Insect Phylogenetics Research Articles

Orthoptera-specific target enrichment (OR-TE) probes resolve relationships over broad phylogenetic scales

Phylogenomic data are revolutionizing the field of insect phylogenetics. One of the most tenable and cost-effective methods of generating phylogenomic data is target enrichment, which has resulted in novel phylogenetic hypotheses and revealed new insights into insect evolution. Orthoptera is the most diverse insect order within polyneoptera and includes many evolutionarily and ecologically interesting species. Still, the order as a whole has lagged behind other major insect orders in terms of transitioning to phylogenomics. In this study, we developed an Orthoptera-specific target enrichment (OR-TE) probe set from 80 transcriptomes across Orthoptera. The probe set targets 1828 loci from genes exhibiting a wide range of evolutionary rates. The utility of this new probe set was validated by generating phylogenomic data from 36 orthopteran species that had not previously been subjected to phylogenomic studies. The OR-TE probe set captured an average of 1037 loci across the tested taxa, resolving relationships across broad phylogenetic scales. Our detailed documentation of the probe design and bioinformatics process is intended to facilitate the widespread adoption of this tool.

Diversity and composition of the bacterial communities associated with the Australian spittlebugs Bathyllus albicinctus and Philagra parva (Hemiptera: Aphrophoridae).

Spittlebugs and froghoppers (Hemiptera: Cercopoidea) are insects feeding on xylem, which potentially can cause significant economic damage worldwide by transmitting plant pathogenic bacteria such as Xylella fastidiosa. Australia and New Zealand are currently free from X. fastidiosa, but they are home to at least 45 native spittlebug species. Among these, the Australian natives Bathyllus albicinctus (Erichson, 1842) and Philagra parva (Donovan, 1805) are particularly widespread and can be found across southern and eastern Australia, with B. albicinctus also in New Zealand. The potential that both species might be capable of vectoring Xylella fastidiosa poses a substantial biosecurity risk if the bacterium were to invade these regions. In this study, we examined 87 spittlebug nymphs collected across 12 different host plant species, in five locations in Victoria, Australia. Our objective was to explore the factors influencing bacterial communities within and between these widespread spittlebug species, considering geographic location, insect phylogenetics, and host plant associations. We employed COI barcoding to assess insect genetic variation and 16S high throughput sequencing (HTS) metabarcoding to analyse bacterial microbiome diversity across various host plants. Our findings revealed minimal genetic divergence among spittlebug individuals in the same species, highlighting conspecificity despite conspicuous morphological divergences. On the other hand, we recorded significant variation in bacterial communities harboured by Bathyllus albicinctus nymphs feeding on different plants, even when these were collected within close proximity to each other. Therefore, host plant association appeared to shape the bacterial communities of spittlebugs more than insect genetic divergence or geographical location. These diverse bacterial communities could potentially facilitate transmission of plant pathogenic bacteria, underscoring the risk of widespread transmission among numerous plant hosts through insect-plant interactions. This study emphasizes the critical need to understand these complex interactions, particularly in the context of biosecurity.

Characterization of the complete mitochondrial genome of the longhorn beetle, Batocerahorsfieldi (Coleoptera, Cerambycidae) and its phylogenetic analysis with suitable longhorn beetles.

Mitochondrial genome analysis is an important tool for studying insect phylogenetics. The longhorn beetle, Batocerahorsfieldi, is a significant pest in timber, economic and protection forests. This study determined the mitochondrial genome of B.horsfieldi and compared it with the mitochondrial genomes of other Cerambycidae with the aim of exploring the phylogenetic status of the pest and the evolutionary relationships among some Cerambycidae subgroups. The complete mitochondrial genome of B.horsfieldi was sequenced by the Illumina HiSeq platform. The mitochondrial genome was aligned and compared with the existing mitochondrial genomes of Batoceralineolata and B.rubus in GenBank (MF521888, MW629558, OM161963, respectively). The secondary structure of transfer RNA (tRNA) was predicted using tRNAScan-SE server v.1.21 and MITOS WebSever. Thirteen protein-coding genes (PCGs) and two ribosomal RNA gene sequences of 21 longhorn beetles, including B.horsfieldi, plus two outgroups, Dryopsernesti (Dryopidae) and Heterocerusparallelus (Heteroceridae), were analyzed. The phylogenetic tree was constructed using maximum likelihood and Bayesian inference methods. In this study, we successfully obtained the complete mitochondrial genome of B.horsfieldi for the first time, which is 15 425 bp in length. It contains 37 genes and an A + T-rich region, arranged in the same order as the recognized ancestor of longhorn beetles. The genome of B.horsfieldi is composed of 33.12% A bases, 41.64% T bases, 12.08% C bases, and 13.16% G bases. The structure, nucleotide composition, and codon usage of the new mitochondrial genome are not significantly different from other longhorn mitochondrial genomes. Phylogenetic analyses revealed that Cerambycidae formed a highly supported single clade, and Vesperidae was either clustered with Cerambycidae or formed a separate clade. Interestingly, B.horsfieldi, B.rubus and B.lineolata were clustered with Monochamus and Anoplophora species in both analyses, with high node support. Additionally, the VesperidaeSpiniphilusspinicornis and Vesperussanzi and the 19 Cerambycidae species formed a sister clade in the Bayesian analysis. Our results have produced new complete mitogenomic data, which will provide information for future phylogenetic and taxonomic research, and provide a foundation for future relevant research.

Biodiversity of Drosophila sp. from the Natural Environment based on the Cytochrome Oxidase subunit 1 Gene

Research on insect phylogenetics is intricated by their similar morphology and significant genetic diversity. The cytochrome oxidase subunit 1 (CO1) gene is the most widely utilized mitochondrial DNA gene in the identification and study of animal molecular biodiversity. This study aims to identify and reconstruct the phylogeny of fruit flies from North Sulawesi using the cytochrome oxidase subunit 1 (CO1) gene. Fruit flies were obtained from 5 (five) areas in North Sulawesi, namely Siau (L1), North Minahasa (L2), Minahasa (L3), Southeast Minahasa (L4), and Bolaang Mongondow (L5). Fruit fly imago limbs were used as a tissue source for genomic DNA extraction. Genomic DNA extraction was carried out using the Quick-DNA™ Miniprep Kit manufacture protocol. The CO1 gene amplification was carried out by the PCR method, and the visualization of the amplicons was carried out by the 1.5% gel electrophoresis method. Nucleotide sequencing used a sequencing service at First BASE Singapore with a bidirectional sequencing method. CO1 gene amplification of each sample was visualized at 690 bp to 702 bp length. After analyzing the CO1 gene concession area using the MEGA XI program, it is found that Drosophila at L1 has 702 bp, L2 has 703 bp, L3 has 698 bp, L4 has 700 bp, and L5 has 697 bp. Based on alignment analysis using the BLAST method, it is found that the L1 fruit fly has a similarity rate of 99.29% (E=0.0) to Drosophila parapallidosa [MK659836.1]. The L2 fruit fly also has a similarity rate of 96.86% with Drosophila parapallidosa [MK659836.1]. The L3 fruit fly has a similarity level of 94.94% with Drosophila parapallidosa [MK659836.1]. The L4 fruit fly has a similarity rate of 94.43% with Drosophila parapallidosa [MK659836.1]. However, the L5 fruit fly shows a similarity rate of 96.86% with Drosophila rubida [EU493593.1]. The reconstruction results with the MEGA XI program using the Minimum Evolution model obtain two monophyletic groups where the fruit fly in Bolaang Mongondow is in a monophyletic group different from other fruit flies. The results of this study prove the variation in fruit fly species in North Sulawesi based on the identification of the CO1 gene.

How well do multispecies coalescent methods perform with mitochondrial genomic data? A case study of butterflies and moths (Insecta: Lepidoptera)

AbstractDespite the broad adoption of multispecies coalescent (MSC) methods for nuclear phylogenomics, they have yet to be applied to mitochondrial (mt) genomic data. As the potential sources of phylogenomic bias that MSC methods can address, such as incomplete lineage sorting, horizontal gene transfer and gene tree heterogeneity, have been found in mt genomic data, these approaches may improve the accuracy of phylogenetic inference with these data. In the present study, we examined the behaviour of MSC methods in reconstructing the phylogeny of Lepidoptera (butterflies and moths), a group for which mt genomic data are known to have strong resolving power. Traditional concatenation methods of analysing mt genomes for Lepidoptera infer topologies highly congruent with those generated from independent nuclear datasets. Individual mt gene trees performed poorly in recovering consensus relationships at deep levels (i.e. superfamily monophyly and inter‐relationships) and only moderately well for shallow relationships (i.e. within Papilionoidea). In contrast, MSC analyses with ASTRAL performed strongly with almost complete concordance to both concatenated mt genome analyses and independent nuclear analyses at both deep and shallow phylogenetic scales. Outgroup choice had a limited impact on tree accuracy, with even phylogenetically distant outgroups still resulting in topologies highly congruent with results from nuclear datasets, although MSC analyses appeared to be marginally more affected by outgroup choice than concatenation analyses. In general, discordance between concatenation and MSC analyses was found at nodes whose resolution varied between previous nuclear phylogenomic studies. The sensitivity of individual relationships to analysis with MSC vs concatenation can thus be used to test the robustness of phylogenetic hypotheses. For insect phylogenetics, MSC is a reliable inference method for mt genomic data and is thus a useful complement to the already widely used concatenation approaches.

Anchored enrichment dataset for true flies (order Diptera) reveals insights into the phylogeny of flower flies (family Syrphidae).

BackgroundAnchored hybrid enrichment is a form of next-generation sequencing that uses oligonucleotide probes to target conserved regions of the genome flanked by less conserved regions in order to acquire data useful for phylogenetic inference from a broad range of taxa. Once a probe kit is developed, anchored hybrid enrichment is superior to traditional PCR-based Sanger sequencing in terms of both the amount of genomic data that can be recovered and effective cost. Due to their incredibly diverse nature, importance as pollinators, and historical instability with regard to subfamilial and tribal classification, Syrphidae (flower flies or hoverflies) are an ideal candidate for anchored hybrid enrichment-based phylogenetics, especially since recent molecular phylogenies of the syrphids using only a few markers have resulted in highly unresolved topologies. Over 6200 syrphids are currently known and uncovering their phylogeny will help us to understand how these species have diversified, providing insight into an array of ecological processes, from the development of adult mimicry, the origin of adult migration, to pollination patterns and the evolution of larval resource utilization.ResultsWe present the first use of anchored hybrid enrichment in insect phylogenetics on a dataset containing 30 flower fly species from across all four subfamilies and 11 tribes out of 15. To produce a phylogenetic hypothesis, 559 loci were sampled to produce a final dataset containing 217,702 sites. We recovered a well resolved topology with bootstrap support values that were almost universally >95 %. The subfamily Eristalinae is recovered as paraphyletic, with the strongest support for this hypothesis to date. The ant predators in the Microdontinae are sister to all other syrphids. Syrphinae and Pipizinae are monophyletic and sister to each other. Larval predation on soft-bodied hemipterans evolved only once in this family.ConclusionsAnchored hybrid enrichment was successful in producing a robustly supported phylogenetic hypothesis for the syrphids. Subfamilial reconstruction is concordant with recent phylogenetic hypotheses, but with much higher support values. With the newly designed probe kit this analysis could be rapidly expanded with further sampling, opening the door to more comprehensive analyses targeting problem areas in syrphid phylogenetics and ecology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0714-0) contains supplementary material, which is available to authorized users.

Which mitochondrial gene (if any) is best for insect phylogenetics?

Despite the benefits of whole-genome analysis, in many instances — particularly in applied entomology — mitochondrial genes continue to offer a reliable, rapid and cheap alternative. To date, most studies using insect mitochondrial DNA have analysed single genes and none have rigorously attempted to assess which genes are best suited for studying particular insect orders; here, we address this issue and use the ability of individual genes to recover ordinal monophyly of various insect orders as a metric. Phylogenies were constructed for nine insect orders and three outgroups, using 12 protein-coding genes and two rRNA genes; 153 genomes were analysed and trees were constructed using PhyML. The importance of gene length and region within the mtDNA genome were explored using correlation and sliding window analyses. No single gene appeared to outperform all others. Accordingly, we recommend that mitochondrial-based reconstructions of insect relationships use a multi-gene approach, using as many genes and taxa as possible.

Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.

What is the phylogenetic signal limit from mitogenomes? The reconciliation between mitochondrial and nuclear data in the Insecta class phylogeny.

BackgroundEfforts to solve higher-level evolutionary relationships within the class Insecta by using mitochondrial genomic data are hindered due to fast sequence evolution of several groups, most notably Hymenoptera, Strepsiptera, Phthiraptera, Hemiptera and Thysanoptera. Accelerated rates of substitution on their sequences have been shown to have negative consequences in phylogenetic inference. In this study, we tested several methodological approaches to recover phylogenetic signal from whole mitochondrial genomes. As a model, we used two classical problems in insect phylogenetics: The relationships within Paraneoptera and within Holometabola. Moreover, we assessed the mitochondrial phylogenetic signal limits in the deeper Eumetabola dataset, and we studied the contribution of individual genes.ResultsLong-branch attraction (LBA) artefacts were detected in all the datasets. Methods using Bayesian inference outperformed maximum likelihood approaches, and LBA was avoided in Paraneoptera and Holometabola when using protein sequences and the site-heterogeneous mixture model CAT. The better performance of this method was evidenced by resulting topologies matching generally accepted hypotheses based on nuclear and/or morphological data, and was confirmed by cross-validation and simulation analyses. Using the CAT model, the order Strepsiptera was recovered as sister to Coleoptera for the first time using mitochondrial sequences, in agreement with recent results based on large nuclear and morphological datasets. Also the Hymenoptera-Mecopterida association was obtained, leaving Coleoptera and Strepsiptera as the basal groups of the holometabolan insects, which coincides with one of the two main competing hypotheses. For the Paraneroptera, the currently accepted non-monophyly of Homoptera was documented as a phylogenetic novelty for mitochondrial data. However, results were not satisfactory when exploring the entire Eumetabola, revealing the limits of the phylogenetic signal that can be extracted from Insecta mitogenomes. Based on the combined use of the five best topology-performing genes we obtained comparable results to whole mitogenomes, highlighting the important role of data quality.ConclusionWe show for the first time that mitogenomic data agrees with nuclear and morphological data for several of the most controversial insect evolutionary relationships, adding a new independent source of evidence to study relationships among insect orders. We propose that deeper divergences cannot be inferred with the current available methods due to sequence saturation and compositional bias inconsistencies. Our exploratory analysis indicates that the CAT model is the best dealing with LBA and it could be useful for other groups and datasets with similar phylogenetic difficulties.

Pisciforma, Setisura, and Furcatergalia (Order: Ephemeroptera) Are Not Monophyletic Based on 18S rDNA Sequences: A Response to Sun et al. (2006)

Sun et al. (2006) recently published an analysis of phylogenetic relationships of the major lineages of maysies (Ephemeroptera). Their study used partial 18S rDNA sequences (583 nucleotides), which were analyzed via parsimony to obtain a molecular phylogenetic hypothesis. Their study included 23 maysy species, representing 20 families. They aligned the DNA sequences via default settings in Clustal and reconstructed a tree by using parsimony in PAUP*. However, this tree was not presented in the article, nor have they made the topology or alignment available despite multiple requests. This molecular tree was compared with previous hypotheses based on morphological data to “test” (but see below) which morphology-based relationships were not signiÞcantly different from the molecular topology. Although molecular data can help shed light on many of the fundamental questions in insect phylogenetics, it is important to perform analyses correctly and to accurately report results in a way that allows subsequent validation. Sun et al. (2006) provided an adequate review of the major traditional hypotheses concerning higher level classiÞcation and relationships among maysies. Some of these hypotheses are based on cladistic analysis of morphology, but others are intuitive phylogenies that were not derived from any formal repeatable and objective analysis. These authors failed to report previously published molecular hypotheses that shed light on maysy phylogeny, many of which disagree with their results. For example, there have been previously published molecular studies investigating higher level relationships among maysies and relatives

Phylogenetic utility of dynamin and triose phosphate isomerase

Abstract Dynamin and triose phosphate isomerase (Tpi), two markers suggested as potentially useful for insect phylogenetics, have been sequenced for 12 scale insect taxa (Hemiptera: Coccoidea). Protocols are given for their amplification using conventional polymerase chain reaction, and their phylogenetic utility has been evaluated using several qualitative criteria and a modified version of the partition addition bootstrap alteration approach. Dynamin and Tpi fragments are easy to amplify and are evolving at a rate comparable with widely used nuclear ribosomal markers. The dynamin fragment has a single short intron. The Tpi fragment has three introns. One possible drawback to the use of the dynamin fragment as a phylogenetic marker is that its short length limits accurate modelling of complex substitution processes.

Evidence from mitochondrial genomics on interordinal relationships in insects

Mitochondrial (mt) genomes are the largest molecular data source for deep level insect phylogenetics that is also obtainable in a reasonable timeframe and for a reasonable cost. Over 100 insect mt genomes have been sequenced, representing 29 of the 30 orders, multiple suborders for a third of the orders, and many representatives of the mega-diverse orders. Genome rearrangements have been found in a third of the insect orders however these rearrangements diagnose groups of ordinal or lower rank. Sequence based phylogenetic hypotheses utilizing mt genomic data are a promising source of data on interordinal relationships however these studies are hampered by base compositional biases, unequal rates of nucleotide substitution across groups and other long-branch effects. Available data from the field of insect mitogenomic phylogenetics is reviewed and future directions in this research outlined.

A mitochondrial genome phylogeny of Diptera: whole genome sequence data accurately resolve relationships over broad timescales with high precision

Abstract Mitochondrial genomes provide a promising new tool for understanding deep‐level insect phylogenetics, but have yet to be evaluated for their ability to resolve intraordinal relationships. We tested the utility of mitochondrial genome data for the resolution of relationships within Diptera, the insect order for which the most data are available. We sequenced an additional three genomes, from a syrphid, nemestrinid and tabanid, representing three additional dipteran clades, ‘aschiza’, non‐heteroneuran muscomorpha and ‘basal brachyceran’, respectively. We assessed the influence of optimality criteria, gene inclusion/exclusion, data recoding and partitioning strategies on topology and nodal support within Diptera. Our consensus phylogeny of Diptera was largely consistent with previous phylogenetic hypotheses of the order, except that we did not recover a monophyletic Muscomorpha (Nesmestrinidae grouped with Tabanidae) or Acalyptratae (Drosophilidae grouped with Calliphoridae). The results were very robust to optimality criteria, as parsimony, likelihood and Bayesian approaches yielded very similar topologies, although nodal support varied. The addition of ribosomal and transfer RNA genes to the protein coding genes traditionally used in mitochondrial genome phylogenies improved the resolution and support, contrary to previous suggestions that these genes would evolve too quickly or prove too difficult to align to provide phylogenetic signal at deep nodes. Strategies to recode data, aimed at reducing homoplasy, resulted in a decrease in tree resolution and branch support. Bayesian analyses were highly sensitive to partitioning strategy: biologically realistic partitions into codon groups produced the best results. The implications of this study for dipteran systematics and the effective approaches to using mitochondrial genome data are discussed. Mitochondrial genomes resolve intraordinal relationships within Diptera accurately over very wide time ranges (1–200 million years ago) and genetic distances, suggesting that this may be an excellent data source for deep‐level studies within other, less studied, insect orders.

The current state of insect molecular systematics: a thriving Tower of Babel.

Insect molecular systematics has undergone remarkable recent growth. Advances in methods of data generation and analysis have led to the accumulation of large amounts of DNA sequence data from most major insect groups. In addition to reviewing theoretical and methodological advances, we have compiled information on the taxa and regions sequenced from all available phylogenetic studies of insects. It is evident that investigators have not usually coordinated their efforts. The genes and regions that have been sequenced differ substantially among studies and the whole of our efforts is thus little greater than the sum of its parts. The cytochrome oxidase I, 16S, 18S, and elongation factor-1 alpha genes have been widely used and are informative across a broad range of divergences in insects. We advocate their use as standards for insect phylogenetics. Insect molecular systematics has complemented and enhanced the value of morphological and ecological data, making substantial contributions to evolutionary biology in the process. A more coordinated approach focused on gathering homologous sequence data will greatly facilitate such efforts.