Mitochondrial phylogenomics reveal the evolutionary, speciation and biogeographical history of fruit flies (Diptera: Tephritidae)

The Brassicaceae (Mustards, Cruciferae) are a cosmopolitan family comprising 370 genera and around 3660 species assigned to lately 50 tribes. The tribal system was originally based on solely homoplasious morphological character traits and reaches back to the early 19th century. De Candolle introduced the first tribal classification of the family nearly 200 years ago (1821) containing 21 partly still utilised classifications nowadays. Although labelling seems to be up to date, generic delimitations have been under permanent significant substitution and replacement. The tribes are arranged in three major monophyletic lineages and some additional small groups. The relationships within and between these lineages have not been resolved very clearly yet, as the Brassicaceae are characterised by frequently occurring hybridisation and polyploidisation events. This could be either the result of early and rapid radiation events or perhaps be the product of reticulate evolution, lediang to conflicting gene trees (KOCH & AL-SHEHBAZ 2009). This lack of resolution could in parts be resolved via the application of the nuclear encoded chalcone synthase gene (chs) on 39 of the Cruciferous tribes. Several small-scale tribal-specific duplication events, including age estimations, could be detected giving insight into the evolutionary history of this molecular single- or low-copy gene. Most definitely a tendency towards diploidisation is proven by purifying selection as well as accelerated synonymous substitution rates among this family resulting sooner or later in the reduction of preliminary multiplied chs loci. Supposedly, chs is single-copy in most diploid mustard taxa. The determination of orthologous and paralogous gene copies exposed to be of essential cause as it could be proven that yet functional but fluctuating DNA sequences demonstrate a huge impact on divergence time estimates as well as on any other extrapolation applying nucleotide or amino acid data. However, all crown age estimations calculated with diverse approaches resulted in reasonable output, dating the most recent common ancestor (tmrca) of the family to the Late Miocene or Oligocene. Adjustments of the DNA sequences resulted in a well-resolved thoroughgoing gene tree phylogeny facilitating established taxonomic as well as phylogenetic achievements and do, moreover, hint to further ambiguities which have to be clarified by the commitment of additional marker systems.

Discrete morphological data have been widely used to study species evolution, but the use of quantitative (or continuous) morphological characters is less common. Here, we implement a Bayesian method to estimate species divergence times using quantitative characters. Quantitative character evolution is modeled using Brownian diffusion with character correlation and character variation within populations. Through simulations, we demonstrate that ignoring the population variation (or population "noise") and the correlation among characters leads to biased estimates of divergence times and rate, especially if the correlation and population noise are high. We apply our new method to the analysis of quantitative characters (cranium landmarks) and molecular data from carnivoran mammals. Our results show that time estimates are affected by whether the correlations and population noise are accounted for or ignored in the analysis. The estimates are also affected by the type of data analyzed, with analyses of morphological characters only, molecular data only, or a combination of both; showing noticeable differences among the time estimates. Rate variation of morphological characters among the carnivoran species appears to be very high, with Bayesian model selection indicating that the independent-rates model fits the morphological data better than the autocorrelated-rates model. We suggest that using morphological continuous characters, together with molecular data, can bring a new perspective to the study of species evolution. Our new model is implemented in the MCMCtree computer program for Bayesian inference of divergence times.

Australasian orchid biogeography at continental scale: Molecular phylogenetic insights from the Sun Orchids (Thelymitra, Orchidaceae)

Molecular Phylogenetics of the Holly Leafminers (Diptera: Agromyzidae: Phytomyza): Species Limits, Speciation, and Dietary Specialization

Despite its complicated taxonomy, the genus Hesperis has not yet been subjected to any detailed molecular phylogenetic study and little is known about its monophyly, origin and biogeographical history, as well as the evolution of morphological characters. Here, we present for the first time comprehensive molecular analyses (nuclear ribosomal ITS sequences) of approximately 40 Hesperis species which represent the full range of morphological variation and the entire geographic distribution area of the genus. Based on our results, monophyly of the genus Hesperis has been proved. Moreover, our phylogenetic analysis shows that almost all traditionally defined sections are not monophyletic. NeighborNet network analysis was performed in order to visualize conflicting phylogenetic signals. The split-graph corresponds to the major clades represented in the consensus tree and revealed a hybridization signal in the evolutionary history of only a single Hesperis species. Divergence time estimations indicate that the origin of Hesperis (7.66–19.9 Mya) coincides with the expansion of grasslands and the closure of the proto-Mediterranean Sea in the middle Miocene. Therefore, diversification within Hesperis was affected by global climate changes, substantial tectonic rearrangements and the expansion of open vegetation systems in the Miocene, all of which had a great impact on the speciation history of the Irano-Turanian flora and fauna. Furthermore, two new species from West of Iran are described and illustrated here. They are readily distinguished from their closely related species by morphological characters such as stem height, leaf shape, pedicel, sepal and fruit indumentum, petal size, stigma shape and seed characters.

Swallowtail butterflies (Lepidoptera: Papilionidae) have been instrumental in understanding many foundational concepts in biology; despite this, a resolved and robust phylogeny of the group has been a major impediment to elucidating patterns and processes of their ecological and evolutionary history. This study presents a mitogenomic, time‐calibrated phylogeny for all swallowtail genera. A shotgun sequencing approach was performed to obtain 32 complete mitogenomes that were added to available butterfly mitogenomes, resulting in a dataset including 142 butterfly taxa (and four outgroups) representing all butterfly families. Phylogenetic analyses were carried out under maximum likelihood (ML) and Bayesian inferences (BIs) with alternative partitioning strategies and the mixture (CAT) model. To test competing hypotheses about the systematics of Papilionidae, such as the enigmatic position of Baronia brevicornis or the status of the tribe Teinopalpini, we estimated the marginal likelihood of alternative topologies and computed Bayes factors. Estimates of divergence times were assessed using a Bayesian relaxed‐clock approach calibrated with six fossils while testing for the number of clocks. The results recovered a well‐resolved and supported phylogeny confirming that Baroniinae is sister to Parnassiinae + Papilioninae, both recovered as monophyletic. It also laid the foundations for classification at tribe and genus level, suggesting that the tribe Teinopalpini only contains the genus Teinopalpus ( Meandrusa being sister to Papilio ). The number of molecular clocks in dating analyses had a significant impact on divergence times. A single clock recovered an origin of butterflies in the Cretaceous (98, 66–188 Ma) and also for swallowtails (85, 55–163 Ma), while partitioning the clocks yielded an origin of Papilionoidea in the very Late Cretaceous (71, 64–86 Ma), and all butterfly families originated in the aftermath of the Cretaceous–Paleogene extinction. These results challenge previous studies suggesting that butterflies appeared in the Early Cretaceous, 110 Ma, concurrently with the rise of angiosperms.

Paris kwantungensis was proposed initially as a monophyletic entity by Miau in 1982, but its taxonomic status remained contentious. We recently collected and studied this taxon at its original locality in Nujiang, Yunnan province, China. A molecular phylogenetic analysis and morphological observations of P. kwantungensis and related species support its recognition as a distinct species. Phylogenetic evidence shows that P. kwantungensis is a sister to P. polyphylla var. chinensis. Morphologically, P. kwantungensis is distinguished by its petals being nearly twice as long as its sepals (as opposed to petals shorter than sepals), reflexed sepals, and a globose capsule with numerous surface granules. This study provides a comprehensive species description of P. kwantungensis and compares it with related species in the genus Paris. The combination of morphological traits and molecular analyses, particularly of the internal transcribed spacer region sequences, clearly establishes P. kwantungensis as a unique and independent species within the genus.

Nematode species of the genus Contracaecum Railliet & Henry, 1912 have been reported around the world in many species of fish-eating birds and seals. Here, Contracaecum jorgei n. sp. is morphologically described using light and scanning electron microscopy for adults and fourth-stage larvae (L4) found in the bird Nannopterum brasilianus and third-stage larvae (L3) found in the freshwater fish Hoplias argentinensis, both from the province of Córdoba, Argentina. Additionally, sequences of cytochrome c oxidase subunit II were obtained from these specimens and molecular phylogenetic analysis was used to determine its relationships within the genus. The present species is distinguished from other species by the number and disposition of cephalic papillae; shape and size of the interlabia; length of the spicules; and number and arrangement of papillae in the posterior end of the male. Furthermore, in the molecular analyses, sequences obtained from adult L4 and L3 specimens of C. jorgei n. sp. were similar and grouped, forming an independent lineage, thus confirming it as a distinct species. Thus, morphological characteristics associated with molecular data support the proposal of a new species.

Masked gulls are believed, on the basis of morphological and recent molecular work, to be a monophyletic group within the Laridae, but relationships of species within the group are not well resolved. We used sequence data from four mitochondrial DNA genes (ND2, ND5, ATPase6, and ATPase 8) totaling >3,600 base pairs to clarify relationships among the species and test competing hypotheses about their origin and biogeography. Monophyly of the masked gulls was confirmed. We also found strong support for a clade including all Southern Hemisphere masked gulls as well as a lone Northern Hemisphere representative, the Black-headed Gull (Larus ridibundus). The Australasian taxa form a well-supported clade, in which the Black-billed Gull (L. bulleri) is sister to the Red-billed Gull (L. novaehollandiae scopulinus) of New Zealand and the Australian Silver Gull (L. n. novaehollandiae). Another well-supported clade includes the Black-headed Gull as sister to the South African Hartlaub's Gull (L. hartlaubii) and the Gray-hooded Gull (L. cirrocephalus) of Africa and South America. The strongly supported position of L. ridibundus within the “southern clade” suggests that it originated from a Southern Hemisphere ancestor and recently dispersed into the Northern Hemisphere. Estimates of divergence times using rate-smoothing methods are consistent with those from previous molecular work and suggest that (1) masked gulls diverged from other gulls <2 mya and (2) much of the radiation in the group occurred in the last 600,000 years. Preuve moléculaire de la radiation récente chez les mouettes “masquées” de l'hémisphère sud

Amanita curtipes and A. ponderosa are two Mediterranean taxa sharing a number of morphological features as well as their habitat. Their synonymy or variety status has been proposed by several authors. To clarify this taxonomic issue we have sequenced the D1-D2 domains of the 28S rRNA gene as well as the complete ITS1-5.8S-ITS2 region of several specimens of the two species collected in Spain, and aligned these sequences with those from other Amanita species. Molecular phylogenetic analysis based on the two regions revealed that A. ponderosa and A. curtipes are clearly distinct species. The distribution of Amanita species in the phylogenetic trees was consistent with the division of the genus in subgenera and sections as proposed by previous authors. Sequences of A. ponderosa and A. curtipes were grouped in a monophyletic cluster together with other species of the section Amidella. However, A. ponderosa was closer to other species in the section, such as A. peckiana and A. volvata, than to A. curtipes. We also indicate the macromorphological characters that are most useful to reliably distinguish A. ponderosa and A. curtipes.

Recovering the evolutionary history of crowned pigeons (Columbidae: Goura): Implications for the biogeography and conservation of New Guinean lowland birds

A combined morphological and molecular phylogenetic analysis was performed to evaluate the subfamily relationships of the parasitoid wasp family Ichneumonidae (Hymenoptera). Data were obtained by coding 135 morphological and 6 biological characters for 131 exemplar species of ichneumonids and 3 species of Braconidae (the latter as outgroups). The species of ichneumonids represent all of the 42 currently recognized subfamilies. In addition, molecular sequence data (cytochrome oxidase I “DNA barcoding” region, the D2 region of 28S rDNA and part of the F2 copy of elongation factor 1-alpha) were obtained from specimens of the same species that were coded for morphology (1309 base pairs total). The data were analyzed using parsimony and Bayesian analyses. The parsimony analysis using all data recovered previously recognized informal subfamily groupings (Pimpliformes, Ophioniformes, Ichneumoniformes), although the relationships of these three groups to each other differed from previous studies and some of the subfamily relationships within these groupings had not previously been suggested. Specifically, Ophioniformes was the sister group to (Ichneumoniformes + Pimplformes), and Labeninae was placed near Ichneumoniformes, not as sister group to all Ichneumonidae except Xoridinae. The parsimony analysis using only morphological characters was poorly resolved and did not recover any of the three informal subfamily groupings and very few of the relationships were similar to the total-evidence parsimony analysis. The molecular-only parsimony analysis and both Bayesian analyses (total-evidence and molecular-only) recovered Pimpliformes, a restricted Ichneumoniformes grouping and many of the subfamily groupings recovered in the total-evidence parsimony analysis. A comparison and discussion of the results obtained by each phylogenetic method and different data sets is provided. It is concluded that the molecular characters produced results that were relatively consistent with traditional, non-phylogenetic concepts of relationships between the ichneumonid subfamilies, whereas the morphological characters did not (at least not by themselves). The inclusion of both molecular and morphological characters using parsimony produced a topology that was the closest to the traditional subfamily relationships. The method of analysis did not greatly affect the overall topology for the molecular-only analyses, but there were differences between Bayesian and parsimony results for the total-evidence analyses (especially near the root of the tree). The Bayesian results did not seem to be altered very much by the inclusion of morphological characters, unlike in the parsimony analysis. In summary, the following groups were supported in multiple analyses regardless of the characters used or method of tree-building: Pimpliformes, higher Ophioniformes, higher Pimpliformes, (Claseinae + Pedunculinae), (Banchinae + Stilbopinae), Campopleginae, Cremastinae, Diplazontinae, Ichneumoninae (includingAlomya), Labeninae, Ophioninae, Poemeniinae, Rhyssinae, and Tersilochinae sensu stricto. Conversely, Ctenopelmatinae and Tryphoninae were never recovered without inclusion of other taxa. Based on the hypothesis of relationships obtained by the total-evidence parsimony analysis, the following formal taxonomic changes are proposed: Alomyinae Förster (=AlomyaPanzer andMegalomyaUchida) is once again synonymized with Ichneumoninae and is now considered a tribe (Alomyinirev. stat.); andNotostilbopsTownes is transferred from Stilbopinae to Banchinae, tribe Atrophini.

The Madras hedgehog (Paraechinus nudiventris) is an understudied species endemic to southern India, with limited genetic data available for understanding its evolutionary history. Here, we present the complete mitochondrial genome (17,232 bp), annotated and analysed in comparison with other Erinaceidae species. The mitogenome retains the typical vertebrate structure, comprising 13 protein-coding genes, 22 tRNAs, two rRNAs, and a non-coding control region, with an A + T rich nucleotide composition. Phylogenetic analyses using Bayesian and maximum-likelihood approaches confirmed the species’ placement within Paraechinus, with divergence from P. micropus estimated during the Pleistocene. Molecular evolutionary analyses revealed selective constraints on oxidative phosphorylation genes, with atp8 showing the highest variability. Our divergence time estimates, incorporating fossil calibrations, refine the evolutionary timeline of Erinaceidae, providing insights into the diversification of hedgehogs in South Asia. By making this annotated mitochondrial genome publicly available, we provide a foundational resource for future studies on the genetic diversity, phylogeography, and conservation of P. nudiventris and its relatives.

The first empirically supported phylogenetic hypothesis of relationships for the southern African endemic butterfly tribe Dirini is presented. Data derived from the morphology and ecology of the adults and immature stages (33 characters), and portions of the mitochondrial gene cytochrome oxidase I ( COI ) and the nuclear genes elongation factor 1α ( EF1α ) and wingless ( WG ) (totalling 1734 bp) were used to infer the relationships of the in‐group genera. An expanded molecular dataset using four genera from the Nymphalini and Satyrini to root the tree, and three genera from the Melanitini to test the monophyly of the tribe, was analysed using parsimony and Bayesian methods. Estimates of divergence times were calculated using two fossil calibrations under a relaxed molecular clock model. The monophyly of the tribe and each in‐group genus were strongly supported. Key findings are the sister‐taxon relationship of Aeropetes and Tarsocera , the apparent simultaneous or nearly simultaneous radiation of four lineages, the polyphyly of the species within Torynesis , and the apparent trans‐Atlantic dispersal of the ancestors of Manataria about 40 Ma. Estimates of divergence times indicate that the tribe has undergone two major radiations since its origin: the first when they left forest habitats in the mid–late Oligocene, shortly after the radiation of the grasses (Poaceae), and the second in the early‐middle Pliocene, coinciding with the aridification of southern Africa and the spread of conditions that favoured C 4 grasses over the C 3 grasses that dirine larvae prefer to eat. The high species diversity within the tribe appears to be partly a taxonomic artefact that may have resulted from the misinterpretation of climate‐related phenotypic variation within extant species. Relocation and breeding experiments should test this hypothesis.

Nassella is one of the largest genera in the tribe Stipeae with approximately 116 species, distributed from Canada to Uruguay and Argentina. Although there are strong morphological characters that distinguish this genus from the rest of the tribe, molecular phylogenetic analyses have shown that it is a polyphyletic genus closely related to Jarava and Amelichloa. Species boundaries for many Nassella taxa are somewhat diffuse and controversial, mainly due to morphological similarities with just a few morphological characters that differentiate one from the other. Previous chromosome counts in Nassella have shown the existence of a high chromosome number variability ranging from 28 to 60 suggesting that polyploidy might have played an important role in the evolution of the genus. The aim of this study was to assess the relationship between chromosome number variation and genome size focusing in 15 species of Nassella distributed in Uruguay and to establish the relationship among N. megapotamia (Spreng. ex Trin.) Barkworth, N. pauciciliata (Roseng. & Izag.) Barkworth, N. poeppigiana (Trin. & Rupr.) Barkworth and N. quinqueciliata (Roseng. & Izag.) Barkworth & Torres using cytogenetic, morphological, and molecular phylogenetic data. Chromosome numbers ranged from 2n = 26 to 2n = 46 confirming the high chromosome number variability reported for the tribe. This is the first record of nuclear DNA content for the genus and 2C-values range from 1.48 to 2.31 pg representing a 1.5-fold difference. There is no direct correspondence between increase in chromosome number and DNA content. This lack of correspondence could be due to DNA loss after polyploidization, or related to chromosomal rearrangements. Four Nassella species exhibited high cytological and morphological homogeneity. Our data based on chromosome number, molecular phylogenetic analysis, and the morphological approach including both optical and scanning electron microscopy strongly support that these four Nassella species (N. megapotamia, N. pauciciliata, N. poeppigiana, N. quinqueciliata) form a complex of species whose boundaries should be revised.