A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages

A multi‐locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria‐encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear‐encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi‐locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.     

Molecular phylogeny of the Byrrhoidea–Buprestoidea complex (Coleoptera,Elateriformia)

The superfamilies of Elateriformia have been in a state of flux since their establishment. The recent classifications recognize Dascilloidea, Buprestoidea, Byrrhoidea and Elateroidea. The most problematic part of the elateriform phylogeny is the monophyly of Byrrhoidea and the relationships of its families. To investigate these issues, we merged more than 500 newly produced sequences of 18S rRNA, 28S rRNA, rrnL mtDNA and cox1 mtDNA for 140 elateriform taxa with data from GenBank. We assembled an all‐taxa (488 terminals) and a pruned data set, which included taxa with full fragment representation (251 terminals); both were aligned in various programs and analysed using maximum‐likelihood criterion and Bayesian inference. Most analyses recovered monophyletic superfamilies and broadly similar relationships; however, we obtained limited statistical support for the backbone of trees. Dascilloidea were sister to the remaining Elateriformia, and Elateroidea were sister to the clade of byrrhoid lineages including Buprestoidea. This clade mostly consisted of four major lineages, that is (i) Byrrhidae, (ii) Dryopidae + Lutrochidae, (iii) Buprestoidea (Schizopodidae sister to Buprestidae) and (iv) a clade formed by the remaining byrrhoid families. Buprestoidea and byrrhoid lineages, with the exception of Byrrhidae and Dryopidae + Lutrochidae, were usually merged into a single clade. Most byrrhoid families were recovered as monophyletic. Callirhipidae and Eulichadidae formed independent terminal lineages within the Byrrhoidea–Buprestoidea clade. Paraphyletic Limnichidae were found in a clade with Heteroceridae and often also with Chelonariidae. Psephenidae, represented by Eubriinae and Eubrianacinae, never formed a monophylum. Ptilodactylidae were monophyletic only when Paralichas (Cladotominae) was excluded. Elmidae regularly formed a clade with a bulk of Ptilodactylidae; however, elmid subfamilies (Elminae and Larainae) were not recovered. Despite the densest sampling of Byrrhoidea diversity up to date, the results are not statistically supported and resolved only a limited number of relationships. Furthermore, questions arose which should be considered in the future studies on byrrhoid phylogeny.     

Ctenocephalides felis and Ctenocephalides canis: introgressive hybridization?

In the present work, a comparative molecular study of Ctenocephalides felis and Ctenocephalides canis isolated from dogs (Canis lupus familiaris) from different geographical regions (Spain, Iran and South Africa) was carried out. We found morphological variations in C. felis that do not correspond with molecular differences. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S rRNA partial gene, and cytochrome c‐oxidase 1 (cox1) mtDNA partial gene sequences were determined to clarify the taxonomic status of these two species, and to assess interpopulation variation and interspecific sequence differences. In addition, a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony and Maximum Likelihood analysis was performed. The 18S rRNA partial gene fragment was useful neither to discriminate C. canis and C. felis nor to infer phylogenetic relationships at this level, whereas ITS1 and ITS2 assessed for specific determination in the genus Ctenocephalides. The cox1 mtDNA sequences of C. felis revealed three main haplotypes and we suggest that there has been introgression of C. canis cox1 mtDNA into C. felis by Wolbachia pipientis. Based on cox1 sequences, restriction mapping identified many endonucleases that could be used to delineate different haplotypes of C. felis and to differentiate C. felis and C. canis.     

The evolutionary history of the order Antipatharia (Cnidaria: Anthozoa: Hexacorallia) as inferred from mitochondrial and nuclear DNA: implications for black coral taxonomy and systematics

Although black corals inhabit all the world's oceans, they have been relatively understudied as ～185 of 247 species occur at depths > 50 m. Antipatharians have been included in several phylogenetic studies; however, sample sizes are small and taxonomic coverage minimal. Low levels of mitochondrial (mt) sequence divergence within Scleractinia and Octocorallia are assumed to apply to all anthozoans, although no formal study has been conducted on the order Antipatharia. To quantify genetic variation in the black coral mitogenome, we analysed DNA sequences of the two longest intergenic regions (IGRs) and cox3‐cox1 for 26 of 41 genera, representing all families and subfamilies. We also quantified divergence at the intraspecific level using six mtIGRs and their flanking protein‐coding genes and rRNA for 100+ colonies of Antipathes griggi. Utilizing sequence data from the two mtIGRs, cox3‐cox1, as well as nuclear 18S and 28S, we constructed the first multi‐locus phylogenies of the Antipatharia. Reconstructions revealed that species in the genus Stichopathes are split across two families, Sibopathes macrospina groups among North Atlantic Parantipathes (suggesting the actinopharynx and mesenteries were secondarily lost), and three families are polyphyletic. These and other results provide novel, independent insights into the evolutionary history of antipatharians and support placement of species into higher‐level groupings based on microscopic skeletal features rather than gross colony morphology. An illustrated key to the seven currently recognized families is also provided. © 2013 The Linnean Society of London     

Species‐specific mitochondrial gene rearrangements in biting midges and vector species identification

Abstract Partial mitochondrial gene sequences of 16 Culicoides species were determined to elucidate phylogenetic relations among species and to develop a molecular identification method for important virus vector species. In addition, the analysis found mitochondrial gene rearrangement in several species. Sequences of the mitochondrial genome region, cox1–trnL2–cox2 (1940–3785 bp) of 16 Culicoides and additional sequences were determined in some species, including whole mitochondrial genome sequences of Culicoides arakawae. Nine species showed common organization in this region, with three genes cox1–trnL2–cox2 and a small or no intergenic region (0–30 bp) between them. The other seven species showed translocation of tRNA and protein‐coding genes and/or insertion of AT‐rich non‐coding sequences (65–1846 bp) between the genes. The varied gene rearrangements among species within a genus is very rare for mitochondrial genome organization. Phylogenetic analyses based on the sequences of cox1+cox2 suggest a few clades among Japanese Culicoides species. No relationships between phylogenetic closeness and mitochondrial gene rearrangements were observed. Sequence data were used to establish a polymerase chain reaction tool to distinguish three important vector species from other Culicoides species, for which classification during larval stages is not advanced and identification is difficult.     

Evolutionary conservation,diversity and specificity of LTR‐retrotransposons in flowering plants: insights from genome‐wide analysis and multi‐specific comparison

The availability of complete or nearly complete genome sequences from several plant species permits detailed discovery and cross‐species comparison of transposable elements (TEs) at the whole genome level. We initially investigated 510 long terminal repeat‐retrotransposon (LTR‐RT) families comprising 32 370 elements in soybean (Glycine max (L.) Merr.). Approximately 87% of these elements were located in recombination‐suppressed pericentromeric regions, where the ratio (1.26) of solo LTRs to intact elements (S/I) is significantly lower than that of chromosome arms (1.62). Further analysis revealed a significant positive correlation between S/I and LTR sizes, indicating that larger LTRs facilitate solo LTR formation. Phylogenetic analysis revealed seven Copia and five Gypsy evolutionary lineages that were present before the divergence of eudicot and monocot species, but the scales and timeframes within which they proliferated vary dramatically across families, lineages and species, and notably, a Copia lineage has been lost in soybean. Analysis of the physical association of LTR‐RTs with centromere satellite repeats identified two putative centromere retrotransposon (CR) families of soybean, which were grouped into the CR (e.g. CRR and CRM) lineage found in grasses, indicating that the ‘functional specification’ of CR pre‐dates the bifurcation of eudicots and monocots. However, a number of families of the CR lineage are not concentrated in centromeres, suggesting that their CR roles may now be defunct. Our data also suggest that the envelope‐like genes in the putative Copia retrovirus‐like family are probably derived from the Gypsy retrovirus‐like lineage, and thus we propose the hypothesis of a single ancient origin of envelope‐like genes in flowering plants.     

mRNA EDITING AND SPLICED‐LEADER RNA TRANS‐SPLICING GROUPS OXYRRHIS,NOCTILUCA, HETEROCAPSA,AND AMPHIDINIUM AS BASAL LINEAGES OF DINOFLAGELLATES1

Identification of novel dinoflagellate taxa through molecular analysis is hindered by lack of well‐defined basal lineages. To address this issue, we attempted to reassess the phylogenetic status of Oxyrrhis marina Dujard. as well as other potentially basal taxa. The analysis was based on two newly established premises: (1) editing density of mitochondrial cob and cox1 mRNA increases from basal to later diverging lineages; (2) nuclear‐encoded mRNA in dinoflagellates is trans‐spliced to receive a 22 bp spliced leader (SL) at the 5′‐end. We analyzed these two genetic traits in O. marina, Noctiluca scintillans (Macartney) Kof. et Swezy, Heterocapsa triquetra (Ehrenb.) F. Stein, H. rotundata (Lohmann) Ge. Hansen, Amphidinium carterae Hulburt, and A. operculatum Clap. et J. Lachm. Surprisingly, no editing was detected in cob and cox1 mRNAs in these lineages, except for a small number of editing events in Amphidinium. However, nuclear‐encoded mRNAs in these species contained the SL sequence at the 5′‐end, indicative of SL RNA trans‐splicing. These findings, together with the recent cob‐cox1‐18S rRNA three‐gene phylogeny, suggest the following: (1) O. marina is a basal dinoflagellate; (2) Heterocapsa, Amphidinium, and Noctiluca likely are also early diverging lineages of dinoflagellates, and the position of Heterocapsa is inconsistent with literature and needs further investigation; and (3) the presence of the 22 bp SL and mitochondrial (mt) mRNA editing can be considered a landmark of dinoflagellate splits.     

Multilocus phylogeny and systematics of Iberian endemic Squalius (Actinopterygii,Leuciscidae)

Inferring the evolutionary history of a group of species can be challenging given the many factors involved. In recent years, the increased availability of sequences of multiple genes per species has spurred the development of new methodologies to analyse multilocus data sets. Two approaches that analyse such data are concatenated supermatrix and coalescent-based species-tree analyses. In this study, we used both of these methods to infer the phylogenetic relationships of Iberian species of the genus Squalius from one mitochondrial and six nuclear genes. We found mitonuclear discordance in the phylogenetic relationships of the group. According to the mitochondrial gene analysis, all species were recovered as monophyletic except S. pyrenaicus; besides, in the concatenated supermatrix analysis of the nuclear markers, this species resolved as polyphyletic with three divergent evolutionary lineages. The coalescent-based nuclear species-tree analysis rendered a well-resolved phylogeny compared with the supermatrix analysis, which was unable to discern between S. carolitertii, S. castellanus and one of the evolutionary lineages of S. pyrenaicus. This result is likely due to the better integration of population uncertainty in the coalescent approach. Furthermore, Bayesian multilocus species delimitation analyses based on a BPP approach strongly supported the distinct nuclear lineages as different species. Nevertheless, the supermatrix analysis was able to obtain well-supported relationships in the divergent lineages with low numbers of individuals. Our study highlights the usefulness of different analytical methodologies to obtain a more complete picture of the evolutionary history of taxa, especially when discordant patterns among genes are found.     

A molecular phylogeny of Glaphyridae (Coleoptera: Scarabaeoidea): evolution of pollination and association with ‘Poppy guild’ flowers

Among Scarabaeoidea, pollen feeding occurs in two major lineages, pleurostict Scarabaeidae and Glaphyridae. Here we infer for the first time the phylogeny of the scarabaeoid lineage Glaphyridae (Coleoptera) based on molecular data using partial gene sequences for 28S rRNA, cytochrome oxidase I (cox1) and 16S rRNA (rrnL) for 41 species. Based on the resulting tree topology, we inferred the timing of the origin of pollination and of their coevolution with different flower host taxa, with particular focus on the prominent red-coloured ‘poppy guild’ flowers. All genera of Glaphyridae that were sampled with multiple species were recovered as monophyletic. According to this analysis, the origin of Glaphyridae was around 140 Ma, while crown group divergence was dated to have occurred c. 112 Ma. Pollen feeding originated in Glaphyridae only once and much later than in other important pollinator groups, between 97 and 67 Ma. According to the reconstruction of ancestral feeding traits, Asteraceae (Cicharioidae) were the first hosts of Glaphyridae. Presumably, a further adaptive radiation was triggered by feeding on and pollination of red flowers (poppy guild) which arose at a later stage. It occurred for the first time between 30 and 40 Ma, whereby the clades that use red Ranunculaceae (Pygopleurus spp.) are older than clades using exclusively red Papaveraceae (Eulasia spp.) (25–30 Ma). The rather young age of red Ranunculaceae would imply that Pygopleurus species only subsequently used red Ranunculus species as flower hosts, and that a broad parallel host shift probably from red Papaver spp. to red Ranunculus asiaticus has occurred rather recently.     

Evolutionary geographic relationships among orthocladine chironomid midges from maritime Antarctic and sub‐Antarctic islands

Two species of chironomid midges are currently described in the genus Belgica Jacobs, 1900. Belgica antarctica Jacobs, 1900 is endemic to parts of the maritime Antarctic, and Belgica albipes (Séguy, 1965) is endemic to Îles Crozet, a sub‐Antarctic archipelago in the southern Indian Ocean. The relationships between these species, and their closest known relative (Eretmoptera murphyi Schaeffer, 1914, endemic to sub‐Antarctic South Georgia), were examined by sequencing DNA fragments for domains 1 and 3–5 of 28S ribosomal DNA and the mitochondrial gene cytochrome c oxidase 1 (cox1). The resulting molecular relationships between the three species were unclear, although their position within the subfamily Orthocladiinae of the Chironomidae, as generated by classical taxonomy, was confirmed. Our data reinforce earlier doubts, based on classical morphological approaches, that the generic placement of E. murphyi may be incorrect. Further analyses may indeed confirm that the species represents a third member of the genus Belgica. Genetic distance analysis, limited to the barcode region of cox1, indicated high differentiation between the two populations of B. albipes sampled (one obtained from the type location), suggesting the likely presence of cryptic species within this taxon, and that the taxonomic status of this species should be revised. Analysis of cox1 sequences in B. antarctica highlighted a strong genetic structure between populations obtained from 12 locations along the Antarctic Peninsula and the South Shetland Islands archipelago, with a number of distinctive mtDNA lineages inhabiting geographically distinct areas. In particular, we found four different haplogroups constituting geographically close but genetically distinct populations, a pattern likely to have been encouraged by the brachyptery of the members of this genus. We suggest that the different genetic patterns shown by each haplogroup have probably been determined by historical dispersal and colonization events during the Pleistocene, and are consistent with their survival in refuges in situ during successive glacial maxima over this period. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 258–274.     

Partial small subunit (18S) rRNA gene sequences from fish parasites of the families Lepocreadiidae and Fellodistomidae (Digenea) and their use in phylogenetic analyses

Digeneans of the families Lepocreadiidae and Fellodistomidae are common parasites of shallow and deep-water marine fishes. Worms recovered from these contrasting environments may be indistinguishable or very similar morphologically and little is known concerning their inter-relationships. To investigate the systematic status of these forms, partial sequences from a small subunit 18S rRNA gene were generated from four species of lepocreadiids (Lepidapedon elongatum, L. gaevskayae, L. rachion and Opechona bacillaris) and two fellodistomids (Fellodistomum fellis and Steringophorus agnotus). Approximately 1,637 bases were sequenced for L. elongatum, L. rachion and F. fellis. A comparison of these sequences identified regions which varied between the two families. As a result, two regions were identified as containing potential phylogenetically informative sites and sequenced from the remaining species. Analyses of these regions by both maximum parsimony and distance methods showed that nucleic acid sequence data can be used to resolve the two families. The results indicate that 18S rRNA sequences are likely to be more satisfactory at resolving higher levels of phylogeny, i.e. at the family level, than at the generic level due to the similarity shown between the sequences of congeners.     

Performance of distance-based DNA barcoding in the molecular identification of Primates

For comparative primatology proper recognition of basal taxa (i.e. species) is indispensable, and in this the choice of a suitable gene with high phylogenetic resolution is crucial. For the goals of species identification in animals, the cytochrome c oxidase subunit 1 (cox1) has been introduced as standard marker. Making use of the difference in intra- and interspecific genetic variation – the DNA barcoding gap – cox1 can be used as a fast and accurate marker for the identification of animal species. For the Order Primates we compare the performance of cox1 (166 sequences; 50 nominal species) in species-identification with that of two other mitochondrial markers, 16S ribosomal RNA (412 sequences, 92 species) and cytochrome b (cob: 547 sequences, 72 species). A wide gap exist between intra- and interspecific divergences for both cox1 and cob genes whereas this gap is less apparent for 16S, indicating that rRNA genes are less suitable for species delimitation in DNA barcoding. For those species where multiple sequences are available there are significant differences in the intraspecific genetic distances between different mitochondrial markers, without, however, showing a consistent pattern. We conclude that cox1 allows accurate differentiation of species and as such DNA barcoding may have an important role to play in comparative primatology.     

Phylogenetic community ecology of soil biodiversity using mitochondrial metagenomics

High‐throughput DNA methods hold great promise for the study of taxonomically intractable mesofauna of the soil. Here, we assess species diversity and community structure in a phylogenetic framework, by sequencing total DNA from bulk specimen samples and assembly of mitochondrial genomes. The combination of mitochondrial metagenomics and DNA barcode sequencing of 1494 specimens in 69 soil samples from three geographic regions in southern Iberia revealed >300 species of soil Coleoptera (beetles) from a broad spectrum of phylogenetic lineages. A set of 214 mitochondrial sequences longer than 3000 bp was generated and used to estimate a well‐supported phylogenetic tree of the order Coleoptera. Shorter sequences, including cox1 barcodes, were placed on this mitogenomic tree. Raw Illumina reads were mapped against all available sequences to test for species present in local samples. This approach simultaneously established the species richness, phylogenetic composition and community turnover at species and phylogenetic levels. We find a strong signature of vertical structuring in soil fauna that shows high local community differentiation between deep soil and superficial horizons at phylogenetic levels. Within the two vertical layers, turnover among regions was primarily at the tip (species) level and was stronger in the deep soil than leaf litter communities, pointing to layer‐mediated drivers determining species diversification, spatial structure and evolutionary assembly of soil communities. This integrated phylogenetic framework opens the application of phylogenetic community ecology to the mesofauna of the soil, among the most diverse and least well‐understood ecosystems, and will propel both theoretical and applied soil science.     

Presence of nuclear copies of mitochondrial origin (NUMTs) in two related species of stingless bee genus Melipona (Hymenoptera: Meliponini)

Transferred copies of mitochondrial DNA (mtDNA) into the nuclear genome (numts) have been reported in several Hymenoptera species, even at a high density in the honey bee nuclear genome. The accidental amplification of numts in phylogenetic studies focused on mtDNA highlights the importance of a correct determination of numts and their related mtDNA sequences. We report here the presence of numts derived from a mitochondrial rDNA 16S gene in the genome of the stingless bee species Melipona colimana and M. fasciata (tribe Meliponini) from Western Mexico. PCR products were cloned in both species obtaining thirty paralogous numts. Numts were identified by the presence of insertions and deletions and the disruption of the 16S secondary structure. Further phylogenetic analyses including alternative mitochondrial cox1 and nuclear ITS1 genes have revealed the presence of another numt (cox1) in the nuclear genome of these two species, and place both as sister lineages within the subgenus Michmelia. This is one of the first studies reporting the presence of numts in Meliponini species, and supports previous studies suggesting frequent transfer of mtDNA to the nuclear genome in Hymenoptera.     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Identification of net‐winged beetle larvae (Coleoptera: Lycidae) using three mtDNA fragments: a comparison of their utility

Abstract We investigated the effectiveness of short mitochondrial DNA fragments for the identification of lycid larvae. The rrnL, cox1 and nad5 mtDNA sequences from 17 specimens of immature stages of Lycidae and Lampyridae were combined with a previously published dataset of homologous fragments representing all major lineages of Lycidae and outgroups. Their relationships were analysed under parsimony criteria. We demonstrate that high‐density profiles are necessary for accurate identification of unknown samples to generic and tribal levels and that a multilocus approach is critical for obtaining reliable results. Although widely used, the cox1 mtDNA fragment showed the worst performance for identification at genus level when the query species was not present in the library. Stronger support for deeper branches came from rrnL mtDNA. The neotenic female larvae and male adult stages of Platerodrilus sp. and Macrolibnetis depressus Pic, 1938 were associated by mtDNA fragments. Based on the present identification, larvae of Dictyopterini (Dictyopterini gen. sp., Dictyoptera aurora Herbst, 1784), Sulabanus sp., Leptotrichalus sp. (Metriorrhynchini) and Macrolibnetis depressus Pic, 1938 (Platerodrilini) are described for the first time. Further species of Platycis Thomson, 1859, Plateros Bourgeois, 1979, Macrolycus Waterhouse, 1878, Cautires Waterhouse, 1879 and Lyponia Waterhouse, 1878 are identified by morphology and molecular markers. The data on larval morphology and their usefulness for classification are discussed.