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Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies

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Abstract

Discovering cryptic species in well-studied areas and taxonomic groups can have profound implications in understanding eco-evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa usually treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles, and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity. This article is protected by copyright. All rights reserved.

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... In recent years, several studies that DNA-barcoded the butterfly species occurring in the Mediterranean and in other regions of Europe highlighted the presence of deep mitochondrial lineages within generally accepted species, suggesting ongoing differentiation or even the presence of cryptic species (Dincă, Dapporto, & Vila, 2011;Dincă, Lukhtanov, & Talavera, 2011;Dincă et al., 2015;Dincă, Zakharov, Hebert, & Vila, 2011;Hausmann et al., 2011). Some of these cases have been subsequently studied in more detail by using integrative approaches and occasionally revealed that species diversity is higher than expected even in intensively studied areas such as Europe Dincă, Zakharov, et al., 2011;Habel et al., 2017;Hernández-Roldán et al., 2016). ...
... We sequenced mitochondrial (cytochrome c oxidase subunit I-COI) and nuclear DNA (internal transcribed spacer 2-ITS2) and analyzed phenotypic markers consisting of wing shape (presumably evolving under strong environmental selection) and genitalia structures (mostly evolving under sexual selection) (Habel et al., 2017;Hebert, Cywinska, & Ball, 2003;Hernández-Roldán et al., 2016;Nazari, Hagen, & Bozano, 2010;Zinetti et al., 2013). ...
... In a second phase, we tested the existence of a signature of diversification in genitalia and wing shape among the clades highlighted by COI (Hernández-Roldán et al., 2016). We carried a series of PLSDA with shape variables (PCs) and the hypothesis for clade attribution as grouping variable. ...
Article
Satyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide-scale biodiversity surveys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera. L. paramegaera and L. meadewaldoi were considerably differentiated for all the analyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest paraphyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts.
... The genus Spialia (Hesperiidae) is one of the butterfly groups in which interspecific gene flow could have played a key role in shaping its diversity. Hernández-Roldán et al. (2016) revised this butterfly genus and described Spialia rosae, a species endemic to the Iberian Peninsula. Based on morphology and on two nuclear DNA (nuDNA) markers (wingless and the internal transcribed spacer 2), S. rosae appears virtually indistinguishable from S. sertorius (Hernández-Roldán et al., 2018), a species distributed in western Europe (Tolman & Lewington, 2008;Tshikolovets, 2011). ...
... and is associated with mountain habitats, while S. sertorius feeds on Poterium spp. and can be found both in lowland and highland (Hernández-Roldán et al., 2016). ...
... Regarding Wolbachia infection rates, all S. rosae individuals studied by Hernández-Roldán et al. (2016) were infected by Wolbachia, while all the S. sertorius were uninfected. Wolbachia is a maternally inherited endosymbiont that may cause cytoplasmic incompatibility and thus constitute a partial reproductive barrier between infected and noninfected organisms (Hurst & Jiggins, 2005;Jiggins, 2003;Werren et al., 2008). ...
Article
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The importance of hybridization and introgression is well documented in the evolution of plants, however, in insects, their role is not fully understood. Given the fact that insects are the most diverse group of organisms, assessing the impact of reticulation events on their evolution may be key to comprehend the emergence of such remarkable diversity. Here, we used an insect model, the Spialia butterflies, to gather genomic evidence of hybridization as a promoter of novel diversity. By using the ddRADseq protocol, we explored the phylogenetic relationships between Spialia orbifer, S. rosae and S. sertorius, and documented two independent events of interspecific gene flow. Our data support that the Iberian endemism S. rosae probably received genetic material from S. orbifer in both mitochondrial and nuclear DNA, which could have contributed to a shift in the ecological preferences of S. rosae. We also show that admixture between S. sertorius and S. orbifer likely occurred in Italy. As a result, the admixed Sicilian populations of S. orbifer are differentiated from the rest of populations both genetically and morphologically, and display signatures of reproductive character displacement in the male genitalia. Additionally, our analyses indicated that genetic material from S. orbifer is present in S. sertorius along the Italian Peninsula. Our findings add to the view that hybridization is a pervasive phenomenon in nature and in butterflies in particular, with important consequences for evolution due to the emergence of novel phenotypes.
... A total of 36,883 records of 162 species were obtained during fieldwork and were complemented with 3,101 bibliographic records or from other authors for the period 1920-2009. Ten species are reported for the first time in the province of Palencia: Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016, Leptidea reali Reissinger, 1990, Euchloe tagis (Hübner, 1804 (Boisduval, 1828). The updated checklist of butterflies brings up to 166 the number of species known to occur in the Palencia province, including four species recorded before 2010: Euchloe belemia (Esper, 1800), Phengaris arion, Libythea celtis (Laicharting, 1782) and Melanargia ines. ...
... El listado de especies, localidad de observación, UTM, altitud, fecha y autor/es de las observaciones se relacionan a continuación. Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 Dos citas publicadas en el trabajo donde se describe la especie (Hernández-Roldán et al., 2016) Euchloe tagis (Hübner, 1804) La primera cita de esta especie en Palencia se debe a Michael Taymas, quien observó 11 ejemplares el 9/05/2015 en el Monte El Viejo de Palencia, en la cuadrícula 30T UM64. De nuevo Michael Taymas observa otros cinco ejemplares el día 10/05/2015, también en el Monte El Viejo, esta vez en la cuadrícula 30T UM74. ...
... El listado de especies, localidad de observación, UTM, altitud, fecha y autor/es de las observaciones se relacionan a continuación. Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 Dos citas publicadas en el trabajo donde se describe la especie (Hernández-Roldán et al., 2016) Euchloe tagis (Hübner, 1804) La primera cita de esta especie en Palencia se debe a Michael Taymas, quien observó 11 ejemplares el 9/05/2015 en el Monte El Viejo de Palencia, en la cuadrícula 30T UM64. De nuevo Michael Taymas observa otros cinco ejemplares el día 10/05/2015, también en el Monte El Viejo, esta vez en la cuadrícula 30T UM74. ...
... The need for more comprehensive analysis reflects the occurrence of introgression (e.g. Spialia sertorius-rosae 17 , Iphiclides podalirius-feisthamelii 18 , Melitaea phoebe-ornata 19 ), paraphyly (e.g. Lasiommata spp 20 .) and reproductive compatibility among deeply divergent lineages (e.g. ...
... Several cases have been revealed where Wolbachia infections are correlated with mtDNA genetic structure in European butterflies (e.g. 17,18,42,43 ). ...
... 62 ) with several recent cases documented in European butterflies, many first highlighted by differentiation in mtDNA (e.g. 17,34,63,64 ). ...
Article
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The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.
... mtDNA variants can determine different respiration performances (Toews, Mandic, Richards, & Irwin, 2014) and adaptive mutations are expected to rapidly spread in a population and even across Hernández-Roldán et al., 2016;Ritter et al., 2013). Due to the nonrecombinant transmission of mtDNA, genetic F I G U R E 4 The tree obtained by applying the Ward algorithm to the average G ST distances based on 226 species in the North-Western Mediterranean among a series of 36 areas (a). ...
... The analysis of intraspecific genetic divergence captured this main pattern, the determinants of which are still largely unexplained. The existence of distinct lineages from Iberia, Sicily and the Italian Peninsula is a very common pattern in butterfly phylogeography, while the diversification in the regions of the Alps and the Pyrenees is mainly determined by a different admixture of lineages Hernández-Roldán et al., 2016;Schmitt, 2007). ...
... This endeavour requires cheap, fast and reliable approaches to map and track changes of biological diversity over spatial and temporal scales, as well as linking them with species functional traits (Joly et al., 2014;Kress et al., 2015;Pacifici et al., 2015;Stein, Martinez, Stiles, Miller, & Zakharov, 2014). In the last decades, mitochondrial DNA has become increasingly prominent in biodiversity research, notably for phylogenetics, phylogeography and in the study of divergence processes (Avise, 2009;Burney & Brumfield, 2009;Cameron, 2014;Dincă et al., 2015;Galtier et al., 2009;Hernández-Roldán et al., 2016;Joly et al., 2014;Kress et al., 2015). ...
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Understanding the dynamics of biodiversity, including the spatial distribution of genetic diversity, is critical for predicting responses to environmental changes, as well as for effective conservation measures. This task requires tracking changes in biodiversity at large spatial scales and correlating with species functional traits. We provide three comprehensive resources to understand the determinants for mitochondrial DNA differentiation represented by i) 15,609 COI sequences and ii) 14 traits belonging to 307 butterfly species occurring in Western‐Central Europe and iii) the first multi‐locus phylogenetic tree of all European butterfly species. By applying phylogenetic regressions we show that mitochondrial DNA spatial differentiation (as measured with Gst, G'st, D and Dst) is negatively correlated with species traits determining dispersal capability and colonization ability. Thanks to the high spatial resolution of the COI data, we also provide the first zoogeographic regionalization maps based on intraspecific genetic variation. The overall pattern obtained by averaging the spatial differentiation of all Western‐Central European butterflies shows that the paradigm of long‐term glacial isolation followed by rapid pulses of post‐glacial expansion has been a pervasive phenomenon in European butterflies. The results and the extensive datasets we provide here constitute the basis for genetically‐informed conservation plans for a charismatic group in a continent where flying insects are under alarming decline. This article is protected by copyright. All rights reserved.
... L'any 2016 es va publicar el descobriment d'una nova espècie de papallona a la península Ibèrica, batejada com a Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016. Aquesta espècie havia passat desapercebuda per les semblances morfològiques amb el murri de la pimpinella, Spialia sertorius (Hoffmannsegg, 1804), ja que són virtualment idèntiques en la morfologia alar i en l'aparell genitàlic (Hernández-Roldán et al. 2016). ...
... L'any 2016 es va publicar el descobriment d'una nova espècie de papallona a la península Ibèrica, batejada com a Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016. Aquesta espècie havia passat desapercebuda per les semblances morfològiques amb el murri de la pimpinella, Spialia sertorius (Hoffmannsegg, 1804), ja que són virtualment idèntiques en la morfologia alar i en l'aparell genitàlic (Hernández-Roldán et al. 2016). Tanmateix, les dues espècies tenen diferències genètiques, bioquímiques i també ecològiques: mentre que S. sertorius és força generalista en les preferències d'hàbitat i les seves erugues s'alimenten de Poterium spp., S. rosae prefereix hàbitats de muntanya i utilitza Rosa spp. ...
... Tanmateix, les dues espècies tenen diferències genètiques, bioquímiques i també ecològiques: mentre que S. sertorius és força generalista en les preferències d'hàbitat i les seves erugues s'alimenten de Poterium spp., S. rosae prefereix hàbitats de muntanya i utilitza Rosa spp. com a planta nutrícia (Hernández-Roldán et al. 2016). ...
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Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 is an Iberian endemism found across most of the major mountain ranges and highlands of the Peninsula. However, its exact range is still unknown since it cannot be easily identified due to the morphological similarity to Spialia sertorius, a more widespread species. Despite this, they can be distinguished by the larval host plant and by using DNA data. In this study, we have sequenced the cytochrome c oxidase subunit I (COI) barcode region of several Spialia individuals across Catalonia in order to assess the presence of S. rosae. We additionally included all the available COI sequences and analysed the mitochondrial genetic structure of this species. We show that S. rosae only has three distinct haplotypes separated by one mutation, as well as a low haplotype diversity (Hd) and nucleotide diversity (π). Thus, for this genomic region, it is one of the most homogeneous butterfly species of Europe. DNA-based identification revealed the presence of two populations in Catalonia, one in els Ports mountain range and another in the Pre-Pyrenees. These two populations display two distinct haplotypes separated by one mutation, which suggests that they have independent phylogeographic histories and that they are currently not connected. Overall, we encourage to maintain prospection efforts in order to assess its abundance and full distribution, a key step prior to evaluate its conservation status in Catalonia. Finally, we propose the common Catalan name of ‘murri dels rosers’ to refer to this species.
... To resolve some of these confusions, we set out to investigate a phylogenetic group consisting of four closely related and exclusively Old World genera Spialia, Gomalia Moore, 1879, Carcharodus and Muschampia that we unite in a subtribe Carcharodina Verity, 1940. These genera have received significant attention in literature (Cock, 2016;Coutsis, 2016;de Jong, 1974ade Jong, , 1974bde Jong, , 1977de Jong, , 1978Devyatkin, 1991;Evans, 1937Evans, , 1949Zhdanko, 1992), including some more recent developments based on molecular studies and description of new cryptic species (Hernandez-Roldan et al., 2016). The four genera have been distinguished largely by appearance of adults and their wing patters and shapes (Evans, 1949). ...
... Other plant families have also been used by some species, e.g. Rosaceae, Convolvulaceae, Tiliaceae, Bignoniaceae, and Sterculiaceae (Henning, Henning, Joannou, & Woodhall, 1997;Hernandez-Roldan et al., 2016;Larsen, 1991;Tuzov, 1997). Immature stages of Carcharodina are similar in appearance among species, but some species-specific characters have been discussed by Cock (2016). ...
Article
We obtained whole genome shotgun sequences and phylogenetically analyzed protein-coding regions of representative skipper butterflies from the genus Carcharodus Hübner, [1819] and its close relatives. Type species of all available genus-group names were sequenced. We find that species attributed to four exclusively Old World genera (Spialia Swinhoe, 1912, Gomalia Moore, 1879, Carcharodus Hübner, [1819] and Muschampia Tutt, 1906) form a monophyletic group that we call a subtribe Carcharodina Verity, 1940. In the phylogenetic trees built from various genomic regions, these species form 7 (not 4) groups that we treat as genera. We find that Muschampia Tutt, 1906 is not monophyletic, and the 5th group is formed by currently monotypic genus Favria Tutt, 1906 new status (type species Hesperia cribrellum Eversmann, 1841), which is sister to Gomalia. The 6th and 7th groups are composed of mostly African species presently placed in Spialia. These groups do not have names and are described here as Ernsta Grishin, gen. n. (type species Pyrgus colotes Druce, 1875) and Agyllia Grishin, gen. n. (type species Pyrgus agylla Trimen, 1889). Two subgroups are recognized in Ernsta: the nominal subgenus and a new one: Delaga Grishin, subgen. n. (type species Pyrgus delagoae Trimen, 1898). Next, we observe that Carcharodus is not monophyletic, and species formerly placed in subgenera Reverdinus Ragusa, 1919 and Lavatheria Verity, 1940 are here transferred to Muschampia. Furthermore, due to differences in male genitalia or DNA sequences, we reinstate Gomalia albofasciata Moore, 1879 and Gomalia jeanneli (Picard, 1949) as species, not subspecies or synonyms of Gomalia elma (Trimen, 1862), and Spialia bifida (Higgins, 1924) as a species, not subspecies of Spialia zebra (Butler, 1888). Sequencing of the type specimens reveals 2.2-3.2% difference in COI barcodes, the evidence that combined with wing pattern differences suggests a new status of a species for Spialia lugens (Staudinger, 1886) and Spialia carnea (Reverdin, 1927), formerly subspecies of Spialia orbifer (Hübner, [1823]).
... Assessing the status of potential cryptic species can be a challenging task. It is often recommended to combine morphological, ecological and genetic data, including multiple independent molecular markers of mitochondrial and nuclear DNA (e.g., Hernández-Roldán et al., 2016;Von Helversen et al., 2001). However, multi-copy nuclear markers such as ITS2 may not always display a perfectly concerted evolution (Shapoval & Lukhtanov, 2015) and single-copy nuclear genes with sufficient variability have historically been scarce. ...
... In butterflies from Pakistan, 3.7% of the species analysed exhibited a minimum distance to the nearest neighbour species <3% (Ashfaq et al., 2013). In Hesperiidae, Hebert, Penton, Burns, Janzen, and Hallwachs (2004) proposed 10 new species derived from Astraptes fulgerator with an average divergence of 2.76% between them and similarly divergent mitochondrial lineages have been shown to represent actual cryptic species in the genus Spialia (Hernández-Roldán et al., 2016). ...
Article
Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double-digest restriction-site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex-biased asymmetries, NUMTs, natural selection, introgression or Wolbachia-mediated genetic sweeps. We suggest that this mito-nuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events. This article is protected by copyright. All rights reserved.
... Both species are morphologically similar as adults, but their larvae feed on different plant genera. Some of the samples in this study were previously used by Hernández-Roldán et al. (2016) and were identified based on the COI mitochondrial DNA marker. The map representing the geographic distribution of S. rosae, in 10 × 10 km squares (MGRS system), was drawn with the automatic cartography software QGIS 2.6. ...
... The white spot in E8 is displaced towards the base of the wing, giving the series a curved shape. The submarginal series has larger spots in E4, E5 and E7 (according to the original description in Hernández-Roldán et al. 2016). Fig. 70 shows the known distribution of S. rosae, which is restricted to the Cantabrian Mountains, the Pyrenees and the Iberian, Central and Betic Mountain Systems. ...
Article
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We present new data on the ecology, natural history and geographic distribution of the recently described skipper Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 and compare its immature stage morphology with the sympatric species S. sertorius (Hoffmannsegg, 1804). Spialia rosae uses species of Rosa L. (Rosaceae) as larval host-plants and prefers montane habitats, while S. sertorius feeds on Sanguisorba minor Scop. (Rosaceae) and inhabits lower altitudes. Rosa corymbifera Borkh. and R. tomentosa Sm. are documented for the first time as foodplants of S. rosae. We report Microgaster australis Thomson, 1895 (Hymenoptera, Braconidae, Microgastrinae) as a larval parasitoid of S. rosae. Details of the immature stages of S. rosae and S. sertorius are shown using scanning electron microscope photographs, confirming the similar immature stage morphology, at least as regards the Iberian S. sertorius. In both species, the egg has high radial ribs, the last instar larva has branched setae covering the head, and the pupa has setae with pointed tips, barrel-like cuticular formations, and hairy mesothoracic tubercles. By extensive sampling of the species of Spialia in the region of Segovia, Central Spain, we extend the previously known geographic distribution of S. rosae to 56 new 100 km2 MGRS squares, which represents a 155 % increase. Spialia rosae is present in the northern part of the interior plateau and in the main mountain systems of the Iberian Peninsula. The main threats to the populations of S. rosae are its limited distribution range and the possible effects of climate change due to its specialization in montane habitats. The conservation status of S. rosae was previously regarded as Data Deficient (DD). With the addition of new data the species can now be evaluated as Least Concern (LC).
... The increasing availability of DNA sequences has facilitated the uncovering of hidden species diversity and research on divergences in ecological traits among them, leading to recognition and understanding of fine-scale species interactions (Ortells et al., 2003;Hebert et al., 2004;Kankare et al., 2005;Cooke et al., 2012;Hernandez-Roldan et al., 2016;Leys et al., 2017). In addition, a vast number of loci obtained using high-throughput sequencing methods such as restriction site-associated DNA (RAD) sequencing has made it possible to estimate the recent divergence of closely related taxa (Baird et al., 2008;Cruaud et al., 2014). ...
... The observation of differences in ecological characteristics such as host use has often been followed by the discovery of cryptic species (Hebert et al., 2004;Kankare et al., 2005;Hernandez-Roldan et al., 2016). In the present study, we revealed that ciid beetles currently identified as O. laminifrons actually comprise four species, each of which use different sets of host fungi in nature. ...
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Insect–host fungus associations are important ecological features that affect population dynamics and interspecific interactions. However, details of host fungus relationships in fungivorous beetles remain unclear due to incompletely revised taxonomy and insufficient host records. We conducted a taxonomic study of the ciid beetle Octotemnus laminifrons (Coleoptera: Ciidae) and its cryptic species in Japan and investigated fungal host‐use by these ciid beetles. Maximum‐likelihood phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I fragments and two nuclear markers recovered four well‐supported, reciprocally monophyletic groups among specimens previously identified as O. laminifrons. Quantitative analyses of genital structures revealed diagnostic differences among these four lineages. These molecular and morphological analyses indicate that the ciid beetles currently identified as O. laminifrons actually comprise four species: O. laminifrons (Motschulsky, 1860), O. assimilis Kobayashi & Sota sp.n., O. crassus Kobayashi & Sota sp.n. and O. kawanabei Kobayashi & Sota sp.n. Here we provide a redescription and lectotype designation for O. laminifrons and descriptions of the three new species. These species showed different patterns of fungal host‐use among four Trametes and one Lenzites species. We also studied the phylogenetic relationships among these four species based on restriction‐site associated DNA (RAD) sequence data, which provided a more reliable phylogenetic tree. We discuss shifts in their fungal host use. Our results demonstrate that closely related, congeneric fungivorous insects exhibit different utility patterns for closely related fungal species, a phenomenon that has rarely been reported previously.
... Además, la aplicación de los avances en la genética al estudio de los lepidópteros ha variado significativamente el contexto taxonómico de los lepidópteros diurnos, con el descubrimiento incluso de especies crípticas (Hernández-Roldán et al., 2016). Este nuevo escenario taxonómico conlleva la necesidad de realizar estudios de campo para reconocer la distribución geográfica y el estado de las poblaciones. ...
... Los muestreos de carácter específico se han llevado a cabo para la búsqueda de nuevas poblaciones de Euchloe tagis (Hübner, 1804), Pieris mannii (Mayer, 1851) y Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016, así como para documentar la distribución regional de Arethusana arethusa (Denis & Schiffermüller, 1775) y A. boabdil (Rambur, 1938). ...
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New records of day-flying Lepidoptera (Papilionoidea, Zygaenoidea) in Euskadi, Navarra and adjacent areas. The first records of Pieris mannii (Mayer, 1851), Euchloe tagis (Hübner, 1804), Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 and Lasiommata petropolitana (Fabricius, 1787) for the autonomous community of Navarra are presented, including a new observation of the latter species in the department of Pyrénées-Atlantiques (France), which extends its known distribution in this country. The first sighting of Zygaena ephialtes (Linnaeus, 1767) for the last forty years in Navarra is also provided. In addition, the presence of P. manii in the neighbouring Basque provinces of Araba/Álava and Gipuzkoa is documented, and the presence of the two Iberian species of the genus Arethusana (de Lesse, 1951) in both territories is confirmed.
... On the other hand, 15 species were added to the list. Another recently discovered species, Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016, has already been added to the Fauna Europaea database. ...
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This paper presents an updated checklist of the butterflies of Europe, together with their original name combinations, and their occurrence status in each European country. According to this checklist, 496 species of the superfamily Papilionoidea occur in Europe. Changes in comparison with the last version (2.6.2) of Fauna Europaea are discussed. Compared to that version, 16 species are new additions, either due to cryptic species most of which have been discovered by molecular methods (13 cases) or due to discoveries of Asian species on the eastern border of the European territory in the Ural mountains (three cases). On the other hand, nine species had to be removed from the list, because they either do not occur in Europe or lost their species status due to new evidence. In addition, three species names had to be changed and 30 species changed their combination due to new evidence on phylogenetic relationships. Furthermore, minor corrections were applied to some authors’ names and years of publication. Finally, the name Polyommatus ottomanus Lefèbvre, 1831, which is threatened by its senior synonym Lycaena legeri Freyer, 1830, is declared a nomen protectum , thereby conserving its name in the current combination Lycaena ottomana.
... Several cases of highly divergent mitochondrial intraspecific lineages have been hypothesized to arise because of Wolbachia-mediated genetic sweeps and cytoplasmic incompatibility (e.g., Ritter et al., 2013). The transfer of Wolbachia infection across species through occasional hybridization may also lead to mitochondrial introgression (Dumas et al., 2013;Hernández-Roldán et al., 2016). ...
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Swallowtail butterflies (Papilionidae) are among the most spectacular and well‐known Lepidoptera in the European fauna, but their systematics is not fully elucidated. A notable case is that of Iphiclides feisthamelii which, after more than 180 years since description, still has a debated status, being often considered as a subspecies of Iphiclides podalirius. To elucidate the relationship between the two taxa and the evolutionary processes that led to their separation, we combine mitochondrial and nuclear DNA (mtDNA and nDNA) data, Wolbachia screening, genitalia morphology and wing UV reflectance. Our results show that the two taxa clearly differ in male and female genital morphology, male wing UV reflectance and nDNA. Two Wolbachia strains were found to widely infect the studied samples, apparently explaining the phylogeographic pattern displayed by mtDNA. The available data point towards a historical Wolbachia infection that spread from I. podalirius to I. feisthamelii and produced a mitochondrial introgression. Currently, a new Wolbachia strain is spreading across mainland populations of I. podalirius, mediating once more a mitochondrial genetic sweep, which has already infected and introgressed I. feisthamelii populations in south‐eastern France. We conclude that, given the marked differences in morphology and nDNA between the two taxa, and the apparent restriction of hybridization to a narrow contact area where non‐hybrid specimens are common, the taxon feisthamelii should be considered as a separate species. Within this species, two well‐differentiated nDNA lineages that represent European and Maghrebian populations are documented, here proposed as subspecies. The case of, presumably, two consecutive Wolbachia‐mediated mitochondrial introgression events, further supports the view that infection by this endosymbiont may be frequently related to mito‐nuclear discordance in insects.
... Precisely this procedure has been the one followed by the authors in the present study. Some recent examples of already published works with a similar methodology and comparable objectives are Hernández-Roldán et al. (2016) and Mally et al. (2016). ...
Article
The Iberian species of the genera Coscinia Hübner, [1819] and Spiris Hübner, [1819], as well as three other species from the Mediterranean area, are revised based on morphological and molecular genetic data. Our results suggest the separation into four morphologically and phylogenetically different genera: Coscinia Hübner, [1819], Lerautia Kemal & Koçak, 2006 stat. rev., Sagarriella Macià, Mally, Ylla, Gastón & Huertas gen. nov. and Spiris Hübner, [1819]. We conclude that there are eight species of the Coscinia genus group present in the studied area: Coscinia cribraria (Linnaeus, 1758), Coscinia chrysocephala (Hübner, [1810]) stat. rev., Coscinia mariarosae Expósito, 1991, Sagarriella libyssa caligans (Turati, 1907) comb. nov., Sagarriella romei (Sagarra, 1924) (= romeii sensu auctorum) comb. nov., Spiris striata Hübner, [1819], Spiris slovenica (Daniel, 1939) and Lerautia bifasciata (Rambur, 1832) comb. rev. We consider Coscinia cribraria benderi (Marten, 1957) stat. nov., Coscinia c. rippertii (Boisduval, 1834) and Coscinia c. ibicenca Kobes, 1991 stat. rev. to be subspecies of C. cribraria. COI Barcodes of C. cribraria diverge by up to 7.99%, and the investigated specimens group into six different COI Barcode BINs. Both the phylogenetic analysis of mitochondrial and nuclear DNA and the morphological examination of different specimens corroborate the changes in taxonomic status and justify the proposed taxonomic categories. We present images of adults and genitalia of both sexes, the immature stages of some of the species and the subspecies studied, as well as phylogenetic results from the analysis of genetic data. We also include data on life history, foodplants and geographical distribution.
... При использовании молекулярных маркеров эта процедура принципиально не отличается от стандартной делимитации видов, поскольку морфологические маркеры не используются. Таким образом, при поиске криптических видов вначале на основании анализа молекулярных маркеров выявляются кластеры генетически сходных особей, а затем делается интерпретация этих кластеров в качестве видов на основании использования дополнительных критериев (Lukhtanov et al., 2006(Lukhtanov et al., , 2014Лухтанов, Шаповал, 2008;Лухтанов, Кузнецова, 2009;Vershinina, Lukhtanov, 2010;Vila et al., 2010;Dinca et al., 2011;Hernández-Roldán et al., 2016;Vishnevskaya et al., 2016;Lukhtanov, Dantchenko, 2017b;Lukhtanov, Shapoval, 2017a). ...
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В статье обсуждаются возможности и ограничения разных подходов к выявлению видовых границ и описанию криптических видов. Вначале рассматриваются более традиционные методы делимитации видов, основанные преимущественно на анализе морфологических особенностей. Затем дается обзор подходов к делимитации видов с использованием молекулярных маркеров. Коротко рассматриваются также возможности подходов, основанных на машинном обучении компьютерных программ, к распознаванию видов по их дигитализированным изображениям. Делается вывод о том, что молекулярные признаки дают важный материал для разграничения видов, ценность которого многократно возрастает, если полученные молекулярные данные сравниваются с информацией по морфологии, географическому распространению и экологическим предпочтениям изучаемых таксонов. В заключение говорится о том, что зачастую только практикующий систематик, досконально знающий свою группу, может правильно интерпретировать получаемые молекулярные данные и вставить их в систему уже существующего знания для принятия таксономического решения.
... Since they are based on different geographical contexts, sympatric speciation is also considered to differ from allopatry because it can directly arise as a consequence of competition for resources [23]. This hypothesis is supported by several models [24], as well as by cases of recently diverging lineages [25,26] and ongoing ecological speciation taking place under natural conditions [27][28][29]. Over small spatial scales, habitat heterogeneity in the form of climatic variation or differences in community composition may initiate niche shifting in response to competition. ...
Article
Understanding how speciation relates to ecological divergence has long fascinated biologists. It is assumed that ecological divergence is essential to sympatric speciation, as a mechanism to avoid competition and eventually lead to reproductive isolation, while divergence in allopatry is not necessarily associated with niche differentiation. The impact of the spatial context of divergence on the evolutionary rates of abiotic dimensions of the ecological niche has rarely been explored for an entire clade. Here, we compare the magnitude of climatic niche shifts between sympatric versus allopatric divergence of lineages in butterflies. By combining next-generation sequencing, parametric biogeography and ecological niche analyses applied to a genus-wide phylogeny of Palaearctic Pyrgus butterflies, we compare evolutionary rates along eight climatic dimensions across sister lineages that diverged in large-scale sympatry versus allopatry. In order to examine the possible effects of the spatial scale at which sympatry is defined, we considered three sets of biogeographic assignments, ranging from narrow to broad definition. Our findings suggest higher rates of niche evolution along all climatic dimensions for sister lineages that diverge in sympatry, when using a narrow delineation of biogeographic areas. This result contrasts with significantly lower rates of climatic niche evolution found in cases of allopatric speciation, despite the biogeographic regions defined here being characterized by significantly different climates. Higher rates in allopatry are retrieved when biogeographic areas are too widely defined-in such a case allopatric events may be recorded as sympatric. Our results reveal the macro-evolutionary significance of abiotic niche differentiation involved in speciation processes within biogeographic regions, and illustrate the importance of the spatial scale chosen to define areas when applying parametric biogeographic analyses.
... Despite morphological similarity, the taxonomic and identification problems within the subgenus Agrodiaetus can be solved if chromosomal (de Lesse 1960a,b, Lukhtanov 1989 or molecular markers (Wiemers 2003, Kandul et al. 2004, Stradomsky and Fomina 2013, or their combination (Lukhtanov et al. 2006, Vila et al. 2010, Lukhtanov and Tikhonov 2015, Shapoval and Lukhtanov 2015a are applied. Although chromosome numbers are invariable in many groups of Lepidoptera (Robinson 1971, Lukhtanov 2014, Hernández-Roldán 2016, a few genera demonstrate chromosomal instability, a situation in which multiple closely related species differ drastically from each other by major chromosomal rearrangements, sometimes resulting in high variability in chromosome number (de Lesse 1960a,b, Talavera et al. 2013b). An unusual diversity of karyotypes is the most remarkable characteristic of the subgenus Agrodiaetus. ...
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Butterflies of the subgenus Agrodiaetus Hьbner, 1822, genus Polyommatus Latreille, 1804, are the model system in studies of speciation and karyotype evolution. A unique feature of the subgenus is the highest diversity in chromosome numbers in the animal kingdom. In Agrodiaetus the number of chromosomes is stable within species and differentiated between species, therefore karyotypes are used for species description, delimitation and identification, although there are exceptions. The use of molecular markers provides an additional information for species delimitation. In our research for testing taxonomic hypotheses we used chromosomal markers in combination with the analysis of two genetically unlinked sequences: fragment of mitochondrial gene COI and nuclear spacer ITS2. This approach resulted in recognition of five cryptic species inhabiting the Balkan Peninsula – P. ripartii Freyer, 1830, P. nephohiptamenos Brown et Coutsis, 1978, P. aroaniensis Brown, 1976, P. orphicus Kolev, 2005 and P. timfristos Lukhtanov, Vishnevskaya et Shapoval, 2016 and three cryptic species inhabiting Azerbaijan and Iran – P. valiabadi Rose et Schurian, 1977, P. rjabovianus Ko􀤊ak, 1980 and P. pseudorjabovi Lukhtanov, Dantchenko, Vishnevskaya et Saifitdinova, 2015 (Vishnevskaya et al. 2016, Lukhtanov et al. 2015). In general, the data obtained indicate that the genetic and taxonomic diversity of the subgenus Agrodiaetus is significantly higher than it was previously thought.
... Wolbachia are bacteria obligately endosymbiotic in arthropods and nematodes (Werren et al., 2008); ants are common Wolbachia hosts (Russell et al., 2017). Wolbachia can trigger reproductive isolation and increase speciation rate in their hosts (e.g., Shoemaker et al., 1999) but has, as far as we know, been included in just one species-delimitation study (Hernández-Roldán et al., 2016). A small-scale pilot study revealed infection of at least some Messor "structor" nests (data not shown), and we therefore screened the entire sample. ...
... When molecular markers are used instead of morphological ones, the procedure of revealing sibling species becomes essentially the same as standard species delimitation. First, clusters of genetically similar specimens are revealed by analysis of molecular markers; then these clusters may be interpreted as species based on additional criteria ( Lukhtanov et al., 2006Lukhtanov et al., , 2014Lukh- tanov andShapoval, 2008, 2017;Lukhtanov and Kuz- netsova, 2009;Vershinina and Lukhtanov, 2010;Vila et al., 2010;Dincă et al., 2011;Hernández-Roldán et al., 2016;Vishnevskaya et al., 2016;Lukhtanov and Dan- tchenko, 2017). ...
Article
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The potentials and limitations of different approaches to revealing species boundaries and describing cryptic species are discussed. Both the traditional methods of species delimitation, mostly based on morphological analysis, and the approaches using molecular markers are considered. Besides, the prospects of species identification using digital image recognition and machine learning are briefly considered. It is concluded that molecular markers provide very important material for species delimitation; the value of these data increases manifold if they can be compared with information on morphology, geographic distribution, and ecological preferences of the studied taxa. In many cases, only a practicing taxonomist who knows his or her group thoroughly can correctly interpret the molecular data and incorporate them into the existing knowledge system in order to make a taxonomic decision.
... Wolbachia infection can rapidly spread into a population, and because of maternal inheritance, can cause a selective sweep that leads to the fixation of the mitochondrial haplotype of the infected specimens. A series of studies report a correlation between patterns of Wolbachia infection and mtDNA structure (Gompert et al. 2006;Nice et al. 2009;Ritter et al. 2013), and a recent study on Spialia butterflies has pinpointed the role Wolbachia likely played in the formation of a cryptic species endemic to Iberia (Hernández-Roldán et al. 2016). ...
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Species reintroductions are increasingly used as means of mitigating biodiversity loss. Besides habitat quality at the site targeted for reintroduction, the choice of source population can be critical for success. The butterfly Melanargia russiae (Esper's marbled white) was extirpated from Hungary over 100 years ago, and a reintroduction program has recently been approved. We used museum specimens of this butterfly, mitochondrial DNA data (mtDNA), endosymbiont screening, and climatic‐similarity analyses to determine which extant populations should be used for its reintroduction. The species displayed 2 main mtDNA lineages across its range: 1 restricted to Iberia and southern France (Iberian lineage) and another found throughout the rest of its range (Eurasian lineage). These 2 lineages possessed highly divergent wsp alleles of the bacterial endosymbiont Wolbachia. The Hungarian specimens represented an endemic haplotype belonging to the Eurasian lineage, differing by one mutation from the Balkan and eastern European populations. The Hungarian populations of M. russiae occurred in areas with a colder and drier climate relative to most sites with extant known populations. Our results suggest the populations used for reintroduction to Hungary should belong to the Eurasian lineage, preferably from eastern Ukraine (genetically close and living in areas with the highest climatic similarity). Materials stored in museum collections can provide unique opportunities to document historical genetic diversity and help direct conservation. This article is protected by copyright. All rights reserved
... Similarly to previous studies 20,27,30,34-36 , our genome-level SNP analyses did not reveal strong genetic differentiation between the three ecotypes but mainly geographical structuring (see Fig. 1). This suggests that ecological shifts within this species are not associated with lineage sorting and do not represent an early stage of speciation, in contrast to processes occurring in many butterflies and other herbivorous arthropods [41][42][43] . Our result is in agreement with previous analyses based on spatially more restricted datasets of M. alcon, showing no significant genetic structure associated with the respective ecotypes and higher local genetic relatedness of the two low-altitude ecotypes of M. alcon, including syntopically occurring forms, than of populations of each ecotype from geographically distant localities 30,35,44,45 . ...
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Biotic interactions are often acknowledged as catalysers of genetic divergence and eventual explanation of processes driving species richness. We address the question, whether extreme ecological specialization is always associated with lineage sorting, by analysing polymorphisms in morphologically similar ecotypes of the myrmecophilous butterfly Maculinea alcon. The ecotypes occur in either hygric or xeric habitats, use different larval host plants and ant species, but no significant distinctive molecular traits have been revealed so far. We apply genome-wide RAD-sequencing to specimens originating from both habitats across Europe in order to get a view of the potential evolutionary processes at work. Our results confirm that genetic variation is mainly structured geographically but not ecologically — specimens from close localities are more related to each other than populations of each ecotype from distant localities. However, we found two loci for which the association with xeric versus hygric habitats is supported by segregating alleles, suggesting convergent evolution of habitat preference. Thus, ecological divergence between the forms probably does not represent an early stage of speciation, but may result from independent recurring adaptations involving few genes. We discuss the implications of these results for conservation and suggest preserving biotic interactions and main genetic clusters.
... Spialia sertorius (Hoffmannsegg, 1804) The taxon living in Sardinia and Corsica was identified in the past as Spialia sertorius therapne. Recent molecular studies showed that S. therapne must be considered as a good species with a diversification higher than 3% respect to S. sertorius (Hernández-Roldán et al, 2016). For this reason, we did not include Sardo-Corsican populations of S. therapne as comparison for S. sertorius. ...
... However, a pace of extinction and colonization on islands (turnover) slower than the dynamics occurring in the surrounding regions can generate intraspecific diversification and relictuality, which creates community distinctiveness among islands and from neighbouring mainland (Dapporto, Bruschini, Dincă, Vila, & Dennis, 2012;Masini, Petruso, Bonfiglio, & Mangano, 2008). Such diversity is usually represented by cryptic species or genetic lineages that can be detected only after in-depth molecular and/or morphologic analyses (Hernández-Roldán et al., 2016;Vodă, Dapporto, Dincă, & Vila, 2015a;Vodă et al., 2016). ...
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Aim: We describe fine-scale diversity patterns of the entire butterfly fauna occurring on the Tuscan Archipelago. By assessing the traits associated with population diversification, haplotype uniqueness and extinction, we aim to identify the factors determining the origin and maintenance of genetic diversity, and population vulnerability to environmental changes. Location: Tuscan Archipelago, Sardinia, Tuscany (Italy) and Corsica (France). Methods: We built a mtDNA dataset (1,303 COI sequences) for the 52 butterfly species reported in the Archipelago, also including specimens from neighbouring areas, and compiled data on 12 species traits and on the apparent extinction of species from the main islands. We calculated indices that measure genetic differentiation, and using phylogenetic regressions we evaluated the relationships between these indices and species traits. Finally, we inferred which traits are associated with disappearance of species on individual islands using phylogenetic regression. Results: The overall spatial pattern of genetic diversity corresponded with the proximity of the areas, but strong contrasts were also identified between geographically close areas. Together with the island endemics, several common and widespread species had a high genetic diversification among islands and mainland. Phylogenetic regressions revealed that smaller-sized, more specialized species, with a preference for drier regions, displayed greater genetic structure and/or haplotype uniqueness. Species that disappeared from islands had a higher population diversification. Capraia has experienced a notable loss of diversity, which significantly affected species with shorter flight periods. Main conclusions: Tuscan island butterflies are characterized by strong genetic contrasts and species differ in their contribution to the overall genetic diversity. By ranking the species for their contribution to genetic diversity and identifying the traits linked to the emergence and maintenance of diversity, we have developed a valuable tool for prioritizing populations as targets for monitoring and conservation action. The dataset constructed also represents a valuable resource for testing biogeographical hypotheses.
... However, a pace of extinction and colonization on islands (turnover) slower than the dynamics occurring in the surrounding regions can generate intraspecific diversification and relictuality, which creates community distinctiveness among 7 islands and from neighbouring mainland (Masini et al., 2008;Dapporto et al., 2012). Such diversity is usually represented by cryptic species or genetic lineages that can be detected only after in-depth molecular and/or morphologic analyses (Hernández-Roldán et al., 2016;Vodă et al., 2015aVodă et al., , 2016. ...
Article
Aim: We describe fine-scale diversity patterns of the entire butterfly fauna occurring on the Tuscan Archipelago. By assessing the traits associated with population diversification, haplotype uniqueness and extinction, we aim to identify the factors determining the origin and maintenance of genetic diversity, and population vulnerability to environmental changes. Location: Tuscan Archipelago, Sardinia, Tuscany (Italy) and Corsica (France). Methods: We built a mtDNA dataset (1,303 COI sequences) for the 52 butterfly species reported in the Archipelago, also including specimens from neighbouring areas, and compiled data on 12 species traits and on the apparent extinction of species from the main islands. We calculated indices that measure genetic differentiation, and using phylogenetic regressions we evaluated the relationships between these indices and species traits. Finally, we inferred which traits are associated with disappearance of species on individual islands using phylogenetic regression. Results: The overall spatial pattern of genetic diversity corresponded with the proximity of the areas, but strong contrasts were also identified between geographically close areas. Together with the island endemics, several common and widespread species had a high genetic diversification among islands and mainland. Phylogenetic regressions revealed that smaller-sized, more specialized species, with a preference for drier regions, displayed greater genetic structure and/or haplotype uniqueness. Species that disappeared from islands had a higher population diversification. Capraia has experienced a notable loss of diversity, which significantly affected species with shorter flight periods. Main conclusions: Tuscan island butterflies are characterized by strong genetic contrasts and species differ in their contribution to the overall genetic diversity. By ranking the species for their contribution to genetic diversity and identifying the traits linked to the emergence and maintenance of diversity, we have developed a valuable tool for prioritizing populations as targets for monitoring and conservation action. The dataset constructed also represents a valuable resource for testing biogeographical hypotheses.
... areas of southern Europe, and they often cause ongoing taxonomic problems (Mutanen et al. 2012). In contrast only moderately few sibling species have been found in sympatry (Hernández-Roldán et al. 2016;Mutanen et al. 2020;Berggren et al. 2022). ...
Article
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The taxonomy of the Caryocolum tricolorella species complex, an informal subsection of the diverse Caryocolum interalbicella species group, is revised and four species are separated from DNA barcodes of the mitochondrial COI (cytochrome c oxidase subunit 1) gene and adult morphology: C. tricolorella (Haworth, 1812), C. fibigerium Huemer, 1988, C. herwigvanstaai sp. nov. , and C. olekarsholti sp. nov. These species show a vicariant distribution pattern, with C. tricolorella widely distributed in Central and Northern Europe, C. fibigerium restricted to the Iberian Peninsula and southern France, C. herwigvanstaai sp. nov. to the Italian Peninsula, and C. olekarsholti sp. nov. to the Balkans. All species are described in detail, and the adults and genitalia of both sexes are illustrated.
... Thus, Wolbachia infection can rapidly spread into a population, and because of maternal inheritance, can cause a selective sweep that favours the mitochondrial haplotype of the infected specimens. An increasing number of studies are reporting correlation between patterns of Wolbachia infection and mtDNA structure, and such cases have been reported for butterflies as well [45,[47][48][49][50]. Furthermore, Wolbachia infections are dynamic and can be lost (e.g. ...
Article
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The Palaearctic butterfly Melitaea didyma stands out as one of the most striking cases of intraspecific genetic differentiation detected in Lepidoptera: 11 partially sympatric mitochondrial lineages have been reported, displaying levels of divergence of up to 7.4%. To better understand the evolutionary processes underlying the diversity observed in mtDNA, we compared mtDNA and genome-wide SNP data using double-digest restriction site-associated DNA sequencing (ddRADseq) results from 93 specimens of M. didyma ranging from Morocco to eastern Kazakhstan. We found that, between ddRADseq and mtDNA results, there is a match only in populations that probably remained allopatric for long periods of time. Other mtDNA lineages may have resulted from introgression events and were probably affected by Wolbachia infection. The five main ddRADseq clades supported by STRUCTURE were parapatric or allopatric and showed high pairwise FST values, but some were also estimated to display various levels of gene flow. Melitaea didyma represents one of the first cases of deep mtDNA splits among European butterflies assessed by a genome-wide DNA analysis and reveals that the interpretation of patterns remains challenging even when a high amount of genomic data is available. These findings actualize the ongoing debate of species delimitation in allopatry, an issue probably of relevance to a significant proportion of global biodiversity.
... The ideal procedure to follow them up with and produce new species identifications would include sequencing nuclear DNA, searching for morphological and life history traits, and (ideally) microbial screening. This array of techniques was used when delineating the European species of Spialia Swinhoe, 1912 (Hernandez-Roldan et al., 2016). In many cases, the issues of barcode non-monophyly can be solved with detailed studies at intraspecific-and closely related species-group levels. ...
Article
Roughly 18,000 species are described annually as new to science, while estimated extinction rates are comparable to or even exceeding these new discoveries. Considering the estimates of up to 15 million extant eukaryotic species on Earth, of which only about 2 million have been described so far, there has been a recent 'boom' of new potential approaches to more quickly discover and describe the millions of unknown species. This deficit is particularly noted in hyperdiverse taxa, as the current rate of species discovery is considered too slow. Recently, a 'minimalist' alpha taxonomic approach was proposed, relying solely on DNA barcoding and a habitus photograph, in a claimed effort to expedite the naming of new species to combat the so-called taxonomic impediment. In this paper, we point to limitations of minimalist taxonomy, present arguments in favour of the integrative approach, and finally explore a number of potential solutions to combat the taxonomic impediment in hyperdiverse taxa without sacrificing utility and quality for apparent speed and quantity.
... Morphological differentiation of genitalia often seems to have taken place along the process of speciation in European species pairs with highly similar external morphology [70][71][72], although cases having virtually indistinguishable genitalia are also known [70,73,74]. The biological species concept, in particular, largely relies on criteria that do not allow splitting populations into separate species until their genetic differentiation is significant enough to impede gene flow and consequent merging of populations [75]. ...
Article
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We studied the evolutionary relationship of two widely distributed parapatric butterfly species, Melitaea athalia and Melitaea celadussa, using the ddRAD sequencing approach, as well as genital morphology and mtDNA data. M. athalia was retrieved as paraphyletic with respect to M. celadussa. Several cases of mito-nuclear discordance and morpho-genetic mismatch were found in the contact zone. A strongly diverged and marginally sympatric clade of M. athalia from the Balkans was revealed. An in-depth analysis of genomic structure detected high levels of admixture between M. athalia and M. celadussa at the contact zone, though not reaching the Balkan clade. The demographic modelling of populations supported the intermediate genetic make-up of European M. athalia populations with regards to M. celadussa and the Balkan clade. However, the dissimilarity matrix of genotype data (PCoA) suggested the Balkan lineage having a genetic component that is unrelated to the athalia-celadussa group. Although narrowly sympatric, almost no signs of gene flow were found between the main M. athalia group and the Balkan clade. We propose two possible scenarios on the historical evolution of our model taxa and the role of the last glacial maximum in shaping their current distribution. Finally, we discuss the complexities regarding the taxonomic delimitation of parapatric taxa.
... The BNM database comprises butterfly occurrence records (unique combinations of species  recorder  location  date) for the United Kingdom, Isle of Man and Channel Islands, many of which are the result of opportunistic, non-standardised sampling by community scientists (verified by a network of expert volunteers). The BNM database was created in 1995 but incorporates a substantial volume of historical records, particularly those gathered by a previous recording scheme that led to the first butterfly atlas of Britain and Ireland (Heath et al., 1984). The extract used for this study contained all records of the 58 breeding species in GB from 1800 to 2014 (see S anchez-Fern andez et al., 2021). ...
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1. The description of how biological information is compiled over time is essential to detect temporal biases in biodiversity data that could directly influence the utility, comparability, and reliability of ecological and biogeographical studies. 2. We explore trends in species recording over time using one of the most spatially and temporally comprehensive country-level databases for any group of insects in the world – the database of butterfly occurrences from Great Britain. 3. Firstly, we used two crucial milestones (the year in which the taxonomic inventory is complete, i.e., when the last species was recorded, the year in which all species are recorded together for the first time) to delimit three main phases in the process of biodiversity recording (taxonomic, faunistic and exhaustive phases). Secondly, we aimed to quantify how far species features (attractiveness and detectability) influence the process of recording through time. 4. During the first stage of biodiversity compilation, when the main aim is to complete the taxonomic inventory (taxonomic period), entomologists tend to record attractive species more frequently. However, once the inventory is complete, particularly in the period during which more spatially and temporally comprehensive information about species distribution is amassed (the exhaustive period), the recording pattern clearly changes to more detectable species. 5. Common, highly detectable species are undersampled in the first phase of biodiversity data compilation and oversampled in the final stages. Awareness of such temporal patterns in recording is necessary in order to correctly interpret and address bias in insect biodiversity trends.
... DNA barcodes are also widely applied in phylogeography and surveys of intraspecific variability. After much initial debate, it is now well established that DNA barcoding (and any other single-marker approach) can be a useful tool to identify potential cryptic species, although an integrative approach is necessary for confirmation (e.g., nuclear markers, morphology, and ecology) DeSalle et al. 2005;Talavera et al. 2013b;Dincǎ et al. 2015;Hernández-Roldán et al. 2016;Lukhtanov et al. 2016;Gaunet et al. 2019). ...
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Taxa are frequently labeled incertae sedis when their placement is debated at ranks above the species level, such as their subgeneric, generic or subtribal placement. This is a pervasive problem in groups with complex systematics due to difficulties in identifying suitable synapomorphies. In this study, we propose combining DNA barcodes with a multi-locus backbone phylogeny in order to assign taxa to genus or other higher-level categories. This sampling strategy generates molecular matrices containing large amounts of missing data that are not distributed randomly: barcodes are sampled for all representatives, and additional markers are sampled only for a small percentage. We investigate the effects of the degree and randomness of missing data on phylogenetic accuracy using simulations for up to 100 markers in 1000-tips trees, as well as a real case: the subtribe Polyommatina (Lepidoptera: Lycaenidae), a large group including numerous species with unresolved taxonomy. Our simulation tests show that when a strategic and representative selection of species for higher-level categories has been made for multi-gene sequencing (approximately one per simulated genus), the addition of this multi-gene backbone DNA data for as few as 5-10% of the specimens in the total dataset can produce high-quality phylogenies, comparable to those resulting from 100% multi-gene sampling. In contrast, trees based exclusively on barcodes performed poorly. This approach was applied to a 1365-specimen dataset of Polyommatina (including ca. 80% of described species), with nearly 8% of representative species included in the multi-gene backbone and the remaining 92% included only by mitochondrial COI barcodes, a phylogeny was generated that highlighted potential misplacements, unrecognized major clades, and placement for insertae sedis taxa. We use this information to make systematic rearrangements within Polyommatina, and to describe two new genera. Finally, we propose a systematic workflow to assess higher-level taxonomy in hyperdiverse groups. This research identifies an additional, enhanced value of DNA barcodes for improvements in higher-level systematics using large datasets.
... Leptidea (Talla et al., 2019) and the recently discovered Spialia rosae (Hernández-Roldán et al., 2016). ...
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The Pleistocene glacial cycles had a profound impact on the ranges and genetic make‐up of organisms. Whilst it is clear that the contact zones that have been described for many sister taxa are secondary and have formed during the last interglacial, it is unclear when the taxa involved began to diverge. Previous estimates based on small numbers of loci are unreliable given the stochasticity of genetic drift and the contrasting effects of incomplete lineage sorting and gene flow on gene divergence. Here we use genome‐wide transcriptome data to estimate divergence for 18 sister species pairs of European butterflies showing either sympatric or contact zone distributions. We find that in most cases species divergence predates the mid‐Pleistocene transition or even the entire Pleistocene period. We also show that although post divergence gene flow is restricted to contact zone pairs, they are not systematically younger than sympatric pairs. This suggests that contact zones are not limited to the initial stages of the speciation process, but can involve notably old taxa. Finally, we show that mitochondrial and nuclear divergence are only weakly correlated and mitochondrial divergence is higher for contact‐zone pairs.
... . Lineages distinguished by mtDNA and allozymes also differ in voltinism. A possibly analogous situation was observed in a cryptic species pair of Hesperidae butterflies, where Spialia rosae Hernández-Roldán, Dapporto, Dincă, Vicente & Vila, 2016 differed from S. sertorius (Hoffmannsegg, 1804) by the COI barcode, specific cuticular hydrocarbons, their host plants, and the presence of Wolbachia58 . Wolbachia presence thus might serve as a further distinguishing trait of the cryptic species pair A. agestis and A. artaxerxes.The mitochondrial diversity of the highly infected A. artaxerxes was low compared to much less affected A. agestis (16 vs. 54 haplotypes, respectively). ...
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The bacterium Wolbachia infects many insect species and spreads by diverse vertical and horizontal means. As co-inherited organisms, these bacteria often cause problems in mitochondrial phylogeny inference. The phylogenetic relationships of many closely related Palaearctic blue butterflies (Lepidoptera: Lycaenidae: Polyommatinae) are ambiguous. We considered the patterns of Wolbachia infection and mitochondrial diversity in two systems: Aricia agestis/Aricia artaxerxes and the Pseudophilotes baton species complex. We sampled butterflies across their distribution ranges and sequenced one butterfly mitochondrial gene and two Wolbachia genes. Both butterfly systems had uninfected and infected populations, and harboured several Wolbachia strains. Wolbachia was highly prevalent in A. artaxerxes and the host's mitochondrial structure was shallow, in contrast to A. agestis. Similar bacterial alleles infected both Aricia species from nearby sites, pointing to a possible horizontal transfer. Mitochondrial history of the P. baton species complex mirrored its Wolbachia infection and not the taxonomical division. Pseudophilotes baton and P. vicrama formed a hybrid zone in Europe. Wolbachia could obscure mitochondrial history, but knowledge on the infection helps us to understand the observed patterns. Testing for Wolbachia should be routine in mitochondrial DNA studies.
... ; (b) Spialia sertoriusand Spialia rosae, largely sympatric in Iberia(Hernández-Roldán et al., 2016); (c) Pyrgus malvae and Pyrgus malvoides, parapatric with a contact zone along central France and the Alps(Koren, Beretta, Črne, & Verovnik, 2013;Litman et al., 2018); (d) Iphiclides podalirius and Iphiclides feisthamelii, parapatric with a contact zone in the Pyrenees and southern France(Gaunet et al., 2019;Wiemers & Gottsberger, 2010); (e) Zerynthia polyxena and Zerynthia cassandra, parapatric with a contact zone in northern Italy(Zinetti et al., 2013); (f) Pontia daplidice and Pontia edusa, parapatric with a contact zone in north-western Italy(Porter, Wenger, Geiger, Scholl, & Shapiro, 1997); (g) Leptidea sinapis/reali/juvernica, with L. reali and L. juvernica being allopatric, but each sympatric with respect to L. sinapis(Dincă, Lukhtanov, et al., 2011, Dinca et al., 2013;(h) Lycaena tityrus and Lycaena bleusei, parapatric in Iberia (Dincă et al., 2015); (i) Polyommatus icarus and Polyommatus celina, parapatric with contact in southern Iberia (Dincă, Dapporto, & Vila, 2011); (j) Lysandra coridon and Lysandra caelestissima, parapatric in central Iberia (Talavera, Lukhtanov, Rieppel, Pierce, & Vila, 2013); (k) Melitaea athalia and Melitaea celadussa, parapatric with a contact zone from southern France through the Alps. We also applied the assignment procedure to a series of species showing almost complete allopatric distribution that were split in Wiemers et al. (2018) but not yet considered in the CLIMBER d: (l) Aglais urticae and Aglais ichnusa; (m) Iolana iolas and Iolana debilitata; (n) Pseudochazara anthelea and Pseudochazara amalthea. ...
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Aim Occurrence data are fundamental to macroecology, but accuracy is often compromised when multiple units are lumped together (e.g., in recently separated cryptic species or in citizen science records). Using amalgamated data leads to inaccuracy in species mapping, to biased beta‐diversity assessments and to potentially erroneously predicted responses to climate change. We provide a set of R functions (biodecrypt) to objectively attribute unidentified occurrences to the most probable taxon based on a subset of identified records. Innovation Biodecrypt assumes that unidentified occurrences can only be attributed at certain distances from areas of sympatry. The function draws concave hulls based on the subset of identified records; subsequently, based on hull geometry, it attributes (or not) unidientified records to a given taxon. Concavity can be imposed with an alpha value and sea or land areas can be excluded. A cross‐validation function tests attribution reliability and another function optimises the parameters (alpha, buffer, distance ratio between hulls). We applied the procedure to 16 European butterfly complexes recently separated into 33 cryptic species for which most records were amalgamated. We compared niche similarity and divergence between cryptic taxa, and re‐calculated and contributed updated climatic niche characteristics of the butterflies in Europe (CLIMBER). Main conclusions Biodecrypt showed a cross‐validated correct attribution of known records always ≥ 98% and attributed more than 80% of unidientified records to the most likely taxon in parapatric species. The functions determined where records can be assigned even for largely sympatric species, and highlighted areas where further sampling is required. All the cryptic taxa showed significantly diverging climatic niches, reflected in different values of mean temperature and precipitation compared to the values originally provided in the CLIMBER database. The substantial fraction of cryptic taxa existing across different taxonomic groups and their divergence in climatic niches highlight the importance of using reliably assigned occurrence data in macroecology.
... Specific associations with different host plants for phytophagous insects are considered as the main ecological features that affect the population dynamics and interactions and, as such, could ultimately involve in the speciation process (Kobayashi & Sota, 2019). Consequently, the observation of differences in host usage has often been followed by the discovery of cryptic species (Hebert, Penton, Burns, Janzen, & Hallwachs, 2004;Hernandez-Roldan et al., 2016;Kankare, Van Nouhuys, & Hanski, 2005;Kobayashi & Sota, 2019). Our results from phylogenetic analysis (particularly in the nuclear gene), morphometric data and statistical tests corroborated to support the presence of one host-specialized lineage on P. salicina and P. cerasifera f. atropurpurea in Central and South China (Figures 2 and S2, Table 1). ...
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The broadly distributed genus Hyalopterus currently comprises three formally recognized species that are highly similar morphologically and hence difficult to be identified with certainty. This group has undergone multiple revisions in the past century, but none of these has assessed species from Asia, which has hampered our understanding of the species diversity within this genus. Based on a comprehensive data set from morphological data and host-associated data, and by coalescent-based delimitation approaches, the Hyalopterus species boundaries, distribution and diversity were clarified here to further reveal the composition of the species. Two single-locus (MLGMYC and mPTP) and two multilocus (BPP and STACEY) delimitation methods were conducted based on extensive sampling. Then, the phylogenetic relationships and morphological divergence were assessed. Our data strongly supported that the number of recognized species in Hyalopterus had likely been underestimated. The phylogenetic analyses recovered four major clades, which corresponded to distinct host-plant preferences. Also, the morphological analyses showed significant differentiation for only one of the newly recognized candidate species uncovered by the delimitation approaches, suggesting the existence of at least two independent evolutionary lineages within Hyalopterus arundiniformis, which showed different patterns of host association. Moreover, based on our data, the taxonomic misidentification of H. arundiniformis in China was corrected here. This study lays the groundwork for the thorough taxonomic revision of Hyalopterus and for future evolutionary studies and underlines the importance of an integrated framework for species determination.
... Host-plant shifts have been suggested to explain speciation in other butterfly taxa (e.g., Fordyce, 2010;Hernández-Roldán et al., 2016), and it seems plausible that diversification of lycaenids in the fynbos might be correlated with shifts in host plant use. However, taxa in the thysbe group are often polyphagous, and many species have been recorded feeding on multiple plant families (Supplementary Table S5). ...
Article
Although best known for its extraordinary radiations of endemic plant species, the South African fynbos is home to a great diversity of phytophagous insects, including butterflies in the genus Chrysoritis (Lepidoptera: Lycaenidae). These butterflies are remarkably uniform morphologically; nevertheless, they comprise 43 currently accepted species and 68 currently valid taxonomic names. While many species have highly restricted, dot-like distributions, others are widespread. Here, we investigate the phylogenetic and biogeographic history underlying their diversification by analyzing molecular markers from 406 representatives of all described species throughout their respective ranges. We recover monophyletic clades for both C. chrysaor and C. thysbe species-groups, and identify a set of lineages that fall between them. The estimated age of divergence for the genus is 32 Mya, and we document significantly rapid diversification of the thysbe species-group in the Pleistocene (∼2 Mya). Using ancestral geographic range reconstruction, we show that West Fynbos is the most likely region of origin for the radiation of the thysbe species-group. The colonization of this region occurred 9 Mya and appears to have been followed by a long period of relative stasis before a recent increase in diversification. Thus, the thysbe radiation does not appear to have resulted from the colonization of new biogeographic areas. Rather, the impact of species interactions (with ants and plants), the appearance of key innovations, and/or the opening of new ecological niche space in the region might explain the sudden burst of speciation that occurred in this group 2 Mya. The biogeographic model suggests two different diversification processes with few historical cross-colonisations, one in eastern South Africa for the C. chrysaor group and the other in western South Africa for the remaining taxa. Distributional range assessments and ecological niche models for each species show important niche overlap, and in a few cases, complete overlap. However, these shared traits are not explained by phylogenetic history. Chrysoritis taxa frequently fly in sympatry and gene tree reticulation appears to be widespread at the species level, suggesting that several episodes of range shifts might have led to secondary sympatries, allowing limited gene flow that challenges species delimitation efforts. In addition, the unusually high diversification rate for the thysbe clade of 1.35 [0.91 – 1.81] lineages per million years also suggests the possibility of taxonomic oversplitting. The phylogeny presented here provides a framework for a taxonomic revision of the genus. We highlight cases of potential synonymy both in allopatry and sympatry, and stress the importance of dedicated studies to assess potential pre- and post-zygotic barriers giving rise to species delimitations of the thysbe group.
... Despite morphological similarity, the taxonomic and identification problems within the subgenus Agrodiaetus can be solved if chromosomal (de Lesse 1960a,b, Lukhtanov 1989 or molecular markers (Wiemers 2003, Kandul et al. 2004, Stradomsky and Fomina 2013, or their combination (Lukhtanov et al. 2006, Vila et al. 2010, Lukhtanov and Tikhonov 2015, Shapoval and Lukhtanov 2015a are applied. Although chromosome numbers are invariable in many groups of Lepidoptera (Robinson 1971, Lukhtanov 2014, Hernández-Roldán 2016, a few genera demonstrate chromosomal instability, a situation in which multiple closely related species differ drastically from each other by major chromosomal rearrangements, sometimes resulting in high variability in chromosome number (de Lesse 1960a,b, Talavera et al. 2013b). An unusual diversity of karyotypes is the most remarkable characteristic of the subgenus Agrodiaetus. ...
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The Balkan Peninsula represents one of the hottest biodiversity spots in Europe. However, the invertebrate fauna of this region is still insufficiently investigated, even in respect of such well-studied organisms as Lepidoptera. Here we use a combination of chromosomal, molecular and morphological markers to rearrange the group of so-called anomalous blue butterflies (also known as ‘brown complex’ of the subgenus Agrodiaetus Hübner, [1822] and as the Polyommatus (Agrodiaetus) admetus (Esper, 1783) species group) and to reveal its cryptic taxonomic structure. We demonstrate that P. aroaniensis (Brown, 1976) is not as widespread in the Balkans as was previously thought. In fact, it has a dot-like distribution range restricted to the Peloponnese Peninsula in South Greece. Polyommatus orphicus Kolev, 2005 is not as closely related to the Turkish species P. dantchenkoi (Lukhtanov & Wiemers, 2003) as was supposed earlier. Instead, it is a Balkan endemic represented by two subspecies: P. orphicus orphicus (Bulgaria) and P. orphicus eleniae Coutsis & De Prins, 2005 (Northern Greece). Polyommatus ripartii (Freyer, 1830) is represented in the Balkans by an endemic subspecies P. ripartii pelopi. The traditionally recognized P. admetus (Esper, 1783) is shown to be a heterogeneous complex and is divided into Polyommatus admetus sensu stricto (the Balkans and west Turkey) and P. yeranyani (Dantchenko & Lukhtanov, 2005) (east Turkey, Armenia, Azerbaijan and Iran). Polyommatus nephohiptamenos (Brown & Coutsis, 1978) is confirmed to be a species with a dot-like distribution range in Northern Greece. Finally, from Central Greece (Timfristos and Parnassos mountains) we describe Polyommatus timfristos Lukhtanov, Vishnevskaya & Shapoval, sp. n. which differs by its haploid chromosome number (n=38) from the closely related and morphologically similar P. aroaniensis (n=47-48) and P. orphicus (n=41-42). We provide chromosomal evidence for three separate south Balkan Pleistocene refugia (Peloponnesse, Central Greece and Northern Greece/South Bulgaria) and stress the biogeographic importance of Central Greece as a place of diversification. Then we argue that the data obtained have direct implications for butterfly karyology, taxonomy, biogeography and conservation.
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The diversity and evolution of host-plant relationships of butterflies has been the subject of much research. In this context, an interesting species is the bog fritillary Boloria eunomia. Across its boreo-montane distribution there are several ecotypes of this butterfly (sometimes distinguished as subspecies), which inhabit distinctly different habitats. However, little is known about the relationships between these forms in terms of host-plant use. We compared larval preferences of two ecotypes in north-eastern Poland, a unique region that is inhabited by different evolutionary lineages of B. eunomia. Larvae of the meadow ecotype reared in captivity were able to grow and develop exclusively on Persicaria bistorta, i.e., their 'native' host plant. In contrast, larvae originating from populations inhabiting raised bogs generally showed poorer survival, though they accepted both Ericaceae species growing in their biotope and P. bistorta, a plant that does not occur in their biotope. They did not show clear food preferences, but mortality was lowest when they were fed Vaccinium uliginosum. This is puzzling as field observations indicate that Vaccinium oxycoccus is the main host plant in the area studied. However, our findings are consistent with the results of phylogeographical studies indicating that meadows were the ancestral habitats of B. eunomia and some populations adapted later to different biotopes. The poor survival of larvae of the bog ecotype recorded in the present study may indicate that this form is at least locally potentially more vulnerable than the other ecotype.
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The Siberian chipmunk is native to northeastern Asia, but alien populations of this squirrel, introduced through the pet trade, occur in many European countries. This rodent has been listed as an invasive species of European concern, being a potential vector of ticks spreading Lyme disease. We aimed to assess its current distribution range and to identify areas of potential invasion. Two sets of species distribution models were conducted, one considering the locations of the species (n = 625 occurrences) and the other with only the occurrences of the Korean sub-species (the invasive one in Europe; n = 255 occurrences), which might be a separate species from the Siberian one. We included 19 uncorrelated predictors (two topographic, nine land cover, five bioclimatic and three anthropogenic variables)
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Cryptic species represent a challenge for documenting global biodiversity. Even in well-studied groups, such as European butterflies, the application of integrative approaches has allowed the recognition of an unexpected number of cryptic taxa. Here, we combine the analysis of mitochondrial (cytochrome c oxidase I, COI) and nuclear (internal transcribed spacer 2, ITS2) markers with geometric morphometrics of the male genitalia to study diversity within the butterfly Muschampia proto. The nuclear marker reveals three well-supported and deeply diverged lineages, which are also detected based on mitochondrial DNA, although the latter recovers one of them as paraphyletic with poor support. These lineages also present distinct male genital characters, which allow blind assignment of > 97% of specimens when applying a jackknife procedure. We conclude that M. proto comprises three cryptic species that started to differentiate ~2 Mya: M. proto, distributed in northern Africa, the Iberian Peninsula and southern France; Muschampia alta comb. & stat. nov., occurring in southern Italy and the Balkan Peninsula; and Muschampia proteides, present in the easternmost part of Europe, the Near East and Iran. This discovery adds two new species to the European butterfly fauna and highlights the necessity to continue investigating potential cryptic diversity.
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Gracillariidae is the most species-rich leaf-mining moth family with over 2,000 described species worldwide. In Europe, there are 263 valid named species recognized, many of which are difficult to identify using morphology only. Here we explore the use of DNA barcodes as a tool for identification and species discovery in European gracillariids. We present a barcode library including 6,791 COI sequences representing 242 of the 263 (92%) resident species. Our results indicate high congruence between morphology and barcodes with 91.3% (221/242) of European species forming monophyletic clades that can be identified accurately using barcodes alone. The remaining 8.7% represent cases of non-monophyly making their identification uncertain using barcodes. Species discrimination based on the Barcode Index Number system (BIN) was successful for 93% of species with 7% of species sharing BINs. We discovered as many as 21 undescribed candidate species, of which six were confirmed from an integrative approach; the other 15 require additional material and study to confirm preliminary evidence. Most of these new candidate species are found in mountainous regions of Mediterranean countries, the South-Eastern Alps and the Balkans, with nine candidate species found only on islands. In addition, 13 species were classified as deep conspecific lineages, comprising a total of 27 BINs with no intraspecific morphological differences found, and no known ecological differentiation. Double-digest restriction-site associated DNA sequencing (ddRAD) analysis showed strong mitonuclear discrepancy in four out of five species studied. This discordance is not explained by Wolbachia-mediated genetic sweeps. Finally, 26 species were classified as “unassessed species splits” containing 71 BINs and some involving geographical isolation or ecological specialization that will require further study to test whether they represent new cryptic species.
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Trait-based analyses explaining the different responses of species and communities to environmental changes are increasing in frequency. European butterflies are an indicator group that responds rapidly to environmental changes with extensive citizen science contributions to documenting changes of abundance and distribution. Species traits have been used to explain long-and short-term responses to climate, land-use and vegetation changes. Studies are often characterised by limited trait sets being used, with risks that the relative roles of different traits are not fully explored. Butterfly trait information is dispersed amongst various sources and descriptions sometimes differ between sources. We have therefore drawn together multiple information sets to provide a comprehensive trait database covering 542 taxa and 25 traits described by 217 variables and sub-states of the butterflies of Europe and Maghreb (northwest Africa) which should serve for improved trait-based ecological, conservation-related, phylogeographic and evolutionary studies of this group of insects. We provide this data in two forms; the basic data and as processed continuous and multinomial data, to enhance its potential usage.
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The Pleistocene glacial cycles had a profound impact on the ranges and genetic make-up of organisms. Whilst it is clear that the contact zones that have been described for many sister taxa are secondary and have formed during the last interglacial, it is unclear when the taxa involved began to diverge. Previous estimates based on small numbers of loci are unreliable given the stochasticity of genetic drift and the contrasting effects of incomplete lineage sorting and gene flow on gene divergence. Here we use genome-wide transcriptome data to estimate divergence for 18 sister species pairs of European butterflies showing either sympatric or contact zone distributions. We find that in most cases species divergence predates the mid-Pleistocene transition or even the entire Pleistocene period. We also show that although post divergence gene flow is restricted to contact zone pairs, they are not systematically younger than sympatric pairs. This suggests that contact zones are not limited to the embryonic stages of the speciation process, but can involve notably old taxa. Finally, we show that mitochondrial and nuclear divergence are only weakly correlated and mitochondrial divergence is higher for contact-zone pairs. This suggests a possible role of selective sweeps affecting mitochondrial variation in maintaining contact zones. Impact Summary The influence of the Pleistocene glacial cycles on structuring species and genetic diversity in temperate taxa has permeated biogeographic and phylogeographic thinking for decades. Although phylogeographic studies have repeatedly claimed that the Pleistocene acted as a species pump, systematic tests of this idea based on robust estimates of species divergence are lacking. Here we estimate divergence times for all sister species pairs of European butterfly using genome-wide transcriptome data. We find that most species pairs are substantially older than the onset of Pleistocene glacial cycling. We also show that post divergence gene flow is restricted to pairs that form contact-zones. However, in contrast to expectations under a null model of allopatric speciation contract zone pairs are not necessarily younger than sympatric pairs.
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DNA barcodes of 200 different butterfly species from Austria were successfully generated, encompassing 94.5 % of the 211 species ever found in Austria. Sequences of 1263 specimens of the barcode region, the mitochondrial COI gene, longer than 500 bp were recovered and 1224 were fully barcode compliant. 172 species (86.0 % of the inventory) can be identified correctly with the barcode. In 28 species barcode sharing or overlap was found, which may be due to introgression, hybridisation or phylogenetic young species. 164 species or 77.4 % of the Austrian butterfly fauna have unique Barcode Index Numbers (BINs). 13 species cover more than 1 BIN and likely include cryptic diversity, e.g. in Melitaea didyma and Melitaea cinxia. The morphologically hardly separable sister species Leptidea sinapis/Leptidea juvernica, Colias hyale/Colias alfacariensis, Boloria napaea/ Boloria pales and Aricia artaxerxes/Aricia agestis are unequivocally separated by their DNA barcodes. M. athalia celadussa is recorded for Austria based on DNA barcode sequences, which are probably reflecting intogression. In Lycaena tityrus, L. alciphron and Euphydryas aurinia the disputed alpine subspecies cluster separately from lowland populations, thus supporting different taxonomic status
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During efforts to generate DNA barcodes for all European Lepidoptera, Batrachedra pinicolella (Zeller, 1839) was found to comprise two genetically distinct clusters. Morphological investigation and results from two nuclear markers and ddRAD sequencing furthermore support the existence of two distinct taxa which we treat as two separate species, B. pinicolella and B. confusella sp. nov. A lectotype for B. pinicolella is designated. Available data indicate that the biology of both species also differs, with Picea abies (L.) Karsten as a proved host-plant for B. pinicolella and Pinus sylvestris L. for B. confusella sp. nov. Both species are mainly distributed on the European continent with B. pinicolella occurring in boreal parts of North and Central Europe and introduced to Canada, reflecting a boreo-montane distribution pattern. Batrachedra confusella sp. nov. is more widely distributed in temperate Northern and Central Europe.
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The Melitaea phoebe group is constituted by six species distributed throughout the Palearctic. One of the most widespread species is Melitaea ornata Christoph, 1893, present from France (Provence) to Central Asia. Recently, populations of M. ornata were discovered in a mountainous region of southeastern Iberia, although doubts about their taxonomy existed. To clarify the taxonomic status of these populations and to revise the distribution of this taxon in Iberia, we have sequenced mitochondrial (COI barcode region) and nuclear (wg, RPS5, MDH, and EF-1α) markers, and analyzed the male genitalia for 72 Iberian individuals and for all the species of the M. phoebe group. This information was complemented with phenological and ecological data. Our results unveiled that the Iberian M. ornata-like taxon is in fact distributed through most of the Iberian Peninsula, except for the southwest and northeast. In contrast to the univoltine M. ornata, the Iberian taxon can be bivoltine in the wild. The Iberian taxon was retrieved to be related to M. ornata, but the differences in the genetic markers and genitalia were comparable to those found between species in the group. Based on the evidence here presented and according to species delimitation results, we propose to consider the Iberian taxon as a novel species , tentatively named Melitaea pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.
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Successful conservation of butterflies is dependent on knowing which larval foodplants they use. However, many published lists of larval foodplants have been copied from previous lists, which in turn have been copied from previous lists. Consequently, errors have crept in, and many plant names have long been superseded. This can result in duplicates in the list, with the same plant being given two different names. Most plant lists do not include the authority, which can make it difficult or impossible to identify which plant is being referred to. For the first time, a list of the current accepted plant names utilised by 471 European butterfly larvae is presented, with references. Where possible, errors in previous lists have been removed. The list of larval foodplants doubled from previous published lists. This has resulted in a list of 1506 different plant species in 72 different families. 86 plant records are only known at the generic level. Larval foodplants of 25 butterfly species are currently unknown. Whilst most plant families are utilised by less than six butterfly species, a few plant families are particularly favoured, with the Poaceae and Fabaceae being the most popular. Similarly, most plant species are only utilised by a few butterfly species, but Festuca ovina and Festuca rubra are favoured by a large number of butterfly species. 20% of European butterfly larvae are monophagous, 50% are oligophagous, and 30% are polyphagous, with Celastrina argiolus able to use plants in 19 different families.
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Molecular approaches have greatly advanced our understanding of species diversity and biogeography in the cladoceran crustaceans. Here, we provide the first large-scale examination of taxonomic diversity in the genus Holopedium Zaddach, 1855, by characterizing patterns of allozyme, mtDNA, and morphological variation from a total of 193 sites from three continents, including collections from near the type localities for the two generally recognized species, Holopedium gibberum Zaddach, 1855, and Holopedium amazonicum Stingelin, 1904. Allozyme data were only available for North American samples but revealed the presence of four species. Divergence patterns in the mitochondrial cytochrome c oxidase subunit I (COI) gene supported those species, as well as a fifth taxon endemic to South America. The five putative species are separated by substantial sequence (8.7–24.5%) and allozyme (0.36–1.54 Nei’s distance) divergences, while intraspecific genetic diversity was generally limited in comparison. Although two of these species exhibited little morphological differentiation from their closest relatives, and diagnostic traits were not found among the characters considered, a population-level approach revealed significant morphological differences among all pairs of taxa. We therefore present both an allozyme key and a morphological/geographic key to all species, as well as new or augmented descriptions for all five species. H. gibberum s.s. is distributed in Europe and across arctic North America, while its cryptic sister species, H. glacialis n. sp., is widely distributed across temperate North America. H. amazonicum s.s. is apparently restricted to the Amazon basin, H. atlanticum n. sp. occurs in lakes along the eastern margin of North America, while H. acidophilum n. sp. occurs sporadically across North America along a narrow band of middle latitudes. Due to high morphological variability within species, as well as the detection of cryptic diversity, we suggest that genetic analyses should be performed on populations from other geographic regions and should always accompany the recognition of new species of Holopedium.
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Molecular approaches have greatly advanced our understanding of species diversity and biogeography in the cladoceran crustaceans. Here, we provide the first large-scale examination of taxonomic diversity in the genus Holopedium Zaddach, 1855, by characterizing patterns of allozyme, mtDNA, and morphological variation from a total of 193 sites from three continents, including collections from near the type localities for the two generally recognized species, Holopedium gibberum Zaddach, 1855, and Holopedium amazonicum Stingelin, 1904. Allozyme data were only available for North American samples but revealed the presence of four species. Divergence patterns in the mitochondrial cytochrome c oxidase subunit I (COI) gene supported those species, as well as a fifth taxon endemic to South America. The five putative species are separated by substantial sequence (8.7-24.5%) and allozyme (0.36-1.54 Nei's distance) divergences, while intraspecific genetic diversity was generally limited in comparison. Although two of these species exhibited little morphological differentiation from their closest relatives, and diagnostic traits were not found among the characters considered, a population-level approach revealed significant morphological differences among all pairs of taxa. We therefore present both an allozyme key and a morphological/geographic key to all species, as well as new or augmented descriptions for all five species. H. gibberum s.s. is distributed in Europe and across arctic North America, while its cryptic sister species, H. glacialis n. sp., is widely distributed across temperate North America. H. amazonicum s.s. is apparently restricted to the Amazon basin, H. atlanticum n. sp. occurs in lakes along the eastern margin of North America, while H. acidophilum n. sp. occurs sporadically across North America along a narrow band of middle latitudes. Due to high mor-phological variability within species, as well as the detection of cryptic diversity, we suggest that genetic analyses should be performed on populations from other geographic regions and should always accompany the recognition of new spe-cies of Holopedium.
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Modern multilocus molecular techniques are a powerful tool in the detection and analysis of cryptic taxa. However, its shortcoming is that with allopatric populations it reveals phylogenetic lineages, not biological species. The increasing power of coalescent multilocus analysis leads to the situation in which nearly every geographically isolated or semi-isolated population can be identified as a lineage and therefore raised to species rank. It leads to artificial taxonomic inflation and as a consequence creates an unnecessary burden on the conservation of biodiversity. To solve this problem, we suggest combining modern lineage delimitation techniques with the biological species concept. We discuss several explicit principles on how genetic markers can be used to detect cryptic entities that have properties of biological species (i.e. of actually or potentially reproductively isolated taxa). Using these principles we rearranged the taxonomy of the butterfly species close to Polyommatus (Agrodiaetus) ripartii. The subgenus Agrodiaetus is a model system in evolutionary research, but its taxonomy is poorly elaborated because, as a rule, most of its species are morphologically poorly differentiated. The taxon P. (A.) valiabadi has been supposed to be one of the few exceptions from this rule due to its accurately distinguishable wing pattern. We discovered that in fact traditionally recognized P. valiabadi is a triplet of cryptic species, strongly differentiated by their karyotypes and mitochondrial haplotypes.
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Citation: Vershinina AO, Anokhin BA, Lukhtanov VA (2015) Ribosomal DNA clusters and telomeric (TTAGG)n repeats in blue butterflies (Lepidoptera, Lycaenidae) with low and high chromosome numbers. Comparative Cytogenetics 9(2): 161–171. Abstract Ribosomal DNA clusters and telomeric repeats are important parts of eukaryotic genome. However, little is known about their organization and localization in karyotypes of organisms with holocentric chromosomes. Here we present first cytogenetic study of these molecular structures in seven blue butterflies of the genus Polyommatus Latreille, 1804 with low and high chromosome numbers (from n=10 to n=ca.108) using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG) n telomeric probes. FISH with the 18S rDNA probe showed the presence of two different variants of the location of major rDNA clusters in Polyommatus species: with one or two rDNA-carrying chromosomes in haploid karyotype. We discuss evolutionary trends and possible mechanisms of changes in the number of ribosomal clusters. We also demonstrate that Polyommatus species have the classical insect (TTAGG) n telomere organization. This chromosome end protection mechanism probably originated de novo in small chromosomes that evolved via fragmentations.
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Sequence-based methods to delimit species are central to DNA taxonomy, microbial community surveys, and DNA meta-barcoding studies. Current approaches either rely on simple sequence similarity thresholds (OTU-picking) or on complex and compute-intensive evolutionary models. OTU-picking methods scale well on large data sets, but the results are highly sensitive to the similarity threshold. Coalescent-based species delimitation approaches often rely on Bayesian statistics and MCMC sampling, and can therefore only be applied to small data sets. We introduce the Poisson Tree Processes (PTP) model to infer putative species boundaries on a given phylogenetic input tree. We also integrate PTP with our Evolutionary Placement Algorithm (EPA-PTP) to count the number of species in phylogenetic placements. We compare our approaches to popular OTU-picking methods and the General Mixed Yule Coalescent (GMYC) model. For de novo species delimitation, the stand-alone PTP model generally outperforms GMYC as well as OTU-picking methods when evolutionary distances between species are small. PTP neither requires an ultrametric input tree, nor a sequence similarity threshold as input. In the open reference species delimitation approach, EPA-PTP yields more accurate results than de novo species delimitation methods. Finally, EPA-PTP scales on large datasets because it relies on the parallel implementations of the EPA and RAxML, thereby allowing to delimit species in high-throughput sequencing data. The code is freely available at www.exelixis-lab.org/software.html. Alexandros.Stamatakis@h-its.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics on-line.
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Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter- and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.
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Estimating genealogical relationships among genes at the population level presents a number of difficulties to traditional methods of phylogeny reconstruction. These traditional methods such as parsimony, neighbour-joining, and maximum-likelihood make assumptions that are invalid at the population level. In this note, we announce the availability of a new software package, TCS, to estimate genealogical relationships among sequences using the method of Templeton et al. (1992) .
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There is increasing evidence that most parapatric cryptic/sister taxa are reproductively compatible across their areas of contact. Consequently, the biological species concept, which assumes absence of interbreeding, is becoming a not so effective criterion in evolutionary ecology. Nevertheless, the few parapatric sister taxa showing complete reproductive barriers represent interesting models to study speciation processes and the evolution of reproductive isolation. In this study, we examined contact populations in northwestern Italy of two butterfly species, Zerynthia polyxena and Z. cassandra, characterized by different genitalic morphotypes. We studied levels of divergence among 21 populations distributed from Sicily to France using three genetic markers (the mitochondrial COI and ND1 genes and the nuclear wingless gene) and genitalic geometric morphometrics. Moreover, we performed species distribution modelling to estimate different climatic requirements of Z. polyxena and Z. cassandra. We projected climatic data into glacial maximum scenarios in order to verify if and to which extent glacial cycles could have contributed to speciation processes. Genetic and morphometric analyses identified two main groups. All specimens showed a concordant pattern of diversification, including those individuals sampled in the contact area. Haplotype distribution and climatic models showed that during glacial maxima both species experienced a strong range contraction and presumably remained separated into different microrefugia in southern France, in the Italian Peninsula and on the islands of Elba and Sicily. Long term separation was probably favoured by reduced dispersal ability and high phylopatry, while genitalic diversification probably favoured interbreeding avoidance. Conversely, the aposematic wing pattern remained almost identical. We compared our results with those obtained in other species and concluded that Z. polyxena and Z. cassandra represent a valuable model in the study of speciation.
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Moths of the family Tortricidae constitute one of the major microlepidopteran groups in terms of species richness and economic importance. Yet, despite their overall significance, our knowledge of their genome organization is very limited. In order to understand karyotype evolution in the family Tortricidae, we performed detailed cytogenetic analysis of Grapholita molesta, G. funebrana, Lobesia botrana, and Eupoecilia ambiguella, representatives of two main tortricid subfamilies, Olethreutinae and Tortricinae. Besides standard cytogenetic methods, we used fluorescence in situ hybridization for mapping of major rRNA and histone gene clusters and comparative genomic hybridization to determine the level of molecular differentiation of the W and Z sex chromosomes. Our results in combination with available data in the codling moth, Cydia pomonella, and other tortricids allow us a comprehensive reconstruction of chromosomal evolution across the family Tortricidae. The emerging picture is that the karyotype of a common ancestor of Tortricinae and Olethreutinae differentiated from the ancestral lepidopteran chromosome print of n = 31 by a sex chromosome-autosome fusion. This rearrangement resulted in a large neo-sex chromosome pair and a karyotype with n = 30 conserved in most Tortricinae species, which was further reduced to n = 28 observed in Olethreutinae. Comparison of the tortricid neo-W chromosomes showed differences in their structure and composition presumably reflecting stochasticity of molecular degeneration of the autosomal part of the neo-W chromosome. Our analysis also revealed conservative pattern of the histone distribution, which is in contrast with high rDNA mobility. Despite the dynamic evolution of rDNA, we can infer a single NOR-chromosome pair as an ancestral state not only in tortricids but probably in all Lepidoptera. The results greatly expand our knowledge of the genome architecture in tortricids, but also contribute to the understanding of chromosomal evolution in Lepidoptera in general.
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DNA barcoding-type studies assemble single-locus data from large samples of individuals and species, and have provided new kinds of data for evolutionary surveys of diversity. An important goal of many such studies is to delimit evolutionarily significant species units, especially in biodiversity surveys from environmental DNA samples. The Generalized Mixed Yule Coalescent (GMYC) method is a likelihood method for delimiting species by fitting within- and between-species branching models to reconstructed gene trees. Although the method has been widely used, it has not previously been described in detail or evaluated fully against simulations of alternative scenarios of true patterns of population variation and divergence between species. Here, we present important reformulations to the GMYC method as originally specified, and demonstrate its robustness to a range of departures from its simplifying assumptions. The main factor affecting the accuracy of delimitation is the mean population size of species relative to divergence times between them. Other departures from the model assumptions, such as varying population sizes among species, alternative scenarios for speciation and extinction, and population growth or subdivision within species, have relatively smaller effects. Our simulations demonstrate that support measures derived from the likelihood function provide a robust indication of when the model performs well and when it leads to inaccurate delimitations. Finally, the so-called single threshold version of the method outperforms the multiple threshold version of the method on simulated data: we argue that this might represent a fundamental limit due to the nature of evidence used to delimit species in this approach. Together with other studies comparing its performance relative to other methods, our findings support the robustness of GMYC as a tool for delimiting species when only single-locus information is available.
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This study provides a DNA barcode library for 1264 of the 1338 species of butterflies and larger moths (Rhopalocera and Macroheterocera) of Germany. These results arise from a research program established by the State of Bavaria which is constructing a DNA barcode library for all animal species within its territorial boundaries. Open access is provided to a data set that includes records for 3467 specimens (957 species) from Germany. An additional 307 species of the German fauna are represented by barcode data specimens collected in other European nations. Most (99 %) of the 957 species from the study area were found to possess diagnostic barcode sequences. A few taxa which apparently share DNA barcodes are discussed in detail. Deep intraspecific sequence divergences (> 2 %) were detected in 32 traditionally recognized species which are undergoing more detailed analysis to ascertain whether they represent cases of cryptic diversity. The study reinforces the effectiveness of DNA barcoding as a tool for monitoring biodiversity and for other applications reliant on species identification.
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The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sexlinked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterize