Phylogeny of the holometabolous insect orders: molecular evidence

Phylogenetic relationships among the holometabolous insect orders were reconstructed using 18S ribosomal DNA data drawn from a sample of 182 taxa representing all holometabolous insect orders and multiple outgroups. Parsimony analysis supports the monophyly of all holometabolous insect orders except for Coleoptera and Mecoptera. Mecoptera is paraphyletic with respect to Siphonaptera, which is nested within Mecoptera. Coleoptera is scattered as a paraphyletic assemblage across the tree topology. These data support a monophyletic Halteria (Strepsiptera + Diptera), Amphiesmenoptera (Trichoptera + Lepidoptera), Neuropterida (Neuroptera + (Megaloptera + Raphidioptera)), but Antliophora (Halteria + Mecoptera + Siphonaptera) and Mecopterida (Antliophora + Amphiesmenoptera) are paraphyletic. The limitations of using 18S ribosomal DNA as the sole phylogenetic marker for reconstructing insect ordinal relationships are discussed.     

Goodbye Halteria? The thoracic morphology of Endopterygota (Insecta) and its phylogenetic implications

Characters of the thorax of 30 representatives of all endopterygote orders and four hemimetabolous outgroup taxa were examined. In total, 126 characters potentially useful for phylogenetic reconstruction are discussed and presented as a data matrix. The thoracic features were analysed with different approaches combined with an additional large set of morphological data. Endopterygota were confirmed as monophyletic and new morphological autapomorphies of the group are suggested. The highly controversial Strepsiptera are not placed as sistergroup of Diptera (Halteria‐concept) but consistently as sistergroup of Coleoptera. This clade was mainly supported by characters associated with posteromotorism. The traditionally proposed relationship of Neuropterida + Coleoptera was not confirmed. Hymenoptera was placed as sistergroup of all remaining orders in parsimony analyses. The inclusion of Strepsiptera + Coleoptera in Mecopterida in parsimony analyses is probably artificial and potential thoracic autapomorphies of Mecopterida in the traditional sense are suggested. Mecopterida are confirmed as a clade in Bayesian analyses. Amphiesmenoptera and Antliophora are well supported. The paraphyly of Mecoptera is due to a clade comprising Nannochoristidae and Siphonaptera + Diptera. The phylogenetic reconstruction using characters of the thorax is impeded by functional constraints, parallel losses, a general trend to reinforce the skeleton and to simplify the muscular apparatus, and also by different specializations occurring in potential outgroup taxa. The addition of a large additional morphological data set only partly compensated for these problems. It is apparent that the inclusion of more outgroup and ingroup taxa is required, notably presumably basal representatives of Mecoptera, Trichoptera, and Diptera. This may reduce the effect of an artificial attraction of branches caused by homoplasy, notably character losses occurring within different lineages.© The Willi Hennig Society 2010.     

Phylogenetic relationships among insect orders based on three nuclear protein-coding gene sequences

Many attempts to resolve the phylogenetic relationships of higher groups of insects have been made based on both morphological and molecular evidence; nonetheless, most of the interordinal relationships of insects remain unclear or are controversial. As a new approach, in this study we sequenced three nuclear genes encoding the catalytic subunit of DNA polymerase delta and the two largest subunits of RNA polymerase II from all insect orders. The predicted amino acid sequences (In total, approx. 3500 amino acid sites) of these proteins were subjected to phylogenetic analyses based on the maximum likelihood and Bayesian analysis methods with various models. The resulting trees strongly support the monophyly of Palaeoptera, Neoptera, Polyneoptera, and Holometabola, while within Polyneoptera, the groupings of Isoptera/"Blattaria"/Mantodea (Superorder Dictyoptera), Dictyoptera/Zoraptera, Dermaptera/Plecoptera, Mantophasmatodea/Grylloblattodea, and Embioptera/Phasmatodea are supported. Although Paraneoptera is not supported as a monophyletic group, the grouping of Phthiraptera/Psocoptera is robustly supported. The interordinal relationships within Holometabola are well resolved and strongly supported that the order Hymenoptera is the sister lineage to all other holometabolous insects. The other orders of Holometabola are separated into two large groups, and the interordinal relationships of each group are (((Siphonaptera, Mecoptera), Diptera), (Trichoptera, Lepidoptera)) and ((Coleoptera, Strepsiptera), (Neuroptera, Raphidioptera, Megaloptera)). The sister relationship between Strepsiptera and Diptera are significantly rejected by all the statistical tests (AU, KH and wSH), while the affinity between Hymenoptera and Mecopterida are significantly rejected only by AU and KH tests. Our results show that the use of amino acid sequences of these three nuclear genes is an effective approach for resolving the relationships of higher groups of insects.     

The Phylogeny of the Extant Hexapod Orders

Morphological and molecular data are marshalled to address the question of hexapod ordinal relationships. The combination of 275 morphological variables, 1000 bases of the small subunit nuclear rDNA (18S), and 350 bases of the large subunit nuclear rDNA (28S) are subjected to a variety of analysis parameters (indel and transversion costs). Representatives of each hexapod order are included with most orders represented multiply. Those parameters that minimize character incongruence (ILD of Mickevich and Farris, 1981, Syst. Zool. 30, 351–370), among the morphological and molecular data sets are chosen to generate the best supported cladogram. A well-resolved and robust cladogram of ordinal relationships is produced with the topology (Crustacea ((Chilopoda Diplopoda) ((Collembola Protura) ((Japygina Campodeina) (Archaeognatha (Zygentoma (Ephemerida (Odonata ((((Mantodea Blattaria) Isoptera) Zoraptera) ((Plecoptera Embiidina) (((Orthoptera Phasmida) (Grylloblattaria Dermaptera)) ((((Psocoptera Phthiraptera) Thysanoptera) Hemiptera) ((Neuropteroidea Coleoptera) (((((Strepsiptera Diptera) Mecoptera) Siphonaptera) (Trichoptera Lepidoptera)) Hymenoptera)))))))))))))).     

18S rDNA sequences and the holometabolous insects

The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

部分具翅昆虫后胸足基骨片的分化与系统发育

从12目具翅昆虫中选出16个代表种,对其后足基骨片的形态特征在不同类群中的衍变进行分析比较,据此构建反映下列初步进化关系的系统树:[Ephemeroptera+(Odonata+Neoptera)]+[Plecoptera+(Megaloptera+Neuroptera+(Orthoptera+(Hemiptera+(...     

Molecular Phylogenetics at the Felsenstein Zone: Approaching the Strepsiptera Problem Using 5.8S and 28S rDNA Sequences

Recent efforts to reconstruct the phylogenetic position of the insect order Strepsiptera have elicited a major controversy in molecular phylogenetics. We sequenced the 5.8S rDNA and major parts of the 28S rDNA 5′ region of the strepsipteran speciesStylops melittae.Their evolutionary dynamics were analyzed together with previously published insect rDNA sequences to identify tree estimation bias risks and to explore additional sources of phylogenetic information. Several major secondary structure changes were found as being autapomorphic for the Diptera, the Strepsiptera, or the Archaeognatha. Besides elevated substitution rates a significant AT bias was present in dipteran and strepsipteran 28S rDNA which, however, was restricted to stem sites in the Diptera while also affecting single-stranded sites in the Strepsiptera. When dipteran taxa were excluded from tree estimation all methods consistently supported the placement of Strepsiptera to within the Holometabola. When dipteran taxa were included maximum likelihood continued to favor a sister-group relationship of Strepsiptera with Mecoptera while remaining methods strongly supported a sister-group relationship with Diptera. Parametric bootstrap analysis revealed maximum likelihood as a consistent estimator if rate heterogeneity across sites was taken into account. Though the position of Strepsiptera within Holometabola remains elusive, we conclude that the evolution of dipteran and strepsipteran rDNA involved similar yet independent changes of substitution parameters.     

Phylogenetic position of the Strepsiptera: Review of molecular and morphological evidence

Molecular evidence of the monophyly of the Halteria (Strepsiptera + Diptera) is reviewed. The majority of morphological characters, which have classically been used to establish a Strepsiptera + Coleoptera sister group, are rejected, because they are based on erroneous interpretations of strepsipteran morphology. The scorings of 31 morphological characters, which directly relate to the phylogenetic position of Strepsiptera, are provided, and their distribution and optimization on the molecular + morphological tree is discussed. Of these, 13 characters specifically support the placement of Strepsiptera within the Mecopterida; seven of which are based on the optimization of inapplicable or missing data, and six of which are based on states that can be scored for Strepsiptera. Only a single character (posteromotorism) suggests a sister group relationship with the Coleoptera. The morphological and molecular data are largely congruent, and suggest that the Strepsiptera are sister group to the Diptera.     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.     

A mitochondrial genome phylogeny of the Neuropterida (lace-wings, alderflies and snakeflies) and their relationship to the other holometabolous insect orders

We present a mitochondrial (mt) genome phylogeny inferring relationships within Neuropterida (lacewings, alderflies and camel flies) and between Neuropterida and other holometabolous insect orders. Whole mt genomes were sequenced for Sialis hamata (Megaloptera: Sialidae), Ditaxis latistyla (Neuroptera: Mantispidae), Mongoloraphidia harmandi (Raphidioptera: Raphidiidae), Macrogyrus oblongus (Coleoptera: Gyrinidae), Rhopaea magnicornis (Coleoptera: Scarabaeidae), and Mordella atrata (Coleoptera: Mordellidae) and compared against representatives of other holometabolous orders in phylogenetic analyses. Additionally, we test the sensitivity of phylogenetic inferences to four analytical approaches: inclusion vs. exclusion of RNA genes, manual vs. algorithmic alignments, arbitrary vs. algorithmic approaches to excluding variable gene regions and how each approach interacts with phylogenetic inference methods (parsimony vs. Bayesian inference). Of these factors, phylogenetic inference method had the most influence on interordinal relationships. Bayesian analyses inferred topologies largely congruent with morphologically‐based hypotheses of neuropterid relationships, a monophyletic Neuropterida whose sister group is Coleoptera. In contrast, parsimony analyses failed to support a monophyletic Neuropterida as Raphidioptera was the sister group of the entire Holometabola excluding Hymenoptera, and Neuroptera + Megaloptera is the sister group of Diptera, a relationship which has not previously been proposed based on either molecular or morphological data sets. These differences between analytical methods are due to the high among site rate heterogeneity found in insect mt genomes which is properly modelled by Bayesian methods but results in artifactual relationships under parsimony. Properly analysed, the mt genomic data set presented here is among the first molecular data to support traditional, morphology‐based interpretations of relationships between the three neuropterid orders and their grouping with Coleoptera.     

Cladistic analysis of Neuroptera and their systematic position within Neuropterida (Insecta: Holometabola: Neuropterida: Neuroptera)

A phylogenetic analysis of Neuroptera using thirty‐six predominantly morphological characters of adults and larvae is presented. This is the first computerized cladistic analysis at the ordinal level. It included nineteen species representing seventeen families of Neuroptera, three species representing two families (Sialidae and both subfamilies of Corydalidae) of Megaloptera, two species representing two families of Raphidioptera and as prime outgroup one species of a family of Coleoptera. Ten equally most parsimonious cladograms were found, of which one is selected and presented in detail. The results are discussed in light of recent results from mental phylogenetic cladograms. The suborders Nevrorthi‐ formia, Myrmeleontiformia and Hemerobiiformia received strong support, however Nevrorthiformia formed the adelphotaxon of Myrmeleontiformia + Hemerobiiformia (former sister group of Myrmeleontiformia only). In Myrmeleontiformia, the sister‐group relationships between Psychopsidae + Nemopteridae and Nymphidae + (Myrmeleontidae + Ascalaphidae) are corroborated. In Hemerobiiformia, Ithonidae + Polystoechotidae is confirmed as the sister group of the remaining families. Dilaridae + (Mantispidae + (Rhachiberothidae + Berothidae)), which has already been proposed, is confirmed. Chrysopidae + Osmylidae emerged as the sister group of a clade comprising Hemerobiidae + ((Coniopterygidae + Sisyridae) + (dilarid clade)). Despite the sister‐group relationship of Coniopterygidae + Sisyridae being only weakly supported, the position of Coniopterygidae within the higher Hemerobiiformia is corroborated. At the ordinal level, the analysis provided clear support for the hypothesis that Megaloptera + Neuroptera are sister groups, which upsets the conventional Megaloptera + Raphidioptera hypothesis.     

Morphological and molecular evidence converge upon a robust phylogeny of the megadiverse Holometabola

We present the largest morphological character set ever compiled for Holometabola. This was made possible through an optimized acquisition of data. Based on our analyses and recently published hypotheses based on molecular data, we discuss higher‐level phylogeny and evolutionary changes. We comment on the information content of different character systems and discuss the role of morphology in the age of phylogenomics. Microcomputer tomography in combination with other techniques proved highly efficient for acquiring and documenting morphological data. Detailed anatomical information (356 characters) is now available for 30 representatives of all holometabolan orders. A combination of traditional and novel techniques complemented each other and rapidly provided reliable data. In addition, our approach facilitates documenting the anatomy of model organisms. Our results show little congruence with studies based on rRNA, but confirm most clades retrieved in a recent study based on nuclear genes: Holometabola excluding Hymenoptera, Coleopterida (= Strepsiptera + Coleoptera), Neuropterida excl. Neuroptera, and Mecoptera. Mecopterida (= Antliophora + Amphiesmenoptera) was retrieved only in Bayesian analyses. All orders except Megaloptera are monophyletic. Problems in the analyses are caused by taxa with numerous autapomorphies and/or inapplicable character states due to the loss of major structures (such as wings). Different factors have contributed to the evolutionary success of various holometabolan lineages. It is likely that good flying performance, the ability to occupy different habitats as larvae and adults, parasitism, liquid feeding, and co‐evolution with flowering plants have played important roles. We argue that even in the “age of phylogenomics”, comparative morphology will still play a vital role. In addition, morphology is essential for reconstructing major evolutionary transformations at the phenotypic level, for testing evolutionary scenarios, and for placing fossil taxa.
© The Willi Hennig Society 2010.     

Phylogeny of the Neuropterida (Insecta: Holometabola)

The Neuropterida, with about 6500 known species — living fossils in a way — at the base of the Holometabola (as a sister group of the Coleoptera), comprise Raphidioptera (about 210 species, two families), Megaloptera (about 300 species, two families) and Neuroptera (6000 species, 17 families). Megaloptera + Neuroptera is argued vs. the traditional Raphidioptera + Megaloptera. Raphidioptera are undisputedly monophyletic. Monophyly of Megaloptera is the operational hypothesis, although occasionally questioned. Sucking tubes of the larvae are the most spectacular autapomorphy of Neuroptera. The construction of larval head capsules indicates three evolutionary lines: Nevrorthiformia, and Myrmeleontiformia + Hemerobiiformia. Traditional Myrmeleontiformia is Psychopsidae + (Nemopteridae + (Nymphidae + (Myrmeleontidae + Ascalaphidae))), the present approach is (Psychopsidae + Nemopteridae) + all other Myrmeleontiformia. Hemerobiiformia are based on the ‘maxillary head’ concept. The ithonid clade Ithonidae/Rapismatidae + Polystoechothidae and the dilarid clade Dilaridae + (Mantispidae + (Rhachiberothidae + Berothidae)) are based on robust criteria. Other relationships remain unclear: Hemerobiidae + Chrysopidae (on similarity) and the ‘early offshoot’ concept of coniopterygidae (on autapomorphies) should not be perpetuated. Chysopidae + Osmylidae and (Hemerobiidae + (Coniopterygidae + Sisyridae)) + dilarid clade are discussed. Aquatic larvae, regarded as independent apomorphies of megaloptera and neuropteran Nevrorthidae and Sisyridae for a long time, are re‐interpreted as a synapomorphy of Megaloptera + Neuroptera and thus plesiomorphic within these groups. Terrestrial larvae (with cryptonephry to solve osmotic problems) are consequently apomorphic. Aquatic Sisyridae with cryptonephry of a single malpighian tubule, is conflicting, but larvae may have become secondarily aquatic, after a terrestrial intermezzo.     

Phylogeny of the Neuropterida: a first molecular approach

Abstract. In a first molecular approach specially dedicated to examining the phylogeny of the Neuropterida, two nuclear and two mitochondrial genes were tested: 18S rRNA, translation elongation factor‐1α, cytochrome c oxidase subunit 3 and 16S rRNA. Molecular results are discussed in the light of a previous holomorphological cladistic analysis. The hypothesis of a sister‐group relationship Raphidioptera + (Neuroptera + Megaloptera) put forward in recent morphological analyses is supported by our data, which is in contrast to the traditional view (Raphidioptera + Megaloptera) + Neuroptera. Furthermore, the Nevrorthidae (constituting the suborder Nevrorthiformia) as a sister group of all other Neuroptera is confirmed. The disruption of the suborder Hemerobiiformia is the most conflicting result of the molecular analysis. Sisyridae and Osmylidae do not cluster within Hemerobiiformia, but represent two distinct and widely separated branches. The remaining Hemerobiiformia emerge as the sister group of the suborder Myrmeleontiformia, which is once more confirmed as monophyletic. Among the genes tested, cytochrome c oxidase subunit 3 proved to be most potent for resolving the phylogenetic relationships among Neuropterida. The nuclear gene for the ribosomal 18S rRNA is too conserved within the alignable regions, whereas the variable sections are too divergent to be applicable within this evolutionary time frame. The elongation factor‐1α gene proved to exist in more than one copy in Neuropterida, and thus is not applicable in the present state of knowledge. With respect to the mitochondrial sequences (cytochrome c oxidase subunit 3, 16S rRNA), saturation impedes the unambiguous resolution of deeper nodes. Apparently, due to early diversification of the heterogeneous Neuroptera, phylogenetic analysis of this group remains a challenge with respect to selection of the proper genes and mutatis mutandis the morphological approach.     

The morphology and evolution of the female postabdomen of Holometabola (Insecta)

In the present article homology issues, character evolution and phylogenetic implications related to the female postabdomen of the holometabolan insects are discussed, based on an earlier analysis of a comprehensive morphological data set. Hymenoptera, the sistergroup of the remaining Holometabola, are the only group where the females have retained a fully developed primary ovipositor of the lepismatid type. There are no characters of the female abdomen supporting a clade Coleopterida + Neuropterida. The invagination of the terminal segments is an autapomorphy of Coleoptera. The ovipositor is substantially modified in Raphidioptera and distinctly reduced in Megaloptera and Neuroptera. The entire female abdomen is extremely simplified in Strepsiptera. The postabdomen is tapering posteriorly in Mecopterida and retractile in a telescopic manner (oviscapt). The paired ventral sclerites of segments VIII and IX are preserved, but valvifers and valvulae are not distinguishable. In Amphiesmenoptera sclerotizations derived from the ventral appendages VIII are fused ventromedially, forming a solid plate, and the appendages IX are reduced. The terminal segments are fused and form a terminal unit which bears the genital opening subapically. The presence of two pairs of apophyses and the related protraction of the terminal unit by muscle force are additional autapomorphies, as is the fusion of the rectum with the posterior part of the genital chamber (cloaca). Antliophora are supported by the presence of a transverse muscle between the ventral sclerites of segment VIII. Secondary egg laying tubes have evolved independently within Boreidae (absent in Caurinus) and in Tipulomorpha. The loss of two muscle associated with the genital chamber are likely autapomorphies of Diptera. The secondary loss of the telescopic retractability of the postabdomen is one of many autapomorphies of Siphonaptera.     

Early Mecopterida and the systematic position of the Microptysmatidae (Insecta: Endopterygota)

In recent times many authors have regarded the Protomeropidae and Microptysmatidae - two essentially Permian groups - as either early trichopteran lineages or members of the stem-group of the Amphiesmenoptera (basically: Trichoptera+Lepidoptera). Actually none of these families possesses, in its ground plan, the most significant derived trait of the amphiesmenopteran forewing, namely a true ‘double-Y loop’ arrangement of the anal veins. Since ‘Carpenter’s organs’, small rounded structures in the costal area of the hindwing, are only known to occur in certain members of the Permochoristidae, Kaltanidae and Protomeropidae, these three families should belong to a fossil clade, which we ascribe to the Mecoptera, suborder Pistillifera sensu lato, mainly on account of a few venational features. Although we maintain the Microptysmatidae in the Mecopterida (=Panorpida, i.e. Amphiesmenoptera, Mecoptera, Diptera, and relatives), we propose to place this family in a separate order: the Permotrichoptera, n. status. Indeed, apparently, Microptysmatidae can be ascribed neither to the Amphiesmenoptera nor to the Antliophora (=Mecoptera-Diptera complex).     

Mecoptera is paraphyletic: multiple genes and phylogeny of Mecoptera and Siphonaptera

Phylogenetic relationships among members of the Mecoptera and Siphonaptera were inferred from DNA sequence data. Four loci (18S and 28S ribosomal DNA, cytochrome oxidase II and elongation factor-1α) were sequenced for 69 taxa selected to represent major flea and mecopteran lineages. Phylogenetic analyses of these data support a paraphyletic Mecoptera with two major lineages: Nannochoristidae + (Siphonaptera + Boreidae) and Meropidae + ((Choristidae + Apteropanorpidae) (Panorpidae + (Panorpidae + Bittacidae))). The flea family Ctenophthalmidae is paraphyletic, and the Ceratophylloidea is monophyletic. Morphological evidence is discussed which is congruent with the placement of Siphonaptera as sister group to Boreidae.