第931回千葉医学会整形外科例会

Background Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. Results Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. Conclusions Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques—Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species’ monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence

Ecological specialization and evolutionary reticulation in extant Hyaenidae

During the Miocene, Hyaenidae was a highly diverse family of Carnivora that has since been severely reduced to four species: the bone-cracking spotted, striped, and brown hyenas, and the specialized insectivorous aardwolf. Previous studies investigated the evolutionary histories of the spotted and brown hyenas, but little is known about the remaining two species. Moreover, the genomic underpinnings of scavenging and insectivory, defining traits of the extant species, remain elusive. Here, we generated an aardwolf genome and analyzed it together with the remaining three species to reveal their evolutionary relationships, genomic underpinnings of their scavenging and insectivorous lifestyles, and their respective genetic diversities and demographic histories. High levels of phylogenetic discordance suggest gene flow between the aardwolf lineage and the ancestral brown/striped hyena lineage. Genes related to immunity and digestion in the bone-cracking hyenas and craniofacial development in the aardwolf showed the strongest signals of selection, suggesting putative key adaptations to carrion and termite feeding, respectively. A family-wide expansion in olfactory receptor genes suggests that an acute sense of smell was a key early adaptation. Finally, we report very low levels of genetic diversity within the brown and striped hyenas despite no signs of inbreeding, putatively linked to their similarly slow decline in effective population size over the last 2 million years. High levels of genetic diversity and more stable population sizes through time are seen in the spotted hyena and aardwolf. Taken together, our findings highlight how ecological specialization can impact the evolutionary history, demographics, and adaptive genetic changes of an evolutionary lineage.The Swedish Research Council and the Knut and Alice Wallenberg Foundation, ERC consolidator grant, an Australian Research Council grant, “Clinician Scientist Programm, Medizinische Fakultat der Universitat Leipzig and Leibniz Competition Fund.https://academic.oup.com/mbeam2022Mammal Research InstituteZoology and Entomolog

Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).

BACKGROUND: Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. RESULTS: Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. CONCLUSIONS: Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects

genomische Ansätze zur Studie von ursprünglicher Herkunft und jüngster Diversifikation

The field of molecular phylogenetics has benefited greatly from the recent advances of modern sequencing approaches that allow for the generation of large genomics data sets Nonetheless a lack of suitable genetic markers and incomplete taxon sampling remain common problems in studies of evolutionary relatedness. Most phylogenetic studies are based on mitochondrial DNA (mtDNA) because information about the nuclear genome and strategies to develop new genetic markers are often not available. The use of appropriate genetic markers and the inclusion of both a geographically and phylogenetically comprehensive taxon sampling are required for adequately reconstructing evolutionary histories among different taxa. This is particularly true for studies of recent diversification. Mayflies (Ephemeroptera) are ancient freshwater insects, dating back more than 300 million years, but at the same time have been reported to successfully colonize and diversify on recently formed Atlantic oceanic islands. This combination of ancient origin and recent diversification makes them a fascinating study system for molecular phylogenetics. In the first part of my thesis, I investigated the recent diversification and colonization history of mayflies on 13 Atlantic oceanic islands of the Azores, Madeira, and the Canary Islands. The island fauna provides an ideal setting to understand how speciation and dispersal shape present-day freshwater biodiversity. A first step in the research was an assessment of the species richness of the island fauna, because current taxonomic estimates are uncertain. Earlier research on mayflies in Europe, Africa, Madagascar, and North America has repeatedly uncovered otherwise cryptic diversity based on analysis of mtDNA. This suggests that past morphological estimates may underestimate species richness, and that a comprehensive understanding of island biodiversity and its evolution requires molecular-based taxonomy. In order to assess the biodiversity and date the origin of the island fauna, I used phylogenetic analyses based on universal mtDNA markers combined with a generalized mixed Yule- coalescent (gmyc) approach. In total, I found twelve island-endemic species within three species groups (Baetis canariensis s.l., B. pseudorhodani s.l., and Cloeon dipterum s.l.) that have diversified within the last 15 million years in parallel throughout the island archipelagos. While intriguing, the results also pointed out the limitations of mtDNA markers for the study of recent diversification events. The study clearly demonstrated a need for the development of new genetic markers that provide increased phylogenetic signal in order to resolve the relationships of closely related species groups. To investigate relationships among newly diverged species, many polymorphisms are needed, and these should ideally be derived from multiple unlinked markers. Since mayflies are a non-model organism i.e. no reference genome is available, I generated a whole genome draft and used these data to design 59 nuclear DNA (nDNA) markers to establish a basis for inferring the evolutionary history of the C. dipterum s.l. species group. Prior to my work, there were only two suitably variable nuclear markers available, namely 28S ribosomal RNA (rRNA) and PEPCK. I applied species tree reconstruction methods using the multispecies coalescent approach, a phylogenetic framework developed within the last five years and suitable for large nDNA data sets. This model was used to overcome both the lack of phylogenetic signal and the potentially conflicting signal derived from gene tree incongruences. Using this approach, I delineated six different Cloeon species, three on the islands and three on the European mainland. The phylogeny resolved complex colonization routes on a large geographic scale (Macaronesian islands, the European mainland and North America). The three Macaronesian Cloeon species appear to have originated from European source populations and different species co-occur in the same freshwater habitats. The diversification within the C. dipterum s.l. species group was mainly promoted by allopatric speciation, whereby strong natural selection on ecological traits i.e. freshwater habitat adaptations and shifts in life history traits are presumed to play a key role. Future research identifying specific ecological, morphological, or behavioral traits, as well as genes that are under natural selection will be needed to understand the mechanistic basis of speciation. The second part of my thesis focused on evolution over much longer temporal scales, namely ancient origins of the extant winged insects. It remains one of the open questions in the field of insect evolution and systematics, and is thought to act as foundation to understand the evolution of flight as one of the most fascinating evolutionary processes, leading to the development of the most diverse and successful animal group. All winged insects (Pterygota) are placed into one of two groups, based on wing function. The inability to fold back the wings, as seen in the Ephemeroptera and Odonata (dragonflies and damselflies), is considered to be an ancestral condition and these orders are therefore referred to as the Palaeoptera (old wings). In contrast, all other orders are able to fold their wings and as such referred to as Neoptera (new wings). The phylogenetic position of the Palaeoptera within the winged insects is one of the unresolved problems in insect systematics and is thus referred to as the ‘Palaeoptera problem’. Morphological and molecular data have provided support for three competing hypotheses: (1) the Palaeoptera hypothesis, stating the Ephemeroptera + Odonata as sister group to the Neoptera, (2) the basal Ephemeroptera hypothesis (Ephemeroptera + (Odonata + Neoptera)), and (3) the basal Odonata hypothesis (Odonata + (Ephemeroptera + Neoptera)). To date molecular phylogenetic reconstructions have been inferred with a limited number of genes, mostly mitochondrial and ribosomal genes, or a limited number of mayfly taxa (i.e. phylogenomic studies). To resolve the ‘Palaeoptera problem’, I increased the taxon sampling to a total of 93 insect taxa, including 19 mayflies and I used as marker the protein-coding regions of the mitochondrial genomes (mitogenomes) in order to overcome the highly sensitive sequence alignment step. I applied two different phylogenetic tree reconstruction methods, namely Bayesian inference and maximum-likelihood. I identified taxa with unstable topological positions under the different statistical models, and tested the effects of excluding these taxa on the overall phylogenetic accuracy. First, I sequenced and annotated the mitogenomes of the three mayfly species Baetis rutilocylindratus, Cloeon dipterum, and Habrophlebiodes zijinensis. A comparison among mayfly mitogenomes showed that the gene content and gene orientation was conserved, including 37 protein- coding genes and low AT content. I found that the pruning of identified problematic taxa greatly improved the node support values of the tree reconstruction. Interestingly, also the chosen outgroup was identified as being a problematic taxon. The Bayesian inferences provided support for the basal Ephemeroptera hypothesis, whereas the maximum- likelihood phylogeny supported the basal Odondata hypothesis. The increased number of taxa, the exclusion of problematic taxa and the use of mitogenomes proved to be well suited to reconstruct ancient relationships. The contradicting results of the two phylogenetic methods support the growing evidences that phylogenetic methods based on Bayesian inference might be more appropriate for reconstructing ancient relationships. Thus, the relationships of the Palaeoptera remained unresolved but the results point out the need to investigate the suitability of currently used phylogenetic methods for resolving ancient splits. Taken together, my thesis presents one of the first genetically comprehensive studies on aquatic insects, combining molecular phylogenetic approaches based on a large set of nDNA markers and mitogenomes. I found that the increase of nDNA markers and the development of bioinformatics approaches for recently evolved species groups and the use of mitogenomes for ancient taxa are extremely important for understanding evolution because of their capacity to reconstruct well supported phylogenetic trees.Das Feld der molekularen Phylogenetik hat bei der Generierung großer Mengen genomischer Daten stark von den aktuellen Fortschritten moderner Sequenzierungstechnologien profitiert. Dennoch mangelt es oft an geeigneten genetischen Markern und ausreichender Taxon-Abdeckung. Die meisten phylogenetischen Studien basieren auf mitochondrieller DNA (mtDNA), weil genomische Information und Strategien zur Entwicklung neuer genetischer Marker oft nicht verfügbar sind. Die Verwendung angemessener, genetischer Marker und die Einbeziehung sowohl umfassender geografischer und phylogenetischer Taxon- Proben sind Voraussetzungen zur adäquaten Rekonstruktion evolutionärer Entwicklungen unterschiedlicher Abstammungslinien. Eintagsfliegen (Ephemeroptera) sind Süßwasserinsekten, deren Ursprung über 300 Millionen Jahre zurück liegt, welche sich erfolgreich spezialisieren und atlantische Inseln kolonisieren konnten. Diese Kombination aus ursprünglicher Herkunft und aktueller Diversifikation macht sie zu einem faszinierenden Studiensystem für die molekulare Phylogenetik. Im ersten Teil meiner Arbeit habe ich die aktuelle Diversifikation und Kolonisierungsgeschichte der Eintagsfliegen auf 13 atlantischen Inseln der Azoren, Madeira und der Kanarischen Inseln untersucht. Die Inselfauna bietet ideale Voraussetzungen, um zu verstehen, wie Speziation und Ausbreitung die heutige Süßwasser-Biodiversität geformt haben. Ein erstes Zwischenziel der Forschungsarbeit war die Erfassung der Artenvielfalt der Inselfauna, denn aktuelle taxonomische Einschätzungen sind unsicher und werden hinterfragt. Frühere Untersuchungen über Eintagsfliegen in Europa, Afrika, Madagaskar und Nordamerika, die auf Analysen mittels mtDNA basieren, haben wiederholt eine andernfalls kryptische Diversität aufgedeckt. Dies suggeriert, dass vorherige morphologische Einschätzungen möglicherweise die Artenvielfalt unterschätzten, und dass ein umfassendes Verständnis von Biodiversität und Evolution auf endemischen Inseln molekularbasierte, taxonomische Untersuchungen erfordern. Um die Biodiversität zu bewerten und die Entstehung der Inselfauna zu datieren, habe ich phylogenetische Analysen durchgeführt, basierend auf universeller mtDNA Markern in Kombination mit einem "generalized mixed Yule-coalescent" (gmyc) Ansatz. Insgesamt fand ich zwölf insel-endemische Spezies in drei Spezies-Gruppen (Baetis canariensis s.l., B. pseudorhodani s.l. und Cloeon dipterum s.l.), die sich innerhalb der letzten 15 Millionen Jahre parallel auf den Insel-Archipelen diversifiziert haben. Obwohl aufschlussreich, unterstreichen die Ergebnisse dennoch die Notwendigkeit der Entwicklung neuer genetischer Marker, die ausreichende, phylogenetische Informationen enthalten, um die Verwandtschaftsverhältnisse der identifizierten, nahverwandten Speziesgruppen zu rekonstruieren. Um die Verwandtschaft zwischen neu-divergierenden Spezies zu untersuchen, sind viele Polymorphismen nötig, und diese sollten idealerweise von einer Vielzahl unabhängigen Marker abstammen. Da Eintagsfliegen keinen Modellorganismus darstellen, weil kein Referenzgenom existiert, erstellte ich einen Ganzgenom- Draft. Dieses benutzte ich als Basis für 59 nukleäre DNA (nDNA) Marker zur Inferenz der Evolutionsgeschichte der C. dipterum s.l. Speziesgruppe). Vor meiner Arbeit gab es lediglich zwei geeignete nDNA Marker: 28S ribosomale RNA (rRNA) und PEPCK. Ich wendete Artbaum Rekonstruktionsmethoden mit einem "multispecies coalescent"-Modell an, ein Ansatz, der in den letzten 5 Jahren entwickelt wurde und zur Analyse von großen nDNA Daten geeignet ist. Dieses Modell wurde gewählt, um sowohl den Mangel an phylogenetischem Signal zu bewältigen als auch um das widersprüchliche phylogenetische Signal aufgrund von Genbaum-Inkongruenzen zu überwinden. Ich grenzte sechs verschiedene Cloeon-Spezies ab, drei auf den Inseln und drei auf dem europäischen Festland. Die Phylogenetik konnte Kolonisationsrouten im großen geographischen Maßstab (Makaronesische Inseln, europäisches Festland und Nordamerika) rekonstruieren. Dabei scheint es, dass die drei makaronesischen Cloeon Spezies von europäischen Ursprungspopulationen abstammen, und Speziespaare in den selben Süßwasserhabitaten vorkommen. Die Diversifizierung innerhalb der C. dipterum s.l. Speziesgruppe wurde wesentlich durch allopatrische Speziation angetrieben, wobei starke natürliche Selektion ökologischer Merkmale (d.h. Süßwasserhabitat-Anpassung) und Verschiebungen von Lebenszyklus-Merkmalen vermutlich eine Schlüsselrolle spielten. Zukünftige Forschung zur Identifikation spezifischer Gene, die unter natürlicher Selektion stehen, und vergleichende Studien einschließlich morphometrischer und ökologischer Analysen werden nötig sein, um die grundlegende Basis von Speziationsmustern zu verstehen. Der zweite Teil ist fokussiert auf den historischen Ursprung der heute lebenden geflügelten Insekten als eine der verbleibenden offenen Fragen in der Insektensystematik. Die Beantwortung bietet gleichsam das Fundament zum Verständnis der Evolution des Fluges als einem der faszinierendsten evolutionären Prozesse, der zur Entwicklung einer außerordentlich mannigfaltigen und erfolgreichen Tiergruppe geführt hat. Die geflügelten Insekten (Pterygota) werden in zwei Gruppen eingeteilt, basierend auf der Funktion ihrer Flügel. Die Unfähigkeit, ihre Flügel zurück zu falten, wie bei den Ephemeroptera und Odonata (Libellen) vorkommend, wird als ursprüngliche Eigenschaft betrachtet; sie werden daher als Palaeoptera (Altflügler) bezeichnet, im Gegensatz zu den Neoptera (Neuflügler), die jene Fähigkeit besitzen. Dabei ist die phylogenetische Stellung der Palaeoptera innerhalb der geflügelten Insekten eines der ungeklärten Probleme der Insektensystematik und wird daher als sogenanntes "Palaeoptera-Problem" bezeichnet. Morphologische und molekulare Daten haben Anhaltspunkte für drei konkurrierende Hypothesen geliefert: (1) die "Palaeoptera"-Hypothese, die Ephemeroptera + Odonata als Schwestergruppe zu den Neoptera beschreibt, (2) die "basale Ephemeroptera"-Hypothese (Ephemeroptera + (Odonata + Neoptera)) und (3) die "basale Odonata"-Hypothese (Odonata + (Ephemeroptera + Neoptera)). Bisher wurden molekular-phylogenetische Rekonstruktionen mit einer begrenzten Anzahl an Genen, hauptsächlich mitochondrielle und ribosomale Gene, oder mit einer geringen Anzahl an Taxa (d.h. phylogenomische Studien) durchgeführt. Bisher wurden molekular-phylogenetische Rekonstruktionen mit einer begrenzten Anzahl an Genen, hauptsächlich mitochondrielle und ribosomale Gene, oder mit einer geringen Anzahl an Taxa (d.h. phylogenomische Studien) durchgeführt. Um das "Palaeoptera-Problem" aufzuklären, intensivierte ich das Taxon-Sampling zu einer Gesamtzahl von 93 Insekten-Taxa, inklusive 19 Eintagsfliegen. Als Marker wählte ich mitochondrielle Genome (Mitogenome), um den kritischen Schritt des Sequenz- Alignment zu bewältigen. Ich wendete zwei verschiedene phylogenetische Baum- Rekonstruktionsmethoden an, und zwar die Bayesian Inference und die Maximum Likelihood Methoden. Ich identifizierte Taxa mit unbeständiger, topologischer Positionierung unter den verschiedenen, statistischen Modellen und untersuchte die Effekte des Entfernens dieser Taxa auf die insgesamte phylogenetische Präzision. Zuerst sequenzierte und annotierte ich die Mitogenome der drei Eintagsfliegen-Spezies Baetis rutilocylindratus, Cloeon dipterum und Habrophlebiodes zijinensis. Ein Vergleich mit bekannten Eintagsfliegen-Mitogenomen zeigte die Konservierung von Genumfang und - orientierung, mit 37 proteinkodierenden Genen und geringem AT-Gehalt. Ich fand heraus, dass das Entfernen der als problematisch identifizierten Taxa den Knoten-Support der Baum-Rekonstruktion stark verbessert. Interessanterweise wurde auch die gewählte Außengruppe (outgroup) als problematisches Taxon identifiziert. Die Bayesian Inference Methode unterstützte die "basale Ephemeroptera"-Hypothese, wohingegen die Maximum Likelihoood Phylogenie die "basale Odonata"-Hypothese unterstützte. Die höhere Anzahl an Taxa, das Ausschließen problematischer Taxa und die Verwendung von Mitogenomen erwies sich als gut geeignet, um ursprüngliche Verwandtschaftsverhältnisse zu rekonstruieren. Die widersprüchlichen Ergebnisse der beiden phylogenetischen Methoden verstärken weiter die zunehmenden Hinweise, dass auf Bayesian Inference basierende Methoden angemessener sind für die Rekonstruktion historischer Artverwandtschaften. Insgesamt können meine Ergebnisse das "Palaeoptera Problem" nicht lösen. Die Ergebnisse belegen aber das Potential und die Notwendigkeit, heute genutzte, phylogenetische Methoden zur Aufklärung ursprünglicher Aufspaltungen einzusetzen. Zusammenfassend stellt meine Arbeit eine der ersten, genetisch umfassenden Studien über aquatische Insekten dar, die molekulare, phylogenetische Ansätze basierend auf einer großen Anzahl kombinierter nDNA Marker und Mitogenomen einsetzt. Die Ergebnisse zeigen eindrücklich, dass die Hinzunahme von nDNA Markern und die Entwicklung von bioinformatischen Methoden innerhalb nah verwandter Arten sowie die Verwendung von Mitogenomen für alte Gruppen, aufgrund ihrer Fähigkeit zur Rekonstruierung von phylogenetischen Stammbäumen, sehr wichtig sind, um die Evolution zu verstehen

Three mitochondrial genomes of early-winged insects (Ephemeroptera: Baetidae and Leptophlebiidae)

Mayflies (Ephemeroptera) are a semi-aquatic insect order with comparatively few genomic data available despite their phylogenetic position at the root of the winged-insects and possession of ancestral traits. Here, we provide three mitochondrial genomes (mtgenomes) from representatives of the two most species-rich families, Baetis rutilocylindratus and Cloeon dipterum (Baetidae), and Habrophlebiodes zijinensis (Leptophlebiidae). All mtgenomes had a complete set of 13 protein-coding genes and a conserved orientation except for two inverted tRNAs in H. zijinensis. Phylogenetic reconstructions using 21 mayfly mtgenomes and representatives of seven additional orders recovered both Baetidae and Leptophlebiidae as well supported monophyletic clades, with Ephemeroptera as the sister-taxon to all other winged insects (i.e. Odonata and Neoptera)

16S *BEAST tree file

The mitochondrial 16S *BEAST output tre

mitochondrial MrBayes tree file

The concatenated mitochondrial (cox1 + 16S) MrBayes output tre

Data from: Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae)

Background Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. Results Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. Conclusions Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects

concatenated MrBayes input file

The concatenated (cox1 + 16S + PEPCK + EF-1α + wg) Mrbayes input file in nexus forma