Reza Zahiri

A new molecular phylogeny offers hope for a stable family level classification of the Noctuoidea (Lepidoptera)

Zahiri, R., Kitching, I. J., Lafontaine, J. D., Mutanen, M., Kaila, L., Holloway, J. D. & Wahlberg, N. (2010). A new molecular phylogeny offers hope for a stable family level classification of the Noctuoidea (Lepidoptera). —Zoologica Scripta, 40, 158–173.

Molecular phylogenetics of Erebidae (Lepidoptera, Noctuoidea)

As a step towards understanding the higher‐level phylogeny and evolutionary affinities of quadrifid noctuoid moths, we have undertaken the first large‐scale molecular phylogenetic analysis of the moth family Erebidae, including almost all subfamilies, as well as most tribes and subtribes. DNA sequence data for one mitochondrial gene (COI) and seven nuclear genes (EF‐1α, wingless, RpS5, IDH, MDH, GAPDH and CAD) were analysed for a total of 237 taxa, principally type genera of higher taxa. Data matrices (6407 bp in total) were analysed by parsimony with equal weighting and model‐based evolutionary methods (maximum likelihood), which revealed a well‐resolved skeleton phylogenetic hypothesis with 18 major lineages, which we treat here as subfamilies of Erebidae. We thus present a new phylogeny for Erebidae consisting of 18 moderate to strongly supported subfamilies: Scoliopteryginae, Rivulinae, Anobinae, Hypeninae, Lymantriinae, Pangraptinae, Herminiinae, Aganainae, Arctiinae, Calpinae, Hypocalinae, Eulepidotinae, Toxocampinae, Tinoliinae, Scolecocampinae, Hypenodinae, Boletobiinae and Erebinae. Where possible, each monophyletic lineage is diagnosed by autapomorphic morphological character states, and within each subfamily, monophyletic tribes and subtribes can be circumscribed, most of which can also be diagnosed by morphological apomorphies. All additional taxa sampled fell within one of the four previously recognized quadrifid families (mostly into Erebidae), which are now found to include two unusual monobasic taxa from New Guinea: Cocytiinae (now in Erebidae: Erebinae) and Eucocytiinae (now in Noctuidae: Pantheinae).

A Transcontinental Challenge — A Test of DNA Barcode Performance for 1,541 Species of Canadian Noctuoidea (Lepidoptera)

This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or “owlet” moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity.

Species-Level Para- and Polyphyly in DNA Barcode Gene Trees: Strong Operational Bias in European Lepidoptera

The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service “Monophylizer” to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric—conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.

Relationships among the basal lineages of Noctuidae (Lepidoptera, Noctuoidea) based on eight gene regions

In this study, we clarify the relationships between the basal lineages in the moth family Noctuidae using DNA sequence data from eight independent gene regions. Data matrices (6.4 kbp) are analysed using parsimony and model‐based methods (maximum likelihood and Bayesian inference). Our results support the family Noctuidae as a monophyletic group in which most subfamilies have hindwing vein M2 reduced or absent. Our phylogenetic hypothesis suggests that in the Noctuidae, the plesiomorphic condition is that in which vein M2 arises about one‐third of the way up the discocellular vein between the origins of M1 and M3, mainly parallel to M3, and is of thickness similar to vein M3. Most Noctuidae lineages possess an apomorphic (derived) condition in which hindwing vein M2 is markedly reduced or totally absent, so that the cubital vein appears to be three‐branched and these lineages are hence referred to as ‘trifine’. However, Noctuidae also include a number of lineages in which vein M2 is unreduced, or only slightly reduced, and these are more problematic for morphological association with the family Noctuidae. Our results also show that the subfamily Acronictinae is not closely related to Pantheinae, but instead shows a closer association with Amphipyrinae. Among the major lineages of Noctuidae, we postulate a general trend, with numerous exceptions, in larval host plants from woody plants in the basal groups towards herb feeding in derived groups. Similarly, the major radiations of monocot‐feeding groups within the family Noctuidae are in the higher trifines. The following taxonomic changes are proposed: Thiacidinae, syn. nov., a junior synonym of Pantheinae, and Dyopsinae, stat. nov., are reinstated as a subfamily.

Phylogeny and Evolution of Pharmacophagy in Tiger Moths (Lepidoptera: Erebidae: Arctiinae)

The focus of this study was to reconstruct a phylogenetic hypothesis for the moth subfamily Arctiinae (tiger moths, woolly bears) to investigate the evolution of larval and adult pharmacophagy of pyrrolizidine alkaloids (PAs) and the pathway to PA chemical specialization in Arctiinae. Pharmacophagy, collection of chemicals for non-nutritive purposes, is well documented in many species, including the model species Utetheisa ornatrix L. A total of 86 exemplar ingroup species representing tiger moth tribes and subtribes (68 genera) and nine outgroup species were selected. Ingroup species included the most species-rich generic groups to represent the diversity of host-plant associations and pharmacophagous behaviors found throughout Arctiinae. Up to nine genetic markers were sequenced: one mitochondrial (COI barcode region), one nuclear rRNA (D2 region, 28S rRNA), and seven nuclear protein-coding gene fragments: elongation factor 1-α protein, wingless, ribosomal protein subunit S5, carbamoylphosphate synthase domain regions, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and cytosolic malate dehydrogenase. A total of 6984 bp was obtained for most species. These data were analyzed using model-based phylogenetic methods: maximum likelihood (ML) and Bayesian inference (BI). Ancestral pharmacophagous behaviors and obligate PA associations were reconstructed using the resulting Bayes topology and Reconstructing Ancestral States in Phylogenies (RASP) software. Our results corroborate earlier studies on the evolution of adult pharmacophagous behaviors, suggesting that this behavior arose multiple times and is concentrated in the phaegopterine-euchromiine-ctenuchine clade (PEC). Our results suggest that PA specialization may have arisen early in the phylogeny of the subfamily and that facultative larval pharmacophagous behaviors are the derived condition.

Major lineages of Nolidae (Lepidoptera, Noctuoidea) elucidated by molecular phylogenetics

To elucidate the evolutionary relationships of the major lineages within the moth family Nolidae, we analysed a molecular dataset comprising eight independent gene regions (6.4 kbp), cytochrome c oxidase subunit I (COI) from the mitochondrial genome, and elongation factor‐1α (EF‐1α), ribosomal protein S5 (RpS5), carbamoylphosphate synthase domain protein (CAD), cytosolic malate dehydrogenase (MDH), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), isocitrate dehydrogenase (IDH) and wingless genes from the nuclear genome, using parsimony and model‐based evolutionary methods (maximum likelihood and Bayesian inference). Our analyses revealed a well‐resolved phylogenetic hypothesis, again recovering the six previously recognized families within Noctuoidea (i.e. Oenosandridae, Notodontidae, Euteliidae, Erebidae, Nolidae and Noctuidae), and monophyly of the quadrifid Noctuoidea (i.e. Euteliidae, Erebidae, Nolidae and Noctuidae). The family Nolidae is diagnosed and characterized by two synapomorphies from morphology: construction of a ridged boat‐shaped cocoon that bears a vertical exit slit at one end; and two other morphological character states: elongation of the forewing retinaculum into a bar‐like or digitate condition and possession of a postpiracular counter‐tympanal hood. We present a new phylogenetic hypothesis for Nolidae consisting of eight strongly supported subfamilies, two of which are erected here: Diphtherinae, Risobinae, Collomeninae subfam. nov., Beaninae subfam. nov., Eligminae, Westermanniinae, Nolinae and Chloephorinae. Where we are able, each monophyletic lineage is diagnosed by morphological autapomorphies and within each subfamily, monophyletic tribes and subtribes are circumscribed, most of which are also diagnosable by morphological apomorphies. We also describe two new taxa: Gelastocerini trib. nov. and Etannina subtrib. nov. The Neotropical subfamily Diphtherinae, here newly circumscribed, is considered to be the plesiomorphic sister lineage to the rest of Nolidae. Diphtherinae are characterized by loss of the proximal pair of metatibial spurs in males and by the presence of a frontal tubercle, which is presumably associated with a derived strategy of emergence from the cocoon.

Phylogenetic relationships of Acronictinae with discussion of the abdominal courtship brush in Noctuidae (Lepidoptera)

We present results of an eight‐gene molecular study of the subfamily Acronictinae and related Noctuidae. Amphipyrinae are recovered as sister to Acronictinae, but with weak support – not surprisingly, the content of the two subfamilies has often been mixed in classifications. Balsinae, previously placed near Acronictinae or within Noctuinae, is recovered within an unresolved polytomy of Cuculliinae, Eustrotiinae, Raphiinae and Dilobinae. Gerbathodes Warren, Moma Hübner and Nacna Fletcher are excluded from Acronictinae. Three genera recently transferred into the subfamily – Cerma Hübner, Chloronycta Schmidt & Anweiler and Comachara Franclemont – are confirmed as acronictines. Lophonycta Sugi (the type genus of Lophonyctinae) is returned to the Acronictinae. Sinocharis Püngeler, formerly considered to be Acontiinae or as the basis of its own subfamily Sinocharinae, is nested within early diverging Acronictinae genera. Both subfamilies are formally synonymized: i.e. Lophonyctinae syn.n. and Sinocharinae syn.n. Nine acronictine genus‐level taxa were found to nest within the nominate genus Acronicta Ochsenheimer: Eogena Guenée, Hyboma Hübner, Hylonycta Sugi, Jocheaera Hübner, Oxicesta Hübner, Simyra Ochsenheimer, Subacronicta Kozhanchikov, Triaena Hübner, and Viminia Chapman. Eogena, Oxicesta, and Simyra, currently treated as valid genera, nest within terminal clades of the genus Acronicta and are here subsumed within the genus: Eogena syn.n., Oxicesta syn.n. and Simyra syn.n. Four well‐supported species groups within Acronicta are identified: the alni clade, the leporina clade, the nervosa clade and the psi clade. While many previous treatments have stated explicitly that Acronictinae lack abdominal scent brushes, or excluded genera with brushes from the subfamily, we show that well‐developed brushes are present in three early diverging acronictine genera: Cerma, Lophonycta, and Sinocharis. We illustrate and describe the brushes of all three genera, and briefly review the taxonomic distribution of the anterior abdominal courtship brushes in Noctuidae, emphasizing the labile evolutionary distribution of these structures.

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America.

Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences.