of the mantle fold epithelium to form the notum begins at metamorphosis, as also occurs in nudibranchs, whereas mantle reflection in other pleurobranchoideans begins well before metamorphosis. Dissolution of overgrown shell walls inside the protoconch and formation of the post‐metamorphic notum from the inner epithelium of the larval mantle fold may be synapomorphies of the Nudipleura. The larval shell in B. californica is additionally noteworthy because it acquires bilateral symmetry later in development, which is very unusual among larval opisthobranchs. We demonstrate an osphradium in the larvae of two pleurobranchoideans and one anthobranch nudibranch, although adults lack this trait. We also identified an autapomorphy of cladobranch nudibranchs in the form of five ampullary neurons within the larval apical ganglion, whereas other planktotrophic opisthobranch larvae have only four of these neurons. Although our data provide morphological criteria defining both the Nudipleura and the cladobranch nudibranchs, they are insufficient to resolve sister group relationships within the Nudipleura.     

A preliminary study on the phylogenetic relationships of Copelatus Erichson (Coleoptera: Dytiscidae: Copelatinae) based on larval chaetotaxy and morphology

The phylogenetic relationships of the diving-beetle (Dytiscidae) subfamily Copelatinae are not well known. Some authors postulated a sister-group relationship between Copelatus Erichson and the remaining Dytiscidae, relying mainly on the absence of a mandibular channel in Copelatus. Other authors suggested a more derived position of the genus. Larval characters have been underutilized in phylogenetic studies, mainly because the larvae of many taxa within the family and, in particular, within Copelatinae are unknown. A comprehensive approach aimed to study the phylogenetic placement of a member of this subfamily based on larval characters has not been produced so far. In this study, the phylogenetic relationships of Copelatus are explored based on a cladistic analysis of 24 taxa and 120 chaetotaxic and morphological characters from larvae. For this purpose, larvae of Copelatus longicornis Sharp are described and illustrated in detail for the first time, with particular emphasis on morphometry and chaetotaxy, with the latter being unexplored until now. The results support a derived position of Copelatus within Dytiscidae, with a sister-group relationship between this genus and a clade formed by the subfamilies Lancetinae, Coptotominae, Laccophilinae, Colymbetinae, Matinae, and Dytiscinae, and part of Agabinae. No evidence was found for a sister-group relationship between Copelatus and the remaining Dytiscidae so that the absence of a mandibular channel in this genus is likely a reduction. Copelatus is supported by three apomorphies within Dytiscidae: mandibular channel absent, internal margin of the stipes with three robust spinulae, and seta MX8 inserted subapically on the galea. Handling editor: K. Martens     

The systematic position of Ergasticus (Decapoda,Brachyura) and allied genera,a molecular and morphological approach

The complete larval series (zoea I, zoea II and megalopa) of the crab Ergasticus clouei is described and illustrated based on plankton samples from Mediterranean waters. The zoea II and megalopal stages, previously unknown, are described here for the first time. Nucleotide sequence analysis of two gene regions (16S rDNA and Cox1 genes) confirmed the assignment of these larvae to Ergasticus clouei. The molecular analyses and the morphology of the larval stages revealed large differences between Ergasticus and Inachus, the type genus of the family Inachidae. In fact, E. clouei larvae presented a series of morphological characters, such as antennal shape and mouthparts setation pattern that placed them closer to the family Oregoniidae. The phylogenetic analyses also showed significant support for the monophyly of the Oregoniidae + Ergasticus group. The data argue for removal of Ergasticus and the related genera (Bothromaia, Pleisticanthoides, Parapleisticantha and Pleistacantha) from the Inachidae and their placement within the Oregoniidae as a separate subfamily, Pleistacanthinae ?tev?i?, 2005. Our results demonstrate that larval stages provide reliable morphological traits, independent from those of adults, to help resolving relationships among Majoidea genera.     

Effects of life-history traits and species distribution on genetic structure at maternally inherited markers in European trees and shrubs

Aim To examine relationships between life‐history traits, ecological and chorological characteristics of woody plant species and patterns of genetic differentiation among populations as assessed by chloroplast DNA (cpDNA) markers, and to compare them with patterns previously described from nuclear markers. Location Europe. Methods Data on cpDNA variation were compiled for 29 temperate European broad‐leaved tree and shrub species. Six qualitative and three quantitative characters of the species were tested for their relationship with two parameters of genetic population differentiation (G_ST and N_ST). Both direct species comparisons and phylogenetically independent contrast analyses were performed. Results When the phylogeny was not taken into account, five characters were significantly related to levels of population differentiation. The relationship disappeared in all but two cases (distribution type and seed mass) when analyses controlled for phylogenetic relationships among species. Main conclusions The correlation between distribution type (boreal‐temperate or temperate) and cpDNA differentiation of temperate European woody plant species suggests that their Quaternary history, in particular the location and isolation of their glacial refugia, is an important determinant of their present‐day level of genetic structure. By contrast, the relationship between life‐history traits and genetic differentiation at maternally inherited markers is weaker, especially when phylogenetic effects are controlled for.     

Association of insect life stages using DNA sequences: the larvae of Philodytes umbrinus (Motschulsky) (Coleoptera: Dytiscidae)

Abstract. Insect life stages are known imperfectly in many cases, and classifications are based often on only one or a few semaphoronts of a species. This is unfortunate as information in alternative life stages often is useful for scientific study. Although recent examples of DNA in taxonomy have emphasized the identification of indistinguishable species, such sequence data facilitate the association of life history stages and hold considerable promise in phylogenetic analysis, evolutionary studies, diagnostics, etc. These concepts are discussed here and an example is provided from diving beetles (Dytiscidae: Coleoptera). Three unknown larval specimens of an apparent species of Laccophilinae collected in Namibia were associated with the species Philodytes umbrinus (Motschulsky) using DNA sequence data. An 806-bp portion of the gene cytochrome oxidase I was sequenced from the unknown larvae. Several identified adult specimens of species of Laccophilinae from Namibia were also sequenced, including two P. umbrinus specimens and specimens from four Laccophilus Leach species. Additional species of Laccophilus from other areas of the world also were sequenced, as were specimens of Agabetes acuductus (Harris), Australphilus saltus Watts, Neptosternus boukali Hendrich & Balke and a species of Laccodytes Régimbart. Parsimony analysis resulted in two most parsimonious trees with the unknown larva unambiguously resolved in a group with both adult specimens of P. umbrinus (bootstrap value = 100%). The average pairwise p-distance between the unknown larva and adult P. umbrinus specimens averaged 0.09% (0–0.14%), compared with an average divergence between other conspecifics in the analysis of 0.24% (0–0.82%) and an overall average divergence between species of 13.49% (1.90–19.86%). Based on this, the unknown larvae were assigned to P. umbrinus. The larvae are diagnosed and described and their relationship with other Laccophilinae is discussed.     

Parallel adaptive radiations in arid and temperate Australia: molecular phylogeography and systematics of the Egernia whitii (Lacertilia: Scincidae) species group

It has been an enduring belief that increasing aridity combined with Pliocene‐Pleistocene glacial cycles resulted in the formation of distinct arid zone and temperate zone faunas within Australia. We assembled a molecular phylogeny for the Egernia whitii species group, an endemic group of skinks that comprises representatives from arid and temperate Australia, in order to test several biogeographical hypotheses regarding the origin of the Australian arid zone fauna. Sequence data were obtained from ten of the 11 species within the species group, plus three other Egernia species and an outgroup (Eulamprus heatwolei). We targeted portions of the ND4 (696 base pairs) and 16S rRNA (500 bp) mitochondrial genes and the β‐Fibrinogen 7th Intron nuclear gene (648 bp). The edited alignment comprised 1844 characters, of which 551 (30%) were variable and 382 (69%) were parsimony informative. We analysed the data using maximum likelihood and Bayesian techniques and produced a single optimal tree. Our phylogeny strongly supports two major clades within the species group, corresponding to temperate‐adapted rock‐dwelling species and arid‐adapted obligate burrowing species. However, the phylogenetic affinities of E. pulchra were not resolved. Our topology indicates that the New South Wales population of E. margaretae is actually E. whitii and reveals that E. margaretae margaretae and E. m. personata are distinct species. There also appears to be a major phylogeographical break within E. whitii occurring in eastern Victoria. Although our data supported several previously proposed phylogenetic relationships, Shimodaira–Hasegawa tests soundly rejected several suggested affinities between certain species. The arid zone members of the E. whitii species group had been suggested to have originated as a result of multiple periods of colonization during the Pleistocene glaciation cycles. However, our genetic data suggest a single origin (presumably from a semiarid E. multiscutata‐like ancestor) for the arid zone members of the group prior to the Plio‐Pleistocene, probably during the late Miocene to early Pliocene. Our topology displays substantial sequence divergence between species with short internodes and long terminal branches, indicating rapid adaptive radiations within the arid and temperate zones. The presence of temperate‐adapted species within more mesic refugia of the arid zone suggests that the necessary adaptations to aridity for colonizing the dry interior of the continent have not evolved since the initial period of adaptive radiation, despite the long evolutionary history of the species group. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 157–173.     

Phylogeny of the nominotypical subgenus of Culex (Diptera: Culicidae): insights from analyses of anatomical data into interspecific relationships and species groups in an unresolved tree

The aim of this study was to produce the first objective and comprehensive phylogenetic analysis of the speciose subgenus Culex based on morphological data. We used implied and equally weighted parsimony methods to analyse a dataset comprised of 286 characters of the larval, pupal, and adult stages of 150 species of the subgenus and an outgroup of 17 species. We determined the optimal support by summing the GC supports for each MPC, selecting the cladograms with the highest supports to generate a strict consensus tree. We then collapsed the branches with GC support < 1 to obtain the ‘best’ topography of relationships. The analyses largely failed to resolve relationships among the species and the informal groups in which they are currently placed based on morphological similarities and differences. All analyses, however, support the monophyly of genus Culex. With the exception of the Atriceps Group, the analyses failed to find positive support for any of the informal species groups (monophyly of the Duttoni Group could not be established because only one of the two species of the group was included in the analyses). Since the analyses would seem to include sufficient data for phylogenetic reconstruction, lack of resolution appears to be the result of inadequate or conflicting character data, and perhaps incorrect homology assessments. Molecular and other biological data are needed to gain insights into the evolution of subgenus Culex. Nevertheless, we discuss the placement of several taxa in the current morphology-based classification of the subgenus based on insights realized during the study.