Single Mitochondrial Gene Research Articles

Mystery on the Bounty: The family-level status of Pacificana cockayni Hogg, 1904 (Araneae)

The family-level placement of the species Pacificana cockayni Hogg, 1904 (Araneae, Miturgidae) has been ambiguous for over a century, with the monotypic genus initially placed in Agelenidae, later transferred to Amaurobioidinae (Anyphaenidae), and presently in Miturgidae. A recent work describing the male and molecular data consisting of a single mitochondrial gene, cytochrome c oxidase subunit I, confirmed that the species is part of the marronoid clade; however, these data did not result in conclusive family-level placement. Here, we use low-coverage whole genome sequencing (lcWGS) combined with data from the Sequence Read Archive to infer a phylogeny from ultraconserved elements (UCEs) and six legacy Sanger loci. Indications of potential family placements from prior work and the topologies from this study support a transfer of Pacificana Hogg, 1904 to Cycloctenidae Simon, 1898 (new family placement).

Complete mitochondrial genome and phylogenetic analysis of Dollfustrema vaneyi (Trematoda: Bucephalidae)

BackgroundThe Bucephalidae is a large family of digenean trematodes but most previous analyses of its phylogenetic position have relied on a single mitochondrial gene or morphological features. Mitochondrial genomes (mitogenomes) remain unavailable for the entire family. To address this, we sequenced the complete mitogenome of Dollfustrema vaneyi and analyzed the phylogenetic relationships with other trematodes.ResultsThe circular genome of Dollfustrema vaneyi spanned 14,959 bp and contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a major non-coding region. We used concatenated amino acid and nucleotide sequences of all 36 genes for phylogenetic analyses, conducted using MrBayes, IQ-TREE and PhyloBayes. We identified pronounced topological instability across different analyses. The addition of recently sequenced two mitogenomes for the Aspidogastrea subclass along with the use of a site-heterogeneous model stabilized the topology, particularly the positions of Azygiidae and Bucephalidae. The stabilized results indicated that Azygiidae was the closest lineage to Bucephalidae in the available dataset, and together, they clustered at the base of the Plagiorchiida.ConclusionsOur study provides the first comprehensive description and annotation of the mitochondrial genome for the Bucephalidae family. The results indicate a close phylogenetic relationship between Azygiidae and Bucephalidae, and reveal their basal placement within the order Plagiorchiida. Furthermore, the inclusion of Aspidogastrea mitogenomes and the site-heterogeneous model significantly improved the topological stability. These data will provide key molecular resources for future taxonomic and phylogenetic studies of the family Bucephalidae.

The efficacy of single mitochondrial genes at reconciling the complete mitogenome phylogeny-a case study on dwarf chameleons.

Although genome-scale data generation is becoming more tractable for phylogenetics, there are large quantities of single gene fragment data in public repositories and such data are still being generated. We therefore investigated whether single mitochondrial genes are suitable proxies for phylogenetic reconstruction as compared to the application of full mitogenomes. With near complete taxon sampling for the southern African dwarf chameleons (Bradypodion), we estimated and compared phylogenies for the complete mitogenome with topologies generated from individual mitochondrial genes and various combinations of these genes. Our results show that the topologies produced by single genes (ND2, ND4, ND5, COI, and COIII) were analogous to the complete mitogenome, suggesting that these genes may be reliable markers for generating mitochondrial phylogenies in lieu of generating entire mitogenomes. In contrast, the short fragment of 16S commonly used in herpetological systematics, produced a topology quite dissimilar to the complete mitogenome and its concatenation with ND2 weakened the resolution of ND2. We therefore recommend the avoidance of this 16S fragment in future phylogenetic work.

Bilocus phylogenetic diversity and biogeographic distribution of isopods infesting Egyptian fishes

Phylogenetic diversity studies of parasitic isopods in Egyptian fisheries are very limited, based on a few records of the single mitochondrial gene, COI. Consequently, there has been no mapping study to monitor the phylogenetic diversity and biogeographic distribution of parasitic isopods among a wide range of Egyptian fisheries, leading to gaps in our understanding of the origin and expansion of these parasites. Hence, this study focused on validating the taxonomy and phylogenetic distribution of parasitic isopods using two marker genes, nuclear 18S rDNA and mitochondrial COI. Isopods were collected from seven fish species, Liza ramada, Solea aegyptiaca, Tilapia zillii, Pomadasys sp., Dicentrarchus sp., Atherinomorus lacunosus, and Nemipterus japonicus at four distant Egyptian lakes, Lake Qarun, Bitter Lakes, Lake Burullus, and Lake Manzala; and two marine localities, Red Sea and Suez Gulf, respectively. Sequence analysis revealed 10 and 19 haplotypes of 18S rDNA and COI, respectively. Lake Qarun showed the highest haplotype intra-diversity and is considered a pool of haplotypes with ancestors from other distant lakes. The Red Sea and Suez Gulf haplotypes showed genetic divergence from those recovered from lakes, implicating endemic isopod species. Some haplotypes had mutations in COI-deduced catalytic amino acids. The lake haplotypes belonged to Livoneca redmanii, suggesting a biogeographic expansion of this species among distant Egyptian fisheries. A new genus belonging to the family Cymothoidae was recorded in the Red Sea fish A. lacunosus. Elthusa vulgaris-like haplotypes were recorded in the Suez Gulf fish N. japonicus. Hence, this study clarified the expansion of isopod phylogenetic diversity among varieties of fish species from distant localities.

How many lineages are there of the stingrays genus Hypanus (Myliobatiformes: Dasyatidae) and why does it matter?

Abstract Stingrays genus Hypanus currently encompasses nine valid species from the Atlantic and Pacific oceans, though the phylogenetic relationships amongst some of them were based on a single mitochondrial gene and did not involve all putative Hypanus species. To address the monophyly of the genus and its relationship to other Dasyatinae genera, we sequenced the whole mitochondrial genomes of all species that supposedly belong to this genus and representatives of Dasyatinae, Neotrygoninae, and, as an outgroup, Fontitrygon (Urogymninae). Based on phylogenetic analyses, Hypanus is the sister-genus to all other Dasyatinae, and this subfamily is closely-related to Neotrygoninae within the family Dasyatidae. The species F. geijskesi is closely related to H. guttatus rather than to its congeners and should be allocated to Hypanus as H. geijskesi for the genus monophyly. After lineage delimitation analyses, we identified three species complexes composed of H. americanus, H. guttatus, and H. say, with two distinct evolutionary lineages within each, leaving the genus with 13 evolutionary units, of which six are currently under threat and only H. sabinus is of least concern. The urgency in identifying these new lineages lies in the fact they might already be under threat before being formally described.

Whole mitochondrial genomes reveal the relatedness of the browsing ant incursions in Australia

Global trade and human movements outspread animal species, for example ants, from their native habitats to new areas. This causes biosecurity concerns because an exotic ant might have adverse impacts on agriculture, the environment, or health; thus, incurring economic losses. The browsing ant, Lepisiota frauenfeldi, was first detected in 2013 at the Perth Airport. Since then, more discrete browsing ant infestations have been found in Perth and at the Ports of Darwin and Brisbane. This exotic ant has been deemed a significant pest in Australia and eradication efforts are underway. However, tackling this invasion requires an understanding of how these infestations are related. Are they same or separate or a combination of both? Here, we carried out a phylogenetic analysis using high-throughput sequencing data to determine their relatedness. Our results showed that each interstate incursion was separate. Furthermore, the Western Australian incursions might have two introductions. These findings are critical in devising effective biosecurity measures. However, we discovered that this information could only be revealed by analysing the whole mitochondrial genome; not by a single mitochondrial gene as typically done for species identification. Here, we sequenced 51 whole mitogenomes including three of its congener L. incisa for the first time, for tracing future infestations.

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

BackgroundMitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.ResultsWith the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80–90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.ConclusionsThis method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Genetic perturbation of mitochondrial function reveals functional role for specific mitonuclear genes, metabolites, and pathways that regulate lifespan

Altered mitochondrial function is tightly linked to lifespan regulation, but underlying mechanisms remain unclear. Here, we report the chronological and replicative lifespan variation across 167 yeast knock-out strains, each lacking a single nuclear-coded mitochondrial gene, including 144 genes with human homologs, many associated with diseases. We dissected the signatures of observed lifespan differences by analyzing profiles of each strain’s proteome, lipidome, and metabolome under fermentative and respiratory culture conditions, which correspond to the metabolic states of replicative and chronologically aging cells, respectively. Examination of the relationships among extended longevity phenotypes, protein, and metabolite levels revealed that although many of these nuclear-encoded mitochondrial genes carry out different functions, their inhibition attenuates a common mechanism that controls cytosolic ribosomal protein abundance, actin dynamics, and proteasome function to regulate lifespan. The principles of lifespan control learned through this work may be applicable to the regulation of lifespan in more complex organisms, since many aspects of mitochondrial function are highly conserved among eukaryotes.

The first complete mitochondrial genome of the genus Echinolaelaps reveals mitochondrial genome rearrangement type and evolution of Gamasida.

Echinolaelaps echidninus is a gamasid mite that is of medical and veterinary significance as parasites and vectors of disease agents, which can carry pathogens of zoonosis such as Rickettsia tsutsugamushi, Rickettsia Q fever, Rickettsia mooseri, Rickettsia pox pathogens, Corynebacterium pseudotuberculosis and Leptospira. At present, only single mitochondrial genes have been analysed for E. echidninus in the world, and no complete mitochondrial genome has been reported. However, information carried by a single gene is limited. Therefore, the complete mitochondrial genome of E. echidninus was determined for the first time by Illumina Hiseq X-Ten platform in this study. The mitochondrial genome is 15 736 bp in length and contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region of 1561 bp in length. Codon analysis of 13 protein-coding genes revealed that UUU, UUA, AUU, AUA and AAU were the most frequently used, while cox2 had the fastest evolutionary rate and cob the slowest. Comparative analysis of genome structure and breakpoint distances of the mitochondrial genomes of 23 species in 17 genera from 10 families of Gamasida deposited in GenBank revealed a novel gene arrangement type of the E. echidninus mitochondrial genome, and different degrees of rearrangement among different taxa of Gamasida. Phylogenetic analyses of Gamasida were performed using the maximum likelihood and Bayesian inference methods. Echinolaelaps echidninus was clustered with Dermanyssoidea and formed a more supportive sister group with Varroa destructor. This study provides novel insights into rearrangement patterns and evolution of mitochondrial genomes of Gamasida.

Characterisation and phylogenetic analysis of the complete mitogenome of the edible insect bamboo wormOmphisa fuscidentalis in Yunnan Province, China

The bamboo wormOmphisa fuscidentalis is a kind of popular edible insect in Indochina Peninsula, however, the genus affiliation of this insect is controversial. Previous taxonomic studies from China and Thailand using limited morphology and single mitochondrial gene evidence indicated that, those samples distributed in different areas from Asia could belong to different species in different genera. To understand its taxonomic status and phylogenetic relationship with related taxa, we first sequenced and characterised the complete mitochondrial genome (mitogenome) of the mothO. fuscidentalis’ mitogenome is 15,347 bp in size, and containing 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs, and a control region. Further, 23 of the 37 genes are encoded by the J-strand while 14 are encoded by the N-strand. Most of PCGs are initiated by ATN codons, but 3 of the 13 PCGs harboured the incomplete termination codon by T. In the gene order ofO. fuscidentalis, the ancestor of Lepidoptera sequencetrnI-trnQ-trnM morphed intotrnM-trnI-trnQ, as observed in other Pyraloidea species. A phylogenetic tree was then constructed based on the nucleotide sequences of mitochondrial 13 PCGs from 41 Pyraloidea species and two Tortricoidea species (outgroups) using Bayesian inference and maximum likelihood, which determined thatO. fuscidentalis belongs to Crambidae. Accordingly, the bamboo worm does not belong to the genusChilo in the subfamily Crambinae, or to the subfamily Spilomelinae. This study provides fundamental data to clarify the taxonomic status and better understand the biology of this edible insect.

Mitochondrial gene defects in Arabidopsis can broadly affect mitochondrial gene expression through copy number.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

Neuroradiological findings in a young patient bearing a new single mitochondrial gene mutation (case report)

BackgroundWe present the clinical, MRI and CT findings in a case of a new mitochondrial genome mutation (tRNA arginine gene), characterized by brain calcifications which are indicative of Kearns–Sayre syndrome (KSS). Some radiological features resembled those of Fahr’s disease (affecting the PDGFRB gene).Case presentationA 36-year-old male presented some typical clinical features of KSS, including onset before 20 years of age, pigmentary retinopathy, progressive external ophthalmoplegia and ptosis. However, the hallmark radiological finding of diffuse calcifications in the nuclear ganglia resembles some cases related to the PDGRFB mutation. Genetic investigation revealed a new mutation in the mitochondrial tRNA-arginine gene.ConclusionsBrain calcifications are a common feature of mitochondrial diseases, but little is known about their pathophysiology. Here, we describe radiological similarities between a new mitochondrial DNA mutation and other genetic conditions, which are related to Fahr’s disease. These similarities could provide new insights into putative genotype–phenotype correlations.

First evidence of White-footed Deer Mouse (<i>Peromyscus leucopus</i>) on mainland New Brunswick, Canada

White-footed Deer Mouse (Peromyscus leucopus) and the closely related, and more northerly ranging, Deer Mouse (Peromyscus maniculatus) broadly overlap in distribution and are often difficult to distinguish from each other. Based on molecular genetic data (cytochrome b gene), we report two new distribution records for P. leucopus for New Brunswick, Canada, the first mainland localities for this species in the province. Previous sampling of Peromyscus in New Brunswick may have overlooked the presence of P. leucopus, possibly because the specimens collected were all assumed to be P. maniculatus. However, current detection in New Brunswick may be part of a broader recent northward range expansion documented to be underway in P. leucopus. Although our use of a single mitochondrial gene to identify P. leucopus does not eliminate the possibility that the New Brunswick specimens are of hybrid origin, our results support the presence of P. leucopus in New Brunswick and suggest more detailed analyses will be required to determine the nature of any genetic interaction between P. leucopus and P. maniculatus in the province. Recognition of morphologically cryptic Peromyscus in southern New Brunswick also emphasizes the need to incorporate comprehensive methods to ensure the correct identification of specimens of this genus in Maritime Canada. We also note the potential implications of this discovery with respect to the incidence of Lyme disease in New Brunswick.

Complete mitochondrial genomes reveal robust phylogenetic signals and evidence of positive selection in horseshoe bats

BackgroundIn genus Rhinolophus, species in the Rhinolophus philippinensis and R. macrotis groups are unique because the horseshoe bats in these group have relatively low echolocation frequencies and flight speeds compared with other horseshoe bats with similar body size. The different characteristics among bat species suggest particular evolutionary processes may have occurred in this genus. To study the adaptive evidence in the mitochondrial genomes (mitogenomes) of rhinolophids, especially the mitogenomes of the species with low echolocation frequencies, we sequenced eight mitogenomes and used them for comparative studies of molecular phylogeny and adaptive evolution.ResultsPhylogenetic analysis using whole mitogenome sequences produced robust results and provided phylogenetic signals that were better than those obtained using single genes. The results supported the recent establishment of the separate macrotis group. The signals of adaptive evolution discovered in the Rhinolophus species were tested for some of the codons in two genes (ND2 and ND6) that encode NADH dehydrogenases in oxidative phosphorylation system complex I. These genes have a background of widespread purifying selection. Signals of relaxed purifying selection and positive selection were found in ND2 and ND6, respectively, based on codon models and physicochemical profiles of amino acid replacements. However, no pronounced overlap was found for non-synonymous sites in the mitogenomes of all the species with low echolocation frequencies. A signal of positive selection for ND5 was found in the branch-site model when R. philippinensis was set as the foreground branch.ConclusionsThe mitogenomes provided robust phylogenetic signals that were much more informative than the signals obtained using single mitochondrial genes. Two mitochondrial genes that encoding proteins in the oxidative phosphorylation system showed some evidence of adaptive evolution in genus Rhinolophus and the positive selection signals were tested for ND5 in R. philippinensis. These results indicate that mitochondrial protein-coding genes were targets of adaptive evolution during the evolution of Rhinolophus species, which might have contributed to a diverse range of acoustic adaptations in this genus.

Phylogenomic Analysis Reveals Dispersal-Driven Speciation and Divergence with Gene Flow in Lesser Sunda Flying Lizards (Genus Draco).

The Lesser Sunda Archipelago offers exceptional potential as a model system for studying the dynamics of dispersal-driven diversification. The geographic proximity of the islands suggests the possibility for successful dispersal, but this is countered by the permanence of the marine barriers and extreme intervening currents that are expected to hinder gene flow. Phylogenetic and species delimitation analyses of flying lizards (genus Draco) using single mitochondrial genes, complete mitochondrial genomes, and exome-capture data sets identified 9-11 deeply divergent lineages including single-island endemics, lineages that span multiple islands, and parapatrically distributed nonsister lineages on the larger islands. Population clustering and PCA confirmed these genetic boundaries with isolation-by-distance playing a role in some islands or island sets. While gdi estimates place most candidate species comparisons in the ambiguous zone, migration estimates suggest 9 or 10 species exist with nuclear introgression detected across some intra-island contact zones. Initial entry of Draco into the archipelago occurred at 5.5-7.5 Ma, with most inter-island colonization events having occurred between 1-3 Ma. Biogeographical model testing favors scenarios integrating geographic distance and historical island connectivity, including an initial stepping-stone dispersal process from the Greater Sunda Shelf through the Sunda Arc as far eastward as Lembata Island. However, rather than reaching the adjacent island of Pantar by dispersing over the 15-km wide Alor Strait, Draco ultimately reached Pantar (and much of the rest of the archipelago) by way of a circuitous route involving at least five overwater dispersal events. These findings suggest that historical geological and oceanographic conditions heavily influenced dispersal pathways and gene flow, which in turn drove species formation and shaped species boundaries. [Biogeography; genomics, Indonesia; lizards; phylogeography; reptiles].

Improving the phylogenetic resolution of Malaysian and Javan mahseer (Cyprinidae), Tor tambroides and Tor tambra: Whole mitogenomes sequencing, phylogeny and potential mitogenome markers.

The true mahseer (Tor spp.) is one of the highest valued fish in the world due to its high nutritional value and great unique taste. Nevertheless, its morphological characterization and single mitochondrial gene phylogeny in the past had yet to resolve the ambiguity in its taxonomical classification. In this study, we sequenced and assembled 11 complete mahseer mitogenomes collected from Java of Indonesia, Pahang and Terengganu of Peninsular Malaysia as well as Sarawak of East Malaysia. The mitogenome evolutionary relationships among closely related Tor spp. samples were investigated based on maximum likelihood phylogenetic tree construction. Compared to the commonly used COX1 gene fragment, the complete COX1, Cytb, ND2, ND4 and ND5 genes appear to be better phylogenetic markers for genetic differentiation at the population level. In addition, a total of six population-specific mitolineage haplotypes were identified among the mahseer samples analyzed, which this offers hints towards its taxonomical landscape.

New insights into the taxonomic status, distribution and natural history of De Witte’s Clicking Frog (Kassinula wittei Laurent, 1940)

Kassinula is a monotypic genus of small frog in the family Hyperoliidae, only represented by Kassinula wittei. This species morphologically resembles both Kassina Girard, 1853 and Afrixalus Laurent, 1944, and its taxonomic status has been debated for decades. It has previously been subsumed within Kassina, and is currently placed as a sister genus to Afrixalus, although it has not been included in any phylogenetic studies until now. This species is poorly represented in museum collections and is only known from fewer than 35 specimens from southern Democratic Republic of the Congo and adjacent Zambia. Newly collected material from central Angola, a range extension of 400–800 km west of previously known localities, allowed us to revisit the taxonomic placement of the genus with the aid of phylogenetic analysis and shed light on its geographic distribution, morphology and natural history. Although our phylogenetic analysis is limited to a single mitochondrial gene (16S), we place Kassinula in the subfamily Hyperoliinae and closely related to Afrixalus, with a high degree of confidence. Further phylogenetic studies are needed before formally synonymising Afrixalus with Kassinula.

Single nucleotide polymorphism‐based species phylogeny of greater fritillary butterflies (Lepidoptera: Nymphalidae: Speyeria) demonstrates widespread mitonuclear discordance

AbstractThe systematics of Speyeria butterflies has historically been complicated by intraspecific variability that has challenged efforts to delimit species and reconstruct phylogenies. Our study presents a phylogenetic comparison of genomic single nucleotide polymorphisms (SNPs) and mitochondrial COI gene sequences, with comprehensive taxon sampling that includes 15 species and 46 subspecies. Increased sampling of genetic markers and taxa improved the match between genetic clusters, obtained with both phylogenetic and cluster‐based analyses, and species previously detected using morphology, as well as showing two species delimitations that may need revision. We also recovered extensive mitonuclear discordance between genomic SNPs and the COI gene, confirming that mitochondrial DNA does not reliably identify several species at broad geographic scales. Resolution of the relationships of Speyeria species demonstrates the importance of sampling variation across the whole genome, and provides an essential foundation for understanding the evolution of this charismatic clade of North American butterflies.

Characterizing patterns of introgressive hybridization between two species ofTyrannusfollowing concurrent range expansion

Hybridization between two species typically occurs when allopatric or ecologically dissimilar species expand into areas of secondary contact or habitat transitions. However, as species' ranges shift rapidly in response to environmental change, the potential for novel types of ephemeral hybrid zones exists. Here, we document and describe the occurrence, prevalence and symmetry of a previously undocumented hybrid zone involving two sympatric, ecologically similar sister species that have been expanding their ranges eastward in the central USA: Scissor‐tailed FlycatchersTyrannus forficatusand Western KingbirdsTyrannus verticalis. We identified cases of hybridization and introgression using analyses of eight microsatellite DNA loci and a single mitochondrial gene. We also evaluated short‐term reproductive consequences of hybridization for both species by surveying for both species and potential hybrids at the periphery of their ranges in northeastern Arkansas and western Tennessee, USA. Genetic data revealed bi‐directional backcrossing at the periphery of the species’ ranges, including a cryptic hybrid. We also analysed DNA of putative ‘pure’ individuals from other parts of their ranges and detected two cryptic admixed individuals, suggesting backcrossed individuals from the periphery may be dispersing to breed or that hybridization events have occurred in the core. Finally, our results suggest that there are no short‐term reproductive consequences of hybridization for the two species. In total, hybrid zones that occur at the edges of expanding, sympatric ranges may be ephemeral; we suggest they play an important role in introgression and may have long‐standing impacts for sympatric sister species. Exploring the extent of hybrid zones such as this for other range‐expanding taxa will elucidate whether this type of hybrid zone is unique or a common occurrence.

Mitochondrial phylogeny, divergence history and high-altitude adaptation of grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) inhabiting the Tibetan Plateau

Grassland caterpillars (Lepidoptera: Lymantriinae: Gynaephora) are the most important pests in alpine meadows of the Tibetan Plateau (TP) and have well adapted to high-altitude environments. To further understand the evolutionary history and their adaptation to the TP, we newly determined seven complete TP Gynaephora mitogenomes. Compared to single genes, whole mitogenomes provided the best phylogenetic signals and obtained robust results, supporting the monophyly of the TP Gynaephora species and a phylogeny of Arctiinae + (Aganainae + Lymantriinae). Incongruent phylogenetic signals were found among single mitochondrial genes, none of which recovered the same phylogeny as the whole mitogenome. We identified six best-performing single genes using Shimodaira-Hasegawa tests and found that the combinations of rrnS and either cox1 or cox3 generated the same phylogeny as the whole mitogenome, indicating the phylogenetic potential of these three genes for future evolutionary studies of Gynaephora. The TP Gynaephora species were estimated to radiate on the TP during the Pliocene and Quaternary, supporting an association of the diversification and speciation of the TP Gynaephora species with the TP uplifts and associated climate changes during this time. Selection analyses revealed accelerated evolutionary rates of the mitochondrial protein-coding genes in the TP Gynaephora species, suggesting that they accumulated more nonsynonymous substitutions that may benefit their adaptation to high altitudes. Furthermore, signals of positive selection were detected in nad5 of two Gynaephora species with the highest altitude-distributions, indicating that this gene may contribute to Gynaephora’s adaptation to divergent altitudes. This study adds to the understanding of the TP Gynaephora evolutionary relationships and suggests a link between mitogenome evolution and ecological adaptation to high-altitude environments in grassland caterpillars.