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DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity
Scientific Reports
Abstract and Figures
How common are cryptic species-those overlooked because of their morphological similarity? Despite its wide-ranging implications for biology and conservation, the answer remains open to debate. Butterflies constitute the best-studied invertebrates, playing a similar role as birds do in providing models for vertebrate biology. An accurate assessment of cryptic diversity in this emblematic group requires meticulous case-by-case assessments, but a preview to highlight cases of particular interest will help to direct future studies. We present a survey of mitochondrial genetic diversity for the butterfly fauna of the Iberian Peninsula with unprecedented resolution (3502 DNA barcodes for all 228 species), creating a reliable system for DNA-based identification and for the detection of overlooked diversity. After compiling available data for European butterflies (5782 sequences, 299 species), we applied the Generalized Mixed Yule-Coalescent model to explore potential cryptic diversity at a continental scale. The results indicate that 27.7% of these species include from two to four evolutionary significant units (ESUs), suggesting that cryptic biodiversity may be higher than expected for one of the best-studied invertebrate groups and regions. The ESUs represent important units for conservation, models for studies of evolutionary and speciation processes, and sentinels for future research to unveil hidden diversity.
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Supplementary resource (1)
... The correct delineation and identification of species is not always possible based on characters of external morphology alone, especially in such taxonomically challenging groups as Colias [23][24][25][26]. Over the last decades, the rapid development and implementation of new molecular techniques and approaches validated mitochondrial (e.g. ...
... Over the last decades, the rapid development and implementation of new molecular techniques and approaches validated mitochondrial (e.g. DNA barcodes) and nuclear DNA markers as a useful and efficient tool for species identification, detecting and analyzing cryptic diversity, revealing interspecific and deep-level relationships and phylogenetic structure in taxonomically challenging groups of insects, solving long-standing taxonomic problems [23,[27][28][29][30][31][32][33][34][35][36][37][38][39]. Despite broad and constantly growing usage of DNA-based techniques in butterfly taxonomy and molecular systematics, surprisingly few studies focused so far on the genus Colias [6, [42][43][44][45][46]. ...
... Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 November 2023 doi:10.20944/preprints202311.0363.v123 ...
The genus Colias Fabricius, 1807 includes numerous taxa and forms with uncertain status and taxonomic position. Among such taxa are Colias mongola Alphéraky, 1897 and Colias tamerlana Staudinger, 1897, interpreted in the literature either as conspecific forms, as subspecies of different but morphologically somewhat similar Colias species, or as distinct species-level taxa. Based on mitochondrial and nuclear DNA markers, we reconstruct a phylogeographic pattern of the taxa in question. We recover and include in our analysis DNA barcodes of the century-old type specimens, the lectotype of C. tamerlana deposited in the Natural History Museum (Museum für Naturkunde), Berlin, Germany (ZMHU), the paralectotype of C. tamerlana and the lectoptype of C. mongola deposited in the Zoological Institute, Russian Academy of Sciences, Saint-Petersburg, Russia (ZISP). Our analysis grouped all specimens within four (HP_I-HP-IV) deeply divergent but geographically poorly structured clades which did not support nonconspecifity of C. mongola–C. tamerlana. We also show that all studied females of the widely distributed haplogroup HP_II were infected with a single Wolbachia strain belonging to the supergroup B, while the males of this haplogroup, as well as all other investigated specimens, were not infected. Our data underline the relevance of large-scale sampling dataset analysis and the need of testing for Wolbachia infection to avoid erroneous phylogenetic reconstructions and species misidentification.
... This approach is particularly effective for animals, where the 5' region of mitochondrial cytochrome c oxidase subunit 1 (COI) aids both specimen identification and species discovery, while also revealing phylogeographic and other eco-evolutionary processes (e.g. [2][3][4][5][6]. Motivated by these applications and aided by cost-effective protocols, 9.1 million DNA barcodes were available on the Barcode of Life Data System 7 by January 2021. ...
... In this context, Lepidoptera, one of the most diverse insect orders, is the taxonomic group which has gained the most intensive analysis with over 1.4 million DNA barcodes (June 2020) and some of the largest regional barcode libraries available (e.g. 4,6,10,12,13 ). This fact reflects the wide interest in Lepidoptera, and their long use as a model system for evolutionary biology, as well as a flagship taxon for insect conservation and for assessing the impact of climate change. ...
... This latter value is certainly at the lower end of interspecific divergence values based on past estimates of interspecific divergence values for COI in Lepidoptera (e.g. 4,10,34,37,50 ). It is also worth emphasising that certain species which share barcodes (i.e. ...
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.
... The correct delineation and identification of species is not always possible based on characters of external morphology alone, especially in such taxonomically challenging groups as Colias [23][24][25][26]. Over the last decades, the rapid development and implementation of new molecular techniques and approaches validated mitochondrial (e.g., DNA barcodes) and nuclear DNA markers as a useful and efficient tool for species identification, detecting and analyzing cryptic diversity, revealing interspecific and deep-level relationships and phylogenetic structure in taxonomically challenging groups of insects, solving longstanding taxonomic problems [23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]. ...
... The correct delineation and identification of species is not always possible based on characters of external morphology alone, especially in such taxonomically challenging groups as Colias [23][24][25][26]. Over the last decades, the rapid development and implementation of new molecular techniques and approaches validated mitochondrial (e.g., DNA barcodes) and nuclear DNA markers as a useful and efficient tool for species identification, detecting and analyzing cryptic diversity, revealing interspecific and deep-level relationships and phylogenetic structure in taxonomically challenging groups of insects, solving longstanding taxonomic problems [23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]. Despite the broad and constantly growing usage of DNA-based techniques in butterfly taxonomy and molecular systematics, surprisingly few studies focused, so far, on the genus Colias [6,[42][43][44][45][46]. ...
The genus Colias Fabricius, 1807 includes numerous taxa and forms with uncertain status and taxonomic position. Among such taxa are Colias mongola Alphéraky, 1897 and Colias tamerlana Staudinger, 1897, interpreted in the literature either as conspecific forms, as subspecies of different but morphologically somewhat similar Colias species or as distinct species-level taxa. Based on mitochondrial and nuclear DNA markers, we reconstructed a phylogeographic pattern of the taxa in question. We recover and include in our analysis DNA barcodes of the century-old type specimens, the lectotype of C. tamerlana deposited in the Natural History Museum (Museum für Naturkunde), Berlin, Germany (ZMHU) and the paralectotype of C. tamerlana and the lectotype of C. mongola deposited in the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia (ZISP). Our analysis grouped all specimens within four (HP_I–HP_IV) deeply divergent but geographically poorly structured clades which did not support nonconspecifity of C. mongola–C. tamerlana. We also show that all studied females of the widely distributed haplogroup HP_II were infected with a single Wolbachia strain belonging to the supergroup B, while the males of this haplogroup, as well as all other investigated specimens of both sexes, were not infected. Our data highlight the relevance of large-scale sampling dataset analysis and the need for testing for Wolbachia infection to avoid erroneous phylogenetic reconstructions and species misidentification.
... For the analyses we used our own sequences as well as published sequences (nuclear sequences 28S, H3, EF1-a, wingless, and ITS2 and mitochondrial genes COI and COII) extracted from GenBank [6,9,16,20,21,[39][40][41][42][43] ( Table 2). The GenBank/BOLD/museum accession numbers of the analyzed sequences are presented in Figures 1-6. ...
... We live in a time when works based on genome-wide data are beginning to appear in insect taxonomy [e.g., 8,9], but at the same time, articles based on multilocus data (phylogenomic approach) [e.g., 6,16,20] or on single mitochondrial gene COI (DNA-barcoding approach) [39,40] still dominate. It seems to us that in between these methodologies is the mixed approach proposed by Talavera et al. [49] who demonstrated that DNA barcodes combined with multilocus data of representative taxa could generate reliable, higher-level phylogenies. ...
The Palearctic blue butterfly genus Pseudophilotes Beuret, 1958 is not homogenous regarding the morphology of its genital structures. For this reason, some of its species have been considered to be representatives of other genera of the subtribe Scolitantidina (subfamily Polyommatinae). Here, we address these taxonomic problems by analyzing the phylogenetic relationships between the genera, subgenera, and species of this subtribe inferred via the analysis of five nuclear and two mitochondrial DNA sequences. We demonstrate that the enigmatic Asian species P. panope (Eversmann, 1851) belongs to the genus Pseudophilotes but not to Praephilotes Forster, 1938 or Palaeophilotes Forster, 1938 and does not represent the independent genus Inderskia Korshunov, 2000, as hypothesized previously. We synonymize P. svetlana Yakovlev, 2003 (syn. nov.) and P. marina Zhdanko, 2004 (syn. nov.) with P. panope. We demonstrate a deep genetic divergence between lineages that were previously considered as subspecies of the single species Iolana iolas (Ochsenheimer, 1816). As a result, we confirm the multispecies concept of the genus Iolana Bethune-Baker, 1914. We show that the Holarctic genus Glaucopsyche can be divided into four subgenera: Glaucopsyche Scudder, 1872 (= Shijimiaeoides Beuret, 1958), Apelles Hemming, 1931, Bajluana Korshunov and Ivonin, 1990, and Phae-drotes Scudder, 1876.
... The primary resources we referred to are as follows: (1) the checklists of Western Palaearctic butterflies by Wiemers et al. (2018) and Middleton-Welling et al. (2020); and (2) a series of DNAbarcode libraries of Eurasiatic butterflies (Dapporto et al., 2019;Dincă et al., 2011Dincă et al., , 2015Dincă et al., , 2021Hausmann et al., 2011;Huemer & Tarmann, 2016;Litman et al., 2018;Lukhtanov et al., 2009;Menchetti et al., 2021). We revised the checklists and added 2541 specimens sequenced de novo, including a comprehensive DNA-barcode library for Maghreb and Macaronesia. ...
... We gathered COI sequences (DNA-barcode region between 400 and 658 bp) from three main sources: (1) de novo sequencing using standard procedures (deWaard et al., 2008) for most of the Maghreb and Macaronesia, but also improving coverage in Europe; (2) published DNA-barcode libraries compiled at the regional level (Dapporto et al., 2019;Dincă et al., 2011Dincă et al., , 2015Dincă et al., , 2021Hausmann et al., 2011;Huemer & Tarmann, 2016;Litman et al., 2018;Menchetti et al., 2021) and other publicly available BOLD projects (e.g., LON-NorBOL for Norway); and (3) studies providing COI of single species or genera (references are available in Appendix S1). In the last case, we checked whether haplotypes, instead of specimens, were included in repositories . ...
Motivation:
Butterflies represent a model in biology and a flagship group for invertebrate conservation. We provide four new resources for the Western Palaearctic butterflies: (1) an updated checklist comprising 552 species; (2) a curated dataset of 32,126 mitochondrial cytochrome c oxidase subunit I (COI) sequences for 532 species, including a de novo reference library for the Maghreb (Morocco, northern Algeria and Tunisia) and Macaronesia (Azores, Madeira and Canary Islands); (3) seven indexes of intraspecific genetic variation (IGV): observed and expected number of haplotypes, haplotype and nucleotide diversity, two fixation indexes and maximum p-distance; and (4) species-level maps illustrating the distribution of COI variability and haplotype networks. The updated checklist will be fundamental for any application dealing with butterfly diversity in the Western Palaearctic. The IGV indexes provide measures for genetic polymorphism and spatial structure and represent proxies for dispersal capacity. These resources will facilitate comparative studies of macrogenetics, foster integrative taxonomy and aid conservation strategies.
Main types of variables contained:
A complete species checklist in table format, 32,126 mitochondrial DNA barcodes provided with metadata (species membership, WGS84 coordinates and sequence length) and a book in PDF format, including the IGV atlas and indexes, are provided.
Spatial location and grain
The checklist encompasses Europe up to the Urals in the east, north Macaronesia (the Azores, Madeira and the Canary Islands) and the Maghreb (Morocco, northern Algeria and Tunisia). COI sequences have been retained in the geographical interval of −31.3 to 67.5° of longitude and 27.5–71.2° of latitude.
Time period and grain:
COI sequences originate from studies published between 1998 and 2022 and from de novo sequencing of 2541 specimens done between 2007 and 2022.
Major taxa and level of measurement:
Butterflies (Lepidoptera: Papilionoidea), analysed from individual to species level.
Software format:
Data and functions to manage the dataset are provided in the iodatabase R package (https://github.com/leondap/iodatabase) and in Dryad (https://doi.org/10.5061/dryad.9w0vt4bjj).
... Given the attractiveness of European butterflies and the long history of their study, we know a lot about their distributional changes, ecologies and life histories. This was followed by the emergence of national and international barcoding programs (e.g., Lukhtanov et al., 2009;Dinc a et al., 2011;Hausmann et al., 2011;Dinc a et al., 2015;Litman et al., 2018;Dinc a et al., 2021). In this well-known group, species delineation methods revealed a portion of polyphyletic, paraphyletic or barcode-sharing species. ...
... For example, Dinc a et al. (2011) showed that in 180 Romanian butterfly species, only 162 species formed separate barcode clusters, three species pairs shared barcodes, four species were paraphyletic, two species pairs were polyphyletic and eight species had intraspecific divergence (p-distances) over 2.0%, which, according to the minimalist approach, would merit their description as a complex of cryptic species for which morphology 'fails'. Similar numbers were obtained for other countries (Hausmann et al., 2011;Dinc a et al., 2015;Litman et al., 2018). Recently, a compilation of 22,306 sequences of 459 European butterfly species (97% of known European species) was analysed by Dinc a et al. (2021). ...
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.
... A major drawback of many phylogeography studies is that they rely solely on mitochondrial DNA markers -usually a 650 base pair (bp) fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene (e.g., Dapporto et al., 2017Dapporto et al., , 2019Dincă et al., 2015;Hebert et al., 2004;Lohman et al., 2010;Mendoza et al., 2016;Scalercio et al., 2020;Smith et al., 2006). Mitochondrial DNA (mtDNA) markers can follow an evolutionary trajectory independent of the nuclear DNA. ...
... Isles also raise the possibility of their influence on mtDNA phylogeography in continental Europe. Recent CO1 analyses reveal that many butterfly species exhibit diverse and complex mtDNA genealogies across Eurasia (Dincă et al., 2015). However, more comprehensive genomic analysis, that have only recently become available, show that differentiation in the nuclear genome is not always concordant with mtDNA variation Hinojosa et al., 2019;Tóth et al., 2017) and the observed mitonuclear discordance could be explained by past ...
The paradigm of isolation in southern refugia during glacial periods followed by expansions during interglacials, producing limited genetic differentiation in northern areas, dominates European phylogeography. However, the existence of complex structured populations in formerly glaciated areas, and islands connected to mainland areas during glacial maxima, call for alternative explanations. We reconstructed the mtDNA phylogeography of the widespread Polyommatus icarus butterfly with an emphasis on the formerly glaciated and connected British Isles. We found distinct geographical structuring of CO1 haplogroups, with an ancient lineage restricted to the marginal European areas, including Northern Scotland and Outer Hebrides. Population genomic analyses, using ddRADSeq genomic markers, also reveal substantial genetic structuring within Britain. However, there is negligble mito-nuclear concordance consistent with independent demographic histories of mitochondrial vs. nuclear DNA. While mtDNA-Wolbachia associations in northern Britain could account for the geographic structuring of mtDNA across most of the British Isles, for nuclear DNA markers (derived from ddRADseq data) butterflies from France cluster between northern and southern British populations – an observation consistent with a scenario of multiple recolonisation. Taken together our results suggest that contemporary mtDNA structuring in the British Isles (and potentially elsewhere in Europe) largely results from Wolbachia infections, however, nuclear genomic structuring suggests a history of at least two distinct colonisations. This two-stage colonisation scenario has previously been put forth to explain genetic diversity and structuring in other British flora and fauna. Additionally, we also present preliminary evidence for potential Wolbachia-induced feminization in the Outer Hebrides.
... We are not aware of any attempts to solve taxonomic problems in the genus Hyponephele using molecular data, although GenBank has scattered information on the mitochondrial DNA barcodes of some species (WAHLBERG et al. 2003;LUKHTANOV et al. 2009;DINCÃ et al. 2015;YANG & ZHANG 2015;FENG & NING 2012;LUKHTANOV & NOVIKOVA 2015;DAPPORTO et al. 2019). Although DNA barcode data alone has a limited value in taxonomy (DASMAHAPATRA et al. 2010;PAZHENKOVA & LUKHTANOV 2019), combining the morphological and DNA barcode data is an efficient method for creating new and testing old species-level taxonomic hypotheses (LUKHTANOV et al. 2016). ...
... We also used 39 published COI sequences (WAHL-BERG et al. 2003;LUKHTANOV et al. 2009;DINCÃ et al. 2015;YANG & ZHANG 2015;FENG & NING 2012;LUKHTANOV & NOVIKOVA 2015;DAPPORTO et al. 2019) which were downloaded from GenBank ( Table 1). The genus Cercyonis was found to be a sister to Hyponephele (ZHANG et al. 2020); therefore, C. pegala was used as an outgroup to root the phylogeny. ...
The genus Hyponephele includes about 40 species distributed throughout the southern part of the Palaearctic area. Within this genus, the taxa of the H. lycaon-H. lupina species complex are similar with respect to the wing pattern and genitalia structure. Here we revise this group using analysis of butterfly morphology, DNA barcodes, and study of the type material. We show that, with a few exceptions, the species in this group are allopatric in distribution. Allopatry in combination with phenotypic similarity may be theoretically interpreted as evidence for the conspecifity of these taxa. Here we falsify this hypothesis by using DNA barcode analysis. We show that the species of this complex are genetically very distant and cannot be combined together as a polytypic species. We also demonstrate that H. lupina consists of two deeply diverged allopatric clades, H. lupina s.s. and H. mauritanica comb. & stat. nov. The barcode p-distance between these taxa (3.4-4.9%) is significantly higher than the generally accepted 'standard' minimum interspecific divergence (2.0-3.0%) threshold. These two clades can also be distinguished by the color of the upperside of the wing in males (brown with conspicuous golden reflection in H. lupina; dark brown without golden reflection in H. mauritanica) and by details in male genitalia and male androconia structures. Syntypes of Hyponephele sifanica, H. cheena cheena, H. cheena iskander, and H. cheena kashmirica are studied and figured.
... Despite the enormous potential of DNA barcoding applications, particularly for assessing cryptic diversity, it is essential not to overlook the potential limitations of this method [6,55]. In many cases, genomic and/or morphological data are discordant with mtDNA genealogies, and barcoding may not always correctly identify species due to historical or ongoing hybridization and introgression, or in some cases, due to incomplete lineage sorting, pseudogenes, or Wolbachia infection [56][57][58][59]. ...
The fauna of Lepidoptera in the Mediterranean is still inadequately documented. As a result, even remotely complete DNA barcode libraries (mt. COI (cytochrome c oxidase 1) gene) are lacking in most areas. This proposed gap is being analyzed for the first time for the fauna of North Cyprus. In the initial phase, 248 morphospecies from 29 families (exclusive Heterocera) were sampled, sequenced and compared with existing DNA reference sequences in the global BOLD database (Barcode of Life Data Systems) via BINs (Barcode Index Numbers). A total of 194 species could be unequivocally assigned to a Linnaean taxon. Additionally, six species previously unidentified in BOLD, as well as fourteen species without reference barcodes, were identified at the species level. Twenty-four of these species were new records for Cyprus. In addition, 25 taxa with new BINs could not be assigned to a valid species due to potential cryptic diversity or the lack of relevant revisions. Furthermore, a few species could not be identified due to barcode sharing and/or potential misidentifications in BOLD. Overall, approximately 20% of the samples could not be identified using the existing DNA barcode libraries, a significant deficit for European standards, which should be addressed as a priority issue in future studies.
... In addition, the optimal similarity thresholds used to assign taxonomy and identify species (Bonin et al., 2023;Clare et al., 2017), as well as the impact of geographical distance and sampling intensity on intraspecific diversity need to be considered. Several studies have indicated that the intraspecific variation of COI tends to increase with larger geographical distributions in certain species (Huemer et al., 2018;Lukhtanov et al., 2009;Mutanen et al., 2012;Smith et al., 2005) and with increased sampling intensity (Dincă et al., 2015;Huemer et al., 2018). ...
For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single‐specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment.
... Among insects, butterflies are one of the best-studied groups (for example, Dincă et al., 2015;Middleton-Welling et al., 2020;Thomas, 2005;Warren, 1992;Warren et al., 2021), can be used as bioindicators of environmental and climate change (Dennis et al., 2003;Hill et al., 2021) and serve as an important invertebrate model for communicating science to society (Preston et al., 2021). Since the 1970s, standardized and systematic monitoring of butterflies across Europe has provided a comprehensive and continuous record of changes in biodiversity. ...
Current rates of habitat degradation and climate change are causing unprecedented declines in global biodiversity. Studies on vertebrates highlight how conservation genomics can be effective in identifying and managing threatened populations, but it is unclear how vertebrate‐derived metrics of genomic erosion translate to invertebrates, with their markedly different population sizes and life histories. The Black‐veined White butterfly ( Aporia crataegi ) was extirpated from Britain in the 1920s. Here, we sequenced historical DNA from 17 specimens collected between 1854 and 1924 to reconstruct demography and compare levels of genomic erosion between extirpated British and extant European mainland populations. We contrast these results using modern samples of the Common Blue butterfly ( Polyommatus icarus ); a species with relatively stable demographic trends in Great Britain. We provide evidence for bottlenecks in both these species around the period of post‐glacial colonization of the British Isles. Our results reveal different demographic histories and N e for both species, consistent with their fates in Britain, likely driven by differences in life history, ecology and genome size. Despite a difference, by an order of magnitude, in historical effective population sizes ( N e ), reduction in genome‐wide heterozygosity in A. crataegi was comparable to that in P. icarus . Symptomatic of A. crataegi 's disappearance were marked increases in runs‐of‐homozygosity (RoH), potentially indicative of recent inbreeding, and accumulation of putatively mildly and weakly deleterious variants. Our results provide a rare glimpse of genomic erosion in a regionally extinct insect and support the potential use of genomic erosion metrics in identifying invertebrate populations or species in decline.
... Despite an absence of in-depth studies focused on this species-pair, the morphological cladistic analysis of Cassulo et al. (1989) first hinted at their distinction at the species level through slight wing-pattern and genitalic differences, which later led (Bozano & Weidenhoffer 2001) to suggest raising this taxon to species rank. Only more recently, while carrying out a barcode meta-analysis of Iberian butterflies, have Dincȃ et al. (2015) provided the first genetic evidence of the two Sooty-Copper distinction. ...
The comparison of closely related taxa is cornerstone in biology, as understanding mechanisms leading up to differentiation in relation to extant shared characters are powerful tools in interpreting the evolutionary process. Hotspots of biodiversity such as the west-Mediterranean, where many lineages meet are ideal grounds to study these processes. We set to explore the interesting example of Sooty Copper butterflies: widespread Eurasian Lycaena tityrus (Poda, 1761) comes into contact in Iberia with closely related and local endemic, L. bleusei (Oberthür, 1884), which hasn’t always been considered a distinct species. An integrative analysis was designed, combining the use of extensive molecular data (five genes), geometric morphometrics analyses, verified and up-to-date distribution data, and environmental niche modelling, aimed at deciphering their true relationship, their placement within European Lycaena and trace their evolutionary history. We revealed several levels of differentiation: L. bleusei and L. tityrus appear to be reciprocally monophyletic independent gene-pools, distinct in all genes analysed, having mutually diverged 4.8 Ma ago. L. tityrus but not L. bleusei, further displays a genetic structure compatible with several glacial refugia, where populations assignable to infraspecific taxa surface. Conversely, L. bleusei shows a loss in mtDNA diversity in relation to nuDNA. Morphological analyses differentiate both species according to size and shape but also discriminate strong seasonal and sexual traits and a geographical phenotype segregation in L. tityrus. Finally, updated distribution and its modelling for current and glacial timeframes reveal both species respond differently to environmental variables, defining a mostly parapatric distribution and an overlapping belt where sympatry was recovered. During the last glacial maximum, a wider expansion in L. bleusei distribution explains current isolated populations. Our study highlights the importance of gathering several lines of evidence when deciphering the relationships between closely related populations in the fringe of cryptic species realm.
... La richesse spécifique la plus grande se concentre au niveau des régions néotropicales et indoaustralienne, avec cinq fois plus d'espèces par unité spatiale que dans le paléarctique (Merckx et al., 2013). Le nombre d'espèces de papillons de jour présentes en Europe n'est pas encore clairement établi, notamment du fait du statut contesté de certaines espèces cryptiques (Dinca et al., 2015). La majorité des évaluations récentes s'accordent sur une richesse comprenant entre 440 et 489 espèces (Van Swaay et al., 2010 ;Karsholt & Nieukerken, 2013 ;Kudrna et al., 2015). ...
Insects are still poorly considered in biodiversity conservation programs, despite their major role in the functioning of terrestrial ecosystems. Because of their great diversity, and a great lack of knowledge of community structuring patterns, the conservation of these organisms faces major challenges related to the evaluation and prioritization of issues as well as the estimation of the species richness of communities. Because they constitute two groups of insects particularly impacted by environmental changes, butterflies and dragonflies are an important part of protected insects in Europe. In this context of low consideration of entomological diversity in conservation biology, this thesis aims to (1) measure the recent dynamics (decline, stability or expansion) of these two taxa in France, and to identify the climatic and ecological factors likely to condition them, (2) for butterflies, to use a fine description of the interactions network between the larvae and their hostplants to question the conservation status according to a systemic approach (3) and finally, with a clearly operational objective, to evaluate the links between the information gains and the efforts / sampling costs mobilized during environmental impact assessments.
The comparative study of the temporal dynamics of the populations of these two taxa was carried out at the scale of three countries of Western Europe through a diachronic analysis of the species distribution patterns on a time step of 35 years. This work has highlighted a strong correlation between the artificialisation of landscapes (urbanization, agricultural intensification, regression of wetlands) and the decline of many species, mainly characterized by strict ecological requirements and currently unprotected. On this same scale, the analysis of the architecture of the interactions network between butterfly larvae and their food plants revealed a modular structure in relation to the taxonomy of the partners, as well as a link between the degree specialization and species vulnerability (although the similarity of the species' diet did not seem to condition their conservation status). A final part was focused on the evaluation of the completeness of entomological surveys carried out during environmental impact assessments. A systematic and calibrated return procedure, on sites sampled in Mediterranean scrublands, allowed highlighting the limits of the current protocols in the estimation of species richness and the detection of protected or vulnerable species.
This thesis contributes to the articulation between fundamental issues and operational needs, by allowing both a better understanding of the structuring mechanisms of insect communities and the formulation of recommendations for a better local application of conservation policies
... The putative reproductive isolation is reflected in the low admixture between these clusters in the STRUCTURE results and in the fact that all the specimens associated with Er. glandulosum shared a highly diverged mitochondrial lineage ( Figure S1), not present in any other sample. The minimum uncorrected genetic distance of this lineage with respect to the rest of specimens analysed was 2.58% for the mitochondrial marker COI, which is a high value for butterflies at intraspecific level (Dincă et al., 2015;Dincă et al., 2021;Huemer, Mutanen, Sefc, & Hebert, 2014); the observed mitochondrial lineage pattern could theoretically be an outcome of Wolbachia infection -Wolbachia bacteria are maternally inherited and may cause male-killing or cytoplasmic incompatibility (Werren, Baldo, & Clark, 2008), which can lead to some degree of reproductive isolation (Bordenstein et al., 2001;Shoemaker et al., 1999)-, but here all specimens analysed were apparently not infected. Considering that populations found over Er. glandulosum and their counterparts that use Geranium live nearby and both plants overlap in the blooming period, it is likely that meetings between individuals of these populations occur. ...
It is widely accepted that the relationship between phytophagous insects and their host plants influences insect diversification. However, studies addressed at documenting host-associated genetic differentiation (HAD) and the mechanisms that drive reproductive isolation in host-associated lineages (or host races) are still rare relative to insect diversity. To uncover further evidence on the HAD processes in Lepidoptera, we investigated the genetic structure of the geranium argus butterfly (Eumedonia eumedon) and tested for isolation by environment (IBE) versus isolation by distance (IBD). Genomic data revealed an array of host races (three of them at the same mountain range, the Cantabrian Mountains, northern Iberia) at apparently distinct levels of reproductive isolation. We found a pattern of IBE mediated by HAD at both local and European scales, in which genetic differentiation between populations and individuals significantly correlated with the taxonomic relatedness of the host plants. IBD was significant only when considered at the wider European scale. We hypothesize that, locally, HAD between Geranium-feeding populations was caused (at least partially) by allochrony, i.e. via adaptation of adult flight time to the flowering period of each host plant species. Nevertheless, the potential reproductive isolation between populations using Erodium and populations using Geranium cannot be explained by allochrony or IBD, and other mechanisms are expected to be at play.
... The Moroccan specimens held two unique haplotypes (Hap 4 and Hap 5) for the 16S rDNA sequences and a unique haplotype (Hap 1) for the cytochrome b gene. Similar results for Iphiclides podalirius L. (the other Papilionidae species), which inhabits both sides of the Gibraltar strait, were obtained by Wiemers and Gottsberger [62] and Dincă et al. [63], based on COI sequence analysis. They showed no significant differences in the sequence of this gene between I. podarilius specimens inhabiting various regions of Europe. ...
The Old World swallowtail Papilio machaon Linnaeus, 1758 is one of the most well-known and most characteristic members of the family Papilionidae. Over the past two centuries, the butterfly has been the subject of many studies. P. machaon is characterised by a tendency to change the wing colour pattern. In turn, due to the great interest of collectors and amateur entomologists, these studies have been converted into the description of over 100 colour forms, aberrations and subspecies. In this study, mitochondrial DNA (mtDNA), 16S rDNA and cytochrome b sequences were used to examine the correlation between the intraspecific classification and genetic structure of P. machaon. The study used 87 specimens from 59 different localities covering the geographic distribution of this species in the palaearctic. The phylogenetic relationships within and between the Old World swallowtail subspecies showed that the intraspecific classification proposed by various authors does not correlate with the variability in mitochondrial DNA sequences. In addition, populations occurring at the species distribution borders in the palaearctic region (i.e., Japan, Kamchatka, Morocco and Sakhalin) are genetically distinct from other species.
... The genus Iphiclides belongs to the tribe Leptocircini (kite swallowtails), which diverged from other taxa in the subfamily Papilioninae about 55 million years ago (Allio, Scornavacca et al. 2020), and only includes two other species: I. podalirinus, which is restricted to Central Asia and Iphiclides feisthamelii, the sister taxon of I. podalirius, which is found in Northern Africa and the Iberian Peninsula. A controversy about the taxonomic status of I. feisthamelii, which has been regarded as a subspecies of I. podalirius (Godart and Duponchel 1832;Tolman and Lewington 2009;Wiemers and Gottsberger 2010), has only recently been resolved; although the two species have no known differences in ecology or life history and share mitochondrial haplotypes (Dinc a et al. 2015), Gaunet et al. (2019) show that they differ consistently in wing patterns (including UV reflectance of males), genital morphology and nuclear DNA and are separated by a narrow hybrid zone in Southern France (Descimon and Mallet 2009). ...
The scarce swallowtail, Iphiclides podalirius (Linnaeus, 1758), is a species of butterfly in the family Papilionidae. Here we present a chromosome-level genome assembly for I. podalirius as well as gene and transposable element annotations. We investigate how the density of genomic features differ between the 30 I. podalirius chromosomes. We find that shorter chromosomes have higher heterozygosity at fourfold-degenerate sites and a greater density of transposable elements. While the first result is an expected consequence of differences in recombination rate, the second suggests a counter-intuitive relationship between recombination and transposable element evolution. This high quality genome assembly, the first for any species in the tribe Leptocircini, will be a valuable resource for population genomics in the genus Iphiclides and comparative genomics more generally.
... The sequences of the COI region available in public databases for L. camilla form two groups differing for ca 2.7% (uncorrected p-distance), so that Dincă et al. (2015) considered a possibility of existence of two sympatric cryptic species. In our study, sequences of both groups were co-amplified from some specimens. ...
Three species of the genus Limenitis (Nymphalidae) (L. camilla, L. helmanni, L. sydyi) have split ranges in the Palearctic. Their disjunction was dated either to the Pleistocene or to the Subboreal time of the Holocene. This genus also exhibits an amphiberingean disjunction, L. populi vs four Nearctic species. To evaluate the disjunction time in Eurasia, we analysed a fragment of the mitochondrial COI gene and a major part of the histone H1 gene. The former was sequenced in 51 specimens of three species with Palearctic disjunctions. We detected a diverged nuclear copy of the COI gene in L. camilla. The histone H1 gene was sequenced in 64 specimens of 8 species. In five species, intra-species and intra-individual nucleotide substitutions and variation in the number of intra-genic repeats were observed and studied by cloning of individual gene copies, with individuals found with more than two variants. With 30–80 copies of histone H1 gene in Limenitis genomes, as estimated by real time PCR, this was interpreted as cis-heterogeneity across the histone gene cluster. No fixed differences between the western and eastern range parts were found in L. helmanni, L. camilla and L. sydyi, although in the former more alleles of both sequences were found in the eastern part. This suggests the range disjunctions to be too recent to be dated by molecular means and they may only be estimated to have taken place not more than 77–100 tya. This fits their provisional dating by Dubatolov and Kosterin (Entomologica Fennica 11(3), 141-161, 2000) to the Subboreal time of the Holocene.
... Butterfly taxonomy is nowadays relatively well studied despite the fact that recent molecular approaches to its study are providing notable novelties that can substantially modify the Fig. 3 Interannual trends of chlorophyll a in Lake La Caldera. Data source Sánchez-Castillo (1986), Carrillo (1989), Villar-Argaiz (1999), traditional taxonomic scenario (Dincă et al. 2015). These are some of the reasons of why butterflies are model organisms to study ecological problems and to be incorporated to a citizen science scheme. ...
Understanding the effects ofClimateclimate changeClimate change and human activities on fragile mountain ecosystems is necessary to successfully managing these environments under future climateClimate scenarios (e.g., global warming, enhanced aridity). This can be done through the study of paleoecological records, which can provide long paleoenvironmental databases containing information on how ecosystems reacted toClimateclimate changeClimate change and human disturbances before the historical record. These studies can be particularly interesting when focusing on especially warm and/or dry past climatic phases. Biotic (pollen, charcoal) and abiotic (physical, geochemistry) analyses from wetland sediment records from the Sierra NevadaSierra Nevada, southern SpainSpainrecordSouthern spain changes in vegetation, fire historyHistory and lake sedimentation since ~11,700 years (cal yr BP). This multiproxy paleoecological study indicates that maxima in temperatureTemperature and humidity occurred in the area in the Early and Middle HoloceneHolocene, with a peak in precipitationPrecipitation between ~10,500 and 7000 cal yr BP. This is deduced by maxima in water runoff, the highest abundance of tree species and algae and high total organic carbon values recorded in the alpine wetland’s sedimentary records of the Sierra NevadaSierra Nevada during that time period. In the last 7000 cal yr BP, and especially after a transition period between ~7000 and 5000 cal yr BP, a progressive aridification process took place, indicated by the decrease in tree species and the increase in xerophytic herbs in this region and a reduction in water runoff evidenced by the decrease in detritic input in the wetland sedimentary records. An increasing trend inSaharan dustSaharan dust depositionSaharan dust deposition in the Sierra NevadaSierra Nevada wetlands is also recorded through inorganic geochemical proxies, probably due to a coetaneous loss of vegetation cover in North Africa. The process of progressive aridification during the Middle and Late HoloceneHolocene was interrupted by millennial-scale climatic oscillations and several periods of relative humid/droughty conditions and warm/cold periods have been identified in different temperatureTemperatureand/or precipitationPrecipitation proxies. Enhanced human impactHuman impact has been observed in the Sierra NevadaSierra Nevada in the last ~3000 cal yr BP through the increase in fires, grazing, cultivation, atmospheric pollution as well as reforestation by Pinus and the massive cultivation of Olea at lower altitudes.
... Butterfly taxonomy is nowadays relatively well studied despite the fact that recent molecular approaches to its study are providing notable novelties that can substantially modify the Fig. 3 Interannual trends of chlorophyll a in Lake La Caldera. Data source Sánchez-Castillo (1986), Carrillo (1989), Villar-Argaiz (1999), González-Olalla et al. (2018) Advancing Open Science in Sierra Nevada … traditional taxonomic scenario (Dincă et al. 2015). These are some of the reasons of why butterflies are model organisms to study ecological problems and to be incorporated to a citizen science scheme. ...
Citizen scienceCitizen science is a very valuable complement to scientific efforts of tracking environmental changeEnvironmental change. The contribution of volunteers in scientific research is growing in Sierra NevadaSierra Nevada, and we here report three pilot experiences that include research on high mountain glacial lakesLakes, butterfliesButterflies, and streamStreammacroinvertebratesMacroinvertebrates. So far, participation has mainly led to the involvement of citizens into routine monitoring programsMonitoring programs, alleviating personnel and resource shortcomings. A necessary step forward in open scienceOpen Science is to develop a roadmap to extend dissemination by storytelling and outreach activities that reach civil society. By doing so, we expect to raise a new alliance, one that closes the gap between the necessary rapprochement among science, citizens, and environment managers.
... García-Barros et al., 2013;Tshikolovets, 2011;Wiemers et al., 2018). Its taxonomic status and its placement within the charlonia group (subgenus Elphinstonia)which, according to Back et al. (2006), includes E. bazae, E. charlonia, E. lucilla Butler, 1886, E. penia (Freyer, 1852 and E. transcaspica (Staudinger 1892)is supported by mitochondrial DNA barcodes (Back et al., 2006;Dinc a et al., 2015Dinc a et al., , 2021, but no nuclear DNA evidence has been published. ...
Although both positive (expansion of thermophilous species) and negative effects (retraction of cold‐adapted species) have been attributed to global warming, range shifts may be constrained by the ecological traits of species.
This can be especially true for highly specialised organisms like the Spanish Greenish Black‐tip ( Euchloe bazae ), an Iberian endemic specialist of semiarid steppes with a debated taxonomic and conservation status.
Here, we first seek to clarify the taxonomic status of this butterfly and its populations by using multilocus phylogenetic inference. Then, we update its distribution range and employ ecological niche modelling, combined with other sources of data, to re‐evaluate its conservation status.
Our results confirm E. bazae as a well‐differentiated species with one of the most restricted distribution ranges among the European butterflies.
We demonstrate that its two disjunct populations, regarded as subspecies, are genetically differentiated and should be treated as independent management units.
Climate models under two future emission scenarios suggest an increase of the area climatically suitable. However, the suitability of the areas currently occupied is estimated to decrease, meaning that rapid range shifts could be required for the survival of the species.
Given the presumably low dispersal capabilities of E. bazae , its current restricted distribution, high degree of population fragmentation (divided in two distant populations), low intrapopulation genetic variability, decline in extension and abundance, low densities, high yearly fluctuations, and numerous threats to its habitat, we consider that their populations are extremely fragile and we propose to consider it ‘Endangered’ [EN B1ab(i,ii,iii,iv); B2ab(i,ii,iii,iv)].
... The genus Iphiclides belongs to the tribe Leptocircini (kite swallowtails), which diverged from other taxa in the subfamily Papilioninae about 55 million years ago (Allio, Scornavacca et al. 2020), and only includes two other species: I. podalirinus, which is restricted to Central Asia and Iphiclides feisthamelii, the sister taxon of I. podalirius, which is found in Northern Africa and the Iberian Peninsula. A controversy about the taxonomic status of I. feisthamelii, which has been regarded as a subspecies of I. podalirius (Godart and Duponchel 1832;Tolman and Lewington 2009;Wiemers and Gottsberger 2010), has only recently been resolved; although the two species have no known differences in ecology or life history and share mitochondrial haplotypes (Dinc a et al. 2015), Gaunet et al. (2019) show that they differ consistently in wing patterns (including UV reflectance of males), genital morphology and nuclear DNA and are separated by a narrow hybrid zone in Southern France (Descimon and Mallet 2009). ...
The scarce swallowtail, Iphiclides podalirius (Linnaeus, 1758), is a species of butterfly in the family Papilionidae. Here we present a chromosome-level genome assembly for I. podalirius as well as gene and transposable element annotations. We estimate heterozygosity within different partitions of the genome and find a negative correlation between chromosome length and heterozygosity at fourfold-degenerate sites. This high quality genome assembly, the first for any species in the tribe Leptocircini, will be a valuable resource for population genomics in the genus Iphiclides and comparative genomics more generally.
... As a direct consequence of only a few indigenous barcodes, we found that the pairwise genetic distance of non-indigenous barcodes is 3 to 5 times that of indigenous barcodes. This directly reduces the ability of DNA-based taxon identification to assess real biodiversity in ecosystems (Dinca et al., 2015;Jackson et al., 2014;Sweeney et al., 2011). Moreover, spatial scale coverage of barcodes (also called barcode maps) is not only important to explain the genetic diversity related to geographical distance (Bergsten et al., 2012;Manel et al., 2020;Millette et al., 2020), but can also at least provide scholars and managers with priority areas to fill the gaps in species barcodes. ...
DNA-based taxon identification is improving the assessment and management of biodiversity in rivers. However, the lack of comprehensive DNA barcode reference libraries and globally highly unequal coverage are still hindering the application prospects of this method worldwide. Here, we analyzed the COI barcode gap in two reference libraries, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on three aquatic animal groups (freshwater fish, aquatic insects and molluscs) in Chinese rivers. Our data show gaps in barcode coverage (e.g., organisms without barcodes) of ca. 40–70% of taxa in these groups in the BOLD or NCBI GenBank database, respectively. These gaps can rise even further if the barcode thresholds are set to contain at least five reference sequences per taxon. Furthermore, most barcodes are from non-local samples, and only 14.4% (BOLD) and 28.8% (NCBI GenBank) of reference sequences were from organisms sampled in China, respectively. The pairwise genetic distance of local barcodes is 3 to 5 times lower than non-local barcodes, indicating that the latter may not be a good substitute. When looking at individual catchments, ca. 60% of the potentially occurring aquatic species have one or more barcodes, yet the barcode coverage varies slightly across ten major river catchments, ranging from 54.3% (Liao River basin) to 68.2% (Huai River basin). The taxa Salmoniformes and Perciformes in freshwater fish, Odonata and Diptera in aquatic insects, and Bivalvia in molluscs have the best barcode coverage in most catchments (mean coverage >70%). This study gives the first overview and current status of barcode reference libraries of three major aquatic animal groups in Chinese rivers. Our results will help to better interpret current metabarcoding studies from China, and also provide a basis to develop a strategy of filling the gaps in the reference libraries of aquatic species in China.
... Melitaea athalia and M. celadussa are among the most widespread and common European butterfly species [18,20,21,40,41]. However, their relationship has been the subject of considerable interest [15,16,42] due to their intermediate feature displays and occasional mito-morphological discordance at the contact zone. ...
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.
... Due to their maternal inheritance, high evolutionary rates and reduced effective population sizes, mitochondrial genes have been widely useful for population genetics studies (Avise 2009;Beheregaray 2008). The mitochondrial genome (mitogenome) also harbors the standard animal DNA barcode marker, which has contributed to the identification and discovery of thousands of animal species (Machado et al. 2017;Iftikhar et al. 2016;Tyagi et al. 2019;Dinca et al. 2015;Bezeng and Van Der Bank 2019). Also, the conserved gene organization of the mitogenome can provide important phylogenetic signal for highest metazoan orders (Boore 1999), whereas the less expansive nature of its content revealed an important tool for phylogenomic studies at different phylogenetic scales (Bernt et al. 2013;Cameron 2014;Junqueira et al. 2016;De Ré et al. 2017). ...
The Zygothrica genus group of Drosophilidae encompasses more than 437 species and five genera. Although knowledge regarding its diversity has increased, uncertainties about its monophyly and position within Drosophilidae remain. Genomic approaches have been widely used to address different phylogenetic questions and analyses involving the mitogenome have revealed a cost-efficient tool to these studies. Thus, this work aims to characterize mitogenomes of three species of the Zygothrica genus group (from the Hirtodrosophila, Paraliodrosophila and Zygothrica genera), while comparing them with orthologous sequences from other 23 Drosophilidae species and addressing their phylogenetic position. General content concerning gene order and overlap, nucleotide composition, start and stop codon, codon usage and tRNA structures were compared, and phylogenetic trees were constructed under different datasets. The complete mitogenomes characterized for H. subflavohalterata affinis H002 and P. antennta present the PanCrustacea gene order with 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein coding genes and an A+T rich region with two T-stretched elements. Some peculiarities such as the almost complete overlap of genes tRNAH/ND4, tRNAF/ND5 and tRNAS2/ND1 are reported for different Drosophilidae species. Non-canonical secondary structures were encountered for tRNAS1 and tRNAY, revealing patterns that apply at different phylogenetic scales. According to the best depiction of the mitogenomes evolutionary history, the three Neotropical species of the Zygothrica genus group encompass a monophyletic lineage sister to Zaprionus, composing with this genus a clade that is sister to the Drosophila subgenus.
... To test the taxonomic status of recovered clades, we used two species delimitations methods, which are commonly used for detection of species-level clusters in taxonomic studies of various groups of insects, including butterflies (Talavera et al., 2013a;Dincă et al., 2015): the automatic barcode gap discovery (ABGD) method (Puillandre et al., 2012) and the generalized mixed Yule coalescent (GMYC) model (Pons et al., 2006;Fujisawa & Barraclough, 2013). For the ABGD test, we used COI alignment from the phylogenetic analysis, excluding outgroups, that was trimmed to the length of the shortest sequence (610 bp) in MEGA7 (Kumar et al., 2016). ...
The tribe Tomarini is represented by the sole genus Tomares, comprising about eight species distributed from the western Mediterranean to Central Asia. We carried out a multilocus phylogenetic and a biogeographical analysis to test the taxonomy of the genus by several molecular species delimitation methods and reveal patterns shaping the current distribution of Tomares. The phylogenetic analysis based on four molecular markers recovered the monophyly of the genus and recovered two deep-branching lineages: an African clade and an Asian clade. Species delimitation analyses suggested six or ten putative species depending on the method applied. The haplotype network analysis of the Tomares nogelii clade revealed no phylogeographical and taxonomic structure. We consider the taxon Tomares nesimachus (syn. nov.) a synonym of T. nogelii and reinstate Tomares callimachus dentata stat. rev. for populations from south-eastern Turkey. Tomares originated between the early Oligocene and the early Miocene, most probably in south-west Asia. The split of the most recent common ancestor of Tomares occurred between the middle-late Miocene and middle-late Pliocene, probably as a response to increasing aridification and habitat fragmentation. Differentiation of the Asian clade took place in south-west Asia during the Pliocene and Pleistocene and coincided temporally with the evolution of Tomares host plants of the genus Astragalus (Fabaceae).
... Molecular diet analysis through DNA metabarcoding has recently overcome this problem (Pompanon et al. 2012, Nielsen et al. 2018, by simultaneously providing species level identification of hundreds of prey and the capacity to processing several hundred samples in a short period, and therefore allowing the reconstruction of insectivore diets with relative ease (Galan et al. 2018, Gordon et al. 2019. Moreover, reduced costs, coupled with technical refinements such as multimarker approaches (da Silva et al. 2019) and the availability of ever more comprehensive barcode databases (e.g., Dinc a et al. 2015, Hendrich et al. 2015, are making this method increasingly powerful and readily available for practical applications. Using DNA metabarcoding and ecological networks to inform landscape and conservation management has already been proposed (Evans et al. 2016), but the approach remains largely unexplored. ...
In multifunctional landscapes, diverse communities of flying vertebrate predators provide vital services of insect pest control. In such landscapes, conservation biocontrol should benefit service‐providing species to enhance the flow, stability and resilience of pest control services supporting the production of food and fiber. However, this would require identifying key service providers, which may be challenging when multiple predators interact with multiple pests. Here we provide a framework to identify the functional role of individual species to pest control in multifunctional landscapes. First, we used DNA metabarcoding to provide detailed data on pest species predation by diverse predator communities. Then, these data were fed into an extensive network analysis, in which information relevant for conservation biocontrol is gained from parameters describing network structure (e.g., modularity) and species roles in such network (e.g., centrality, specialization). We applied our framework to a Mediterranean landscape, where 19 bat species were found to feed on 132 insect pest species. Metabarcoding data revealed potentially important bats that consumed insect pest species in high frequency and/or diversity. Network analysis showed a modular structure, indicating sets of bat species that are required to regulate specific sets of insect pests. A few generalist bats had particularly important roles, either at network or module levels. Extinction simulations highlighted six bats, including species of conservation concern, which were sufficient to ensure that over three‐quarters of the pest species had at least one bat predator. Combining DNA metabarcoding and ecological network analysis provides a valuable framework to identify individual species within diverse predator communities that might have a disproportionate contribution to pest control services in multifunctional landscapes. These species can be regarded as candidate targets for conservation biocontrol, although additional information is needed to evaluate their actual effectiveness in pest regulation.
... Among insects, the order Lepidoptera has received particular intensive study, work which has established that COI is very effective for species discrimination and specimen identification. As a result, DNA barcodes have been used for large-scale assessments of cryptic diversity and geographic patterns of genetic variation in Lepidoptera (Dincă et al., 2015;Hausmann et al., 2013;Huemer, Mutanen, Sefc, & Hebert, 2014;, Gaytán et al., 2020, as well as to enable rapid diversity inventories and to delineate putative species in poorly-known groups (Kekkonen & Hebert, 2014;Zenker et al., 2016). ...
Prior studies of butterfly diversification in the Neotropics have focused on Amazonia and the tropical Andes, while southern regions of the continent have received little attention. To address the gap in knowledge about the Lepidoptera of temperate South America, we analyzed over 3,000 specimens representing nearly 500 species from Argentina for a segment of the mitochondrial COI gene. Representing 42% of the country’s butterfly fauna, collections targeted species from the Atlantic and Andean forests, biodiversity hotspots that were previously connected but are now isolated. We assessed COI effectiveness for species discrimination and identification and how its performance was affected by geographic distances and taxon coverage. COI data also allowed to study patterns of genetic variation across Argentina, particularly between populations in the Atlantic and Andean forests. Our results show that COI discriminates species well, but that identification success is reduced on average by ~20% as spatial and taxonomic coverage rises. We also found that levels of genetic variation are associated with species’ spatial distribution type, a pattern which might reflect differences in their dispersal and colonization abilities. In particular, intraspecific distance between populations in the Atlantic and Andean forests was significantly higher in species with disjunct distributions than in those with a continuous range. All splits between lineages in these forests dated to the Pleistocene, but divergence dates varied considerably, suggesting that historical connections between the Atlantic and Andean forests have differentially affected their shared butterfly fauna. Our study supports the fact that large-scale assessments of mitochondrial DNA variation are a powerful tool for evolutionary studies.
... To date, only a few studies have used DNA barcoding analyses to aid in, identify, and distinguish sponge species in South Africa (Matcher et al., 2017;Samaai et al., 2017;2020;Walmsley et al., 2012). When this molecular tool is linked to authoritatively identified voucher specimens (Bingpeng et al., 2018;Chakraborty & Ghosh, 2014;Deca€ ens et al., 2016;Dinc a et al., 2015;Hebert et al., 2003Hebert et al., , 2004Lakra et al., 2011;Moura et al., 2008;Ratnasingham & Hebert, 2013), it ensures results of the highest quality currently available. ...
DNA barcoding based on a fragment of the cytochrome c oxidase subunit I (COI) gene from the mitochondrial genome is widely applied in species identification, species discovery and biodiversity studies. The aim of this study was to establish a barcoding reference database of sponges collected from South Africa, and evaluate the applicability of the COI gene for aiding in the identification of sponges in combination with tentative morphological identifications. A total of 317 mitochondrial COI barcode sequences, with an additional 21 extended COI fragments and 24 nuclear ITS sequences, were obtained from 11 orders, 38 families, 58 genera and 124 species of spiculated sponges. A Neighbour Joining (NJ) trees that were reconstructed using these sequences in most cases clustered species in accordance with their current taxonomic identification, and we conclude that COI sequencing can be used to aid in the identification of sponge species. We further demonstrate that DNA barcoding analysis has potential to uncover cryptic sponge species, and to reveal dubious morphological identifications. We recommend that future taxonomic studies of South African sponges incorporate multiple sources of information for species identification or discovery.
... Potential cryptic taxa were recognized when the results of the three methods (ABGD, GMYC, and PTP) were identical. This approach for defining potential cryptic butterfly taxa was proposed by Dincă et al. (2015), comparing the species identified using the current taxonomy with those MOTUs recovered by the three different analysis approaches and classifying them into one of three categories: (1) Single entity: species that were recovered as a single entity, indicating correspondence with current taxonomy; (2) Lumped: specimens of two or more species that were recovered as a single entity; (3) Multiple entities: species that were split into two or more entities, which represent potential cryptic biodiversity. ...
Cryptic biological diversity has generated ambiguity in taxonomic and evolutionary studies. Single-locus methods and other approaches for species delimitation are useful for addressing this challenge, enabling the practical processing of large numbers of samples for identification and inventory purposes. This study analyzed one assemblage of high Andean butterflies using DNA barcoding and compared the identifications based on the current morphological taxonomy with three methods of species delimitation (automatic barcode gap discovery, generalized mixed Yule coalescent model, and Poisson tree processes). Sixteen potential cryptic species were recognized using these three methods, representing a net richness increase of 11.3% in the assemblage. A well-studied taxon of the genus Vanessa , which has a wide geographical distribution, appeared with the potential cryptic species that had a higher genetic differentiation at the local level than at the continental level. The analyses were useful for identifying the potential cryptic species in Pedaliodes and Forsterinaria complexes, which also show differentiation along altitudinal and latitudinal gradients. This genetic assessment of an entire assemblage of high Andean butterflies (Papilionoidea), provides baseline information for future research in a region characterized by high rates of endemism and population isolation.
... Beside molecular identification of already known species, species delimitation tools in DNA taxonomy allow inferring hypothetic species and/or evolutionary significant units from molecular data (Puillandre, Lambert, Brouillet, & Achaz, 2012;Tang, Humphreys, Fontaneto, & Barraclough, 2014). Species delimitation molecular methods are used for both biodiversity investigation without a priori hypothesis on the possible species (Dincă et al., 2015;Gómez-Zurita, 2016) or, more frequently, as support to resolve taxonomic issue when other delimitation approaches give uncertain results (e.g., Montagna et al., 2016;Kajtoch, Montagna, & Wanat, 2018;Plewa et al., 2018;García-Melo et al., 2019), in the framework of the so-called "integrative taxonomy" approach (Dayrat, 2005). ...
In the contest of global biodiversity loss, molecular species delimitation approaches can be very useful for accelerating species discovery through DNA taxonomy and inventory through DNA metabarcoding. In this study, the effect of some intrinsic factors on the efficiency of various single‐marker species delimitation methods (fixed and variable nucleotide distance thresholds, ABGD, ASAP, GMYC, mPTP) was tested on more than 90 empirical datasets, derived from a set of 7,237 COI sequences attributed to 542 leaf beetles species (Coleoptera: Chrysomelidae). The considered factors were: i) the number of haplotypes per species (as a proxy for genetic diversity); ii) the geographic distance among conspecific collection localities (as a proxy of sampling width); iii) the difficulty related to morphological identification of species; iv) the taxonomic rank. Distance‐based methods, with on average more than 70% of match with morphological identification, outperformed those relying on phylogenetic trees, with less than 59%. A high number of haplotypes per species was found to have a negative effect on delimitation efficiency, whereas large geographic distances within species had a positive effect. All methods delimitations (except for GMYC) were significantly affected by the presence of species that are difficult to be identified, decreasing their efficiency. Finally, the only method influenced by the taxonomic rank of the dataset was GMYC, showing lower efficiency in datasets at the genus than at higher levels. The observed biases we highlighted affecting efficiency could be accounted for when developing input datasets for species delimitation analyses to obtain a more reliable representation of biological diversity.
Aim
The distribution of within-species lineages has been affected by Quaternary climate changes, and population differentiation has been influenced by species life history traits. We investigated whether the distribution of individual mitochondrial genetic lineages reflects the constituent species' traits. Using the functionally diverse group of butterflies, we examined which lineages are present in Central Europe, an important suture zone.
Location
Czech Republic and Western Palearctic.
Taxon
A total of 140 butterfly species.
Methods
We sequenced DNA barcodes (cytochrome c oxidase 1) (959 sequences) of the entire Czech Republic butterfly fauna and used Barcoding of Life Data System to visualise the species' biogeographic patterns across Europe. We categorised the distribution patterns of lineages inhabiting the Czech Republic, and used multivariate statistics to interpret these categories by the butterflies' habitats, life history traits and threat levels.
Results
Open habitat dwellers with specialist traits belonged to Eastern, Southern and temperate lineages. Habitat generalists and woodland dwellers belonged to the Western lineage, formed several lineages or displayed low genetic diversity; they often developed on woody plants, were large-winged and had long flight periods. The most threatened species were the specialists of Southern, Eastern and temperate lineages.
Main Conclusions
The distribution of lineages in Central Europe reflects the history of Quaternary ecosystems: during cold periods of the Pleistocene, the diverse open habitats prevailed and species could expand westwards. Such species also suffer the most under the current anthropogenic habitat alteration. On the other hand, the mobile generalists and woodland dwellers expanded to Central Europe during the Holocene. Our approach of linking the distribution of lineages with species traits can be transferred to other study systems, and we show that DNA barcoding of under-sampled areas represents a powerful tool for discovering the driving forces of biogeography.
Face aux changements globaux actuels, l’enjeu des suivis de la dynamique de la biodiversité est croissant et entraîne une forte demande d’évaluations rapides et détaillées des changements de biodiversité. L’identification moléculaire des espèces est de plus en plus utilisée pour remplacer ou compléter les méthodes de surveillance écologique plus classiques. Le metabarcoding est considéré comme un outil d’inventaire, de connaissance de la biologie (prédateurs proies, pollinisateurs, etc.) et même de la découverte de l’histoire d’écosystèmes. Il permet de générer des données sur la biodiversité de manière rapide, précise et fiable, sur un large éventail d’organismes. Ce type de méthodologie est particulièrement intéressant pour les observatoires dépourvus de l’expertise nécessaire pour distinguer les nombreuses espèces de groupes hyper diversifiés comme les insectes ou ceux difficiles à inventorier. La reconnaissance des espèces à partir de l’ADN d’échantillons environnementaux (ADNe), tels que l’eau, les sédiments, le sol, l’air ou diverses matières organiques, a un large champ d’application. Par son caractère non invasif et non destructif, ces approches sont importantes pour l’évaluation déontologique de la biodiversité. Les chercheurs intègrent de plus en plus l’ADNe dans leurs études pour la biosurveillance en raison de sa précision et de sa facilité de déploiement. Dans ce document, nous donnons un aperçu des champs d’application des méthodes basées sur l’ADN pour le suivi de la biodiversité, des méthodes d’acquisition des données, de traitement des données pour la classification des espèces, et nous évoquons les défis inhérents à chacune de ces étapes.
1. The association of darker, less reflective insect wings with cooler environments (Bogert’s rule) is thought to be related to adult thermoregulation, but the adaptive explanation and the implications for sensitivity to climate warming are yet to be tested. We re-evaluate the pattern for butterflies using finer resolution data than in previous approaches, both geographically and morphologically, and test its correlation with recent evidence of impacts of warming on butterflies.
2. We compared reflectance–climate relationships at different grid sizes, selected the best subset of reflectance measurements and tested the contribution of the species basking mode, the phylogenetic structure of the data and the correlation between reflectance and published abundance or altitudinal shifts. We used standardised RGB (Red, Green, Blue) values from 222 species from the Iberian Peninsula, and regional mean temperature and precipitation data from the study area (10 and 50-km resolutions) and Europe (50-km resolution).
3. Correlations between reflectance and temperature increased at finer geographical and morphological resolutions. However, the butterfly basking mode did not improve the statistical explanation of the pattern. Reflectance shows a strong phylogenetic structure, while variance partitioning indicated a poor pure contribution of the climate variables in the reflectance–climate correlation.
4. Overall, mean temperature and precipitation were only modest predictors of butterfly reflectance. No correlation between reflectance and recent abundance or altitudinal shifts was found using the hypothesised best estimates of reflectance. Although significant correlations between butterfly shading and altitudinal shifts were found for two of the reflectance measurements, this is interpreted as weak, probably artifactual evidence on the predictive power of this relationship.
5. The strong phylogenetic pattern of the reflectance and the low fraction of the reflectance measures analysed suggest that tests for alternative explanations are still needed to shed light on the meaning of the colour–environment relationships in butterflies, which probably are of a complex nature. From an adaptive point of
view, unravelling the basis of Bogert’s pattern in butterflies requires a closer,abitat-level approach and alternative variables to adult thermoregulatory behaviour to be tested.
The study of ecological interactions between plants, phytophagous insects and their natural enemies is an essential but challenging component for understanding ecosystem dynamics. Molecular methods such as DNA barcoding can help elucidate these interactions. In this study, we employed DNA barcoding to establish hostplant and parasitoid interactions with hesperiid butterflies, using a complete reference library for Hesperiidae of continental Europe and north-western Africa (53 species, 100% of those recorded) based on 2934 sequences from 38 countries. A total of 233 hostplant and parasitoid interactions are presented, some recovered by DNA barcoding larval remains or parasitoid cocoons. Combining DNA barcode results with other lines of evidence allowed 94% species-level identification for Hesperiidae, but success was lower for parasitoids, in part due to unresolved taxonomy. Potential cases of cryptic diversity, both in Hesperiidae and Microgastrinae, are discussed. We briefly analyse the resulting interaction networks. Future DNA barcoding initiatives in this region should focus attention on north-western Africa and on parasitoids, because in these cases barcode reference libraries and taxonomy are less well developed.
Several species of Encyrtus are widely diversified. Frequency of the distances of COI sequences by the presence of multiple picks could indicate to the current speciation process, which makes barcode threshold value unclear. 28s distribution shows diversification among species groups and complexes. E. infidus is composed by several branches and paraphyletic to E. sasakii, which one of its branches. Together they form E. infidus group. Another branch, E. aurantii group, includes up to twenty-one species, forming two clear clusters, E. albidus and E. aurantii complexes (with short and long ovipositor respectively), and one lineage with unclear relation to these complexes. E. albidus and E. manditricolor, previously described as a sister species of E. aurantii, are the branches of E. albidus complex, which could include up to thirteen species, has narrow host range (Coccus sp). E. aurantii complex is less diversified molecularly and morphologically, but has wider host range than E. albidus complex. It includes only two clearly separated branches. One branch sharing same morphology, molecularly is separated. Three singletons have morphology pattern clearly different from E. aurantii group and each other, but not similar to E. infidus group, all have very basal position. One belongs to E. ludmilae and others to the distinct new species. Molecularly very distant E. infelix, combines characters from the both groups. Infected hosts belong to the two subfamilies of scale insects. Host use is according the groups divergence, three basal singletons and E. infelix infects same subfamily host as E. aurantii group.
Biological invasions are a grave threat to ecosystems. The black garden ant (Lasiusniger) is a pest species in Europe. Current literature states that L. niger occupies a disjunct native distribution in the Holarctic, however, based on recent work, we re-evaluate this distribution. The native range of L. niger is reconsidered based on phylogenetic relationships (nine mitochondrial and nuclear markers, 5670 bp), DNA-barcoding (98 Holarctic specimens), morphometry (88 Holarctic specimens, 19 different measurements) and subjective assessment of phenotype. The potential spread of this species is estimated using ecological niche modeling. Lasius niger is more closely related to other Palearctic species than to the Nearctic ants known under this name. The latter are described as a distinct species, L. ponderosae sp. nov. However, DNA-barcoding discovered established populations of L. niger in metropolitan areas in Canada (Vancouver and Halifax). We describe a morphometrical method to delineate L. ponderosae sp. nov. and L. niger. MtDNA diversity and divergence is high within L. ponderosae sp. nov., but low within L. niger. More than 1,000,000 km2 are suitable as a habitat for L. niger in North America. This case emphasizes the critical role of integrative taxonomy to detect cryptic species and identify potential biological invasions in their nascent stages.
Several morphological and mitochondrial lineages of the alpine ringlet butterfly species Erebia pronoe have been described, indicating a complex phylogenetic structure. However, the existing data were insufficient and allow neither a reconstruction of the biogeographic history, nor an assessment of the genetic lineages. Therefore, we analysed mitochondrial (COI, NDI) and nuclear (EF1α, RPS5) gene sequences and compared them with sequences from the sister species Erebia melas. Additionally, we combined this information with morphometric data of the male genitalia and the infection patterns with Wolbachia strains, based on a WSP analysis. We obtained a distinct phylogeographic structure within the E. pronoe-melas complex with eight well-distinguishable geographic groups, but also a remarkable mito-nuclear discordance. The mito-nuclear discordance in E. melas and E. pronoe glottis can be explained by different ages of Wolbachia infections with different Wolbachia strains, associated selective sweeps, and hybridisation inhibition. Additionally, we found indications for incipient speciation of E. pronoe glottis in the Pyrenees and a pronounced range dynamic within and among the other high mountain systems of Europe. Our results emphasize the importance of combined approaches in reconstructing biogeographic patterns and evaluating phylogeographic splits.
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.
The Brown Hairstreak ( Thecla betulae L.) is one of the least observed butterflies of the Palaearctic region, even though its distribution spans from Portugal in the west, to Russia and Korea in the far east. Adults are arboreal and seldom descend to ground level. As a result, this species is mostly monitored via the detection of eggs on the food plant during wintertime. In the Iberian Peninsula, this species was largely unknown until very recently, but a recent burst of regional studies in Spain has begun bridging this gap. However, their focused nature and a still incomplete knowledge on T. betulae in Portugal promoted the need for an integrative study at the Iberian scale.
Here, we carried out a full literature review on the distribution, ecology and behaviour of T. betulae in Portugal and Spain. Complemented with field work in Portugal, we revealed an almost continuous distribution in the northern third of Iberia, whilst populations further south are mostly mountain-bound. In order to help with future discovery of new populations, we built a species-distribution model relating its occurrence with bioclimatic variables. This model accurately explains the current known occupation of the territory and highlights other areas where the species may potentially be found. Finally, we found evidence of a broadening of the species’ niche through the local use of an hitherto unknown food plant. This study sets a new knowledge baseline for future works and conservation of T. betulae through southern Europe.
Keywords Pronophilina, Morpho, Forsterinaria, mitochondrial DNA, molecular taxonomy, andean cloud forest, community ecology Correspondence MA Marín, DNA barcoding is a technique used primarily for the documentation and identification of biological diversity based on mitochondrial DNA sequences. Butterflies have received particular attention in DNA barcoding studies, although varied performance may be obtained due to different scales of geographic sampling and speciation processes in various groups. The montane Andean Satyrinae constitutes a challenging study group for taxonomy. The group displays high richness, with more of 550 species, and remarkable morphological similarity among taxa, which renders their identification difficult. In the present study, we evaluated the effectiveness of DNA barcodes in the identification of montane Andean satyrines and the effect of increased geographical scale of sampling on identification performance. Mitochondrial sequences were obtained from 104 specimens of 39 species and 16 genera, collected in a forest remnant in the northwest Andes. DNA barcoding has proved to be a useful tool for the identification of the specimens, with a well-defined gap and producing clusters with unambiguous identifications for all the morphospecies in the study area. The expansion of the geographical scale with published data increased genetic distances within species and reduced those among species, but did not generally reduce the success of specimen identification. Only in Forsterinaria rustica (Butler, 1868), a taxon with high intraspecific variation , the barcode gap was lost and low support for monophyly was obtained. Likewise, expanded sampling resulted in a substantial increase in the intraspecific distance in Morpho sulkowskyi (Kollar, 1850); Panyapedaliodes drymaea (Hewitson, 1858); Lymanopoda obsoleta (Westwood, 1851); and Lymanopoda labda Hewitson, 1861; but for these species, the barcode gap was maintained. These divergent lineages are nonetheless worth a detailed study of external and genitalic morphology variation, as well as ecological features, in order to determine the potential existence of cryptic species. Even including these cases, DNA barcoding performance in specimen identification was 100% successful based on monophyly, an unexpected result in such a taxonomically complicated group.
Larval and pupal stages are described for Eueides lybia lybia (Nymphalidae: Heliconiinae) in Suriname. Its hostplants are Passiflora nitida and P. glandulosa (Passifloraceae). Based on characters of the habitus of the imago, the male genitalia, the late larval stages and barcodes, it is argued that Eueides lybia consists of two species, Eueides lybia (Fabricius, 1775) and Eueides olympia (Fabricius, 1793) revised status. The most straightforward way to distinguish between the two is the costal streak on the ventral forewing of the imago: red in E. lybia and yellow in E. olympia. E. olympia is considered to have three subspecies, E. olympia olympia (Fabricius, 1793) new status, E. olympia lybioides Staudinger 1876 new status and E. olympia salcedoi K. Brown & F. Fernández Yépez, 1985 new status. E. olympia lybioides may turn out to be a distinct species, as may populations of E. lybia from Peru.
Dorcadionini is a highly speciose tribe within Cerambycidae and is also one of the most challenging in terms of species identification owing to the large number of taxa, intraspecific variability or similarity between species. To support species identification and taxonomic decisions, we generated DNA barcodes for 24 species and 152 specimens. Cases of disagreement between the barcode and taxonomic species were further analysed with the 28S and internal transcribed spacer 2 (ITS2) nuclear markers. The mitochondrial variation is mostly in accordance with the traditional taxonomy except a case of haplotype sharing between Dorcadion murrayi, Dorcadion axillare and Dorcadion pusillum that suggest hybridisation events. We hypothesise that introgression is the likely explanation for part of the large morphological variability found in many other species. In Dorcadion aethiops and Dorcadion fulvum, the taxonomic subspecies are not distinguishable using molecular data, as the haplotypes are identical or very similar, while in Dorcadion equestre, the subspecific structure is in accordance with the genetic data. A low between-species divergence ranging from 0.62 to 2.38% was found in four species, in contrast with the deep intraspecific divergences in D. equestre. We use two methods for single-locus species delimitation (General Mixed Yule Coalescent and Poisson Tree Processes), but none was perfect as both have shortcomings in oversplitting or lumping species. Dorcadion fulvum fulvum is confirmed for Romania.
Central European dry grasslands represent extrazonal patches of the Eurasian steppe biome. They suffer from severe habitat alterations due to land-use changes, abandonment, or inappropriate management. The butterflies Chazara briseis (Linnaeus, 1764) (Lepidoptera: Nymphalidae), Polyommatus damon (Denis & Schiffermüller, 1775) (Lepidoptera: Lycaenidae), and Polyommatus dorylas (Denis & Schiffermüller, 1775) (Lepidoptera: Lycaenidae), specialized inhabitants of these steppe patches, are all swiftly disappearing from Central Europe. We reviewed data on the recent history of their population retractions in the region, including conservation efforts. Using samples from their whole distribution ranges, we sequenced and analyzed COI and wingless genes and together with Species Distribution Modelling reconstructed their biogeographic histories. Populations of C. briseis expanded over the Eurasian steppe biome, where large ungulates maintained extensive grasslands with short open sward. Polyommatus damon became widespread in the steppes during glacial times, and retracted during interglacials, resembling cold-adapted species. It is limited by too dry weather, and it requires disturbed grassland followed by temporal abandonment. Its present genetic structure was induced by the major Pleistocene Mountain glaciations. Polyommatus dorylas prefers an oceanic climate and populated Central Europe from the Balkans during the Holocene. The species depends on disturbed ground. Currently, all three species inhabit only a few remnant sites in Central Europe, and their populations have been further declining in recent years. Targeted conservation actions, including habitat management at remaining sites, ex situ breeding, and (re)introductions, are being taken in Austria, the Czech Republic, and Germany.
This report presents the proposal for an EU Pollinator Monitoring Scheme (EU-PoMS), based on the findings of an expert group of 21 people from 12 European countries. The EU-PoMS delivers of a cost-effective Core Scheme, which includes the most relevant taxa, is able to detect changes in the status of pollinators, has EU-wide coverage, and uses standardised sampling methods. It also provides a set of additional modules for other taxa and measures beyond the Core Scheme. The report proposes a general EU indicator to assess status and trends of pollinators, and a Common Agricultural Policy specific indicator to evaluate the impacts of the CAP, and the measures implemented within, on both pollinators and pollination. Finally, it estimates the costs for establishing and implementing the Core Scheme, considering: staff, equipment, travel, taxonomic, training, data management and coordination costs.
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.
Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.
Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.
Recent multi-locus phylogenetic studies repeatedly showed that what was thought to be the Clouded Apollo butterfly Parnassius mnemosyne (Linnaeus, 1758) represents a cryptic species complex. This complex contains at least three distant species-level phylogenetic lineages. Here, we compile a set of morphology- and DNA-based evidences supporting the distinctiveness of two species in this group, i.e. P. mnemosyne s. str. and P. nebrodensis Turati, 1907 stat. rev. These species can be distinguished from each other based on a combination of diagnostic characters in the male genitalia structure, wing scale patterns, and the forewing venation. The species status of P. nebrodensis is supported based on unique nucleotide substitutions in the mitochondrial (COI, ND1, and ND5) and nuclear (Wg and EF-1a) genes. P. nebrodensis is endemic to the Western Mediterranean Region. This species shares a disjunctive range through the Pyrenees, Western and Central Alps, Apennines, and the Nebrodi and Madonie mountains on Sicily. Altogether 38 nominal taxa initially described as P. mnemosyne subspecies are considered here to be junior synonyms of P. nebrodensis. At first glance, P. nebrodensis can be assessed as an endangered species due to its restricted distribution, narrow range of habitats, and ongoing population decline. Isolated populations of this species scattered through mountain ranges need special management and conservation efforts.
DNA ‘barcoding’ has contributed greatly to resolving species limits in rapidly diverging tropical insect groups. Here, we explored species diversity in the widespread, cryptic Neotropical butterfly genus Hermeuptychia by generating 601 new sequences of the cytochrome c oxidase I (COI) barcode, tripling available information. Our dataset focused in particular on Ecuador, a biogeographically pivotal country, with additional sequences from Brazil, Costa Rica, French Guiana, Guatemala, Mexico, Panama and Peru. We examined the performance of two phylogeny-based approaches for objectively delineating species, Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP), as well as a clustering-based approach, Automatic Barcode Gap Discovery (ABGD), on the combined dataset, including our data and 302 published sequences. In general, GMYC and PTP tended to cluster or split likely species as assessed from morphology, depending on model settings, whereas ABGD was less sensitive and produced a more plausible classification. Numerous sequences formed well-supported clades, putative species, that were unrepresented in previously published datasets. The average diversity across all methods was 45 species, in contrast to the 11 species recognized in the current taxonomy. The resulting COI dataset, in combination with ongoing genomic and morphological research, should significantly clarify our understanding of Hermeuptychia species diversity.
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.
DNA-Barcoding butterflies and moths (Lepidoptera) in forest sites of South Tyrol (IT01 Ritten and IT02 Montiggl). A barcode library based on the mtDNA COI-Gen (Barcodefragment 5´, 658bp) is presented for 597 species of Lepidoptera from two forest sites in the Italian Alps. Altogether 766 specimens have been successfully sequenced, 695 specimens with the full barcode length of 658bp. The medium distance to the nearest neighbour is 8.06 % in the fully barcoded species whereas intraspecific divergence (based on few samples) is low with a mean distance of 0.26 %. Deep genetic splits are restricted to 3 species, but this number may increase with further samples. Only 11 species have identical or overlapping barcodes with a divergence < 1 %. The effectiveness of barcoding for identification of difficult taxa is exemplarily demonstrated and finally potential cases of cryptic diversity are briefly discussed.
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
A leading strategy in international efforts to reverse ongoing losses in biodiversity is the use of protected areas. We use a broad range of data and a review of the literature to show that the effectiveness of existing, and the current pace of the establishment of new, protected areas will not be able to overcome current trends of loss of marine and terrestrial biodiversity. Despite local successes of well-designed and well-managed protected areas proving effective in stemming biodiversity loss, there are significant shortcomings in the usual process of implementation of protected areas that preclude relying on them as a global solution to this problem. The shortcomings include technical problems associated with large gaps in the coverage of critical ecological processes related to individual home ranges and propagule dispersal, and the overall failure of such areas to protect against the broad range of threats affecting ecosystems. Practical issues include budget constraints, conflicts with human development, and a growing human population that will increase not only the extent of anthropogenic stressors but the difficulty in successfully enforcing protected areas. While efforts towards improving and increasing the number and/or size of protected areas must continue, there is a clear and urgent need for the development of additional solutions for biodiversity loss, particularly ones that stabilize the size of the world's human population and our ecological demands on biodiversity.
Coalescent-based species delimitation methods combine population genetic and phylogenetic theory to provide an objective means for delineating evolutionarily significant units of diversity. The generalised mixed Yule coalescent (GMYC) and the Poisson tree process (PTP) are methods that use ultrametric (GMYC or PTP) or non-ultrametric (PTP) gene trees as input, intended for use mostly with single-locus data such as DNA barcodes.
Here, we assess how robust the GMYC and PTP are to different phylogenetic reconstruction and branch smoothing methods. We reconstruct over 400 ultrametric trees using up to 30 different combinations of phylogenetic and smoothing methods and perform over 2000 separate species delimitation analyses across 16 empirical data sets. We then assess how variable diversity estimates are, in terms of richness and identity, with respect to species delimitation, phylogenetic and smoothing methods.
The PTP method generally generates diversity estimates that are more robust to different phylogenetic methods. The GMYC is more sensitive, but provides consistent estimates for BEAST trees. The lower consistency of GMYC estimates is likely a result of differences among gene trees introduced by the smoothing step. Unresolved nodes (real anomalies or methodological artefacts) affect both GMYC and PTP estimates, but have a greater effect on GMYC estimates. Branch smoothing is a difficult step and perhaps an underappreciated source of bias that may be widespread among studies of diversity and diversification.
Nevertheless, careful choice of phylogenetic method does produce equivalent PTP and GMYC diversity estimates. We recommend simultaneous use of the PTP model with any model-based gene tree (e.g. RAxML) and GMYC approaches with BEAST trees for obtaining species hypotheses.
The analysis of DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTUs). This approach accelerates and improves taxonomic workflows by exposing cryptic species and decreasing the risk of synonymy. This study tested the congruence of OTUs resulting from the application of three analytical methods (ABGD, BIN, GMYC) to sequence data for Australian hypertrophine moths. OTUs supported by all three approaches were viewed as robust, but 20% of the OTUs were only recognized by one or two of the methods. These OTUs were examined for three criteria to clarify their status. Monophyly and diagnostic nucleotides were both uninformative, but information on ranges was useful as sympatric sister OTUs were viewed as distinct, while allopatric OTUs were merged. This approach revealed 124 OTUs of Hypertrophinae, a more than two-fold increase from the currently recognized 51 species. Because this analytical protocol is both fast and repeatable, it provides a valuable tool for establishing a basic understanding of species boundaries that can be validated with subsequent studies. This article is protected by copyright. All rights reserved.
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.
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.
Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.
Because many animal species are undescribed, and because the identification of known species is often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By assigning individuals to presumptive species, called operational taxonomic units (OTUs), these systems speed investigations into the patterning of biodiversity and enable studies that would otherwise be impossible. Although OTUs have conventionally been separated through their morphological divergence, DNA-based delineations are not only feasible, but have important advantages. OTU designation can be automated, data can be readily archived, and results can be easily compared among investigations. This study exploits these attributes to develop a persistent, species-level taxonomic registry for the animal kingdom based on the analysis of patterns of nucleotide variation in the barcode region of the cytochrome c oxidase I (COI) gene. It begins by examining the correspondence between groups of specimens identified to a species through prior taxonomic work and those inferred from the analysis of COI sequence variation using one new (RESL) and four established (ABGD, CROP, GMYC, jMOTU) algorithms. It subsequently describes the implementation, and structural attributes of the Barcode Index Number (BIN) system. Aside from a pragmatic role in biodiversity assessments, BINs will aid revisionary taxonomy by flagging possible cases of synonymy, and by collating geographical information, descriptive metadata, and images for specimens that are likely to belong to the same species, even if it is undescribed. More than 274,000 BIN web pages are now available, creating a biodiversity resource that is positioned for rapid growth.
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.
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.
Populations of the Scarce Swallowtail (Iphiclides podalirius Linnaeus, 1758) from the Iberian Peninsula and NW Africa differ in wing pattern characters and therefore have been separated as a distinct subspecies (I. p. feisthamelii Duponchel, 1832) or even attributed species status, although no genetic or experimental studies to assess their status appear to have been conducted. Recent mtDNA studies found a pattern of differentiation incongruent with morphological data: while African and European populations were strongly differentiated, identical haplotypes were found across the Pyrenees within populations of both taxa. Our genetic analyses of nuclear loci indicate a pattern different from mtDNA but concordant with morphology. If placed within the context of historical biogeography, our results suggest that African populations differentiated from European ones about 1.3–1.5 million years ago during the Early Pleistocene, but missing or insufficient reproductive isolation enabled exchange of mtDNA between I. p. podalirius and Spanish I. p. feisthamelii during the Holocene. Although this scenario indicates conspecifity of I. p. feisthamelii with I. p. podalirius despite considerable genetic differentiation, further studies of nuclear loci are needed to study the extent of genetic exchange between both taxa. Our results demonstrate that mitochondrial and nuclear differentiation patterns may be discordant. DNA barcoding results relying only on mtDNA markers therefore need to be interpreted with caution.
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.
Many cold adapted species occur in both montane settings and in the subarctic. Their disjunct distributions create taxonomic complexity because there is no standardized method to establish whether their allopatric populations represent single or different species. This study employs DNA barcoding to gain new perspectives on the levels and patterns of sequence divergence among populations of 122 arctic-alpine species of Lepidoptera from the Alps, Fennoscandia and North America. It reveals intraspecific variability in the barcode region ranging from 0.00-10.08%. Eleven supposedly different species pairs or groups show close genetic similarity, suggesting possible synonymy in many cases. However, a total of 33 species show evidence of cryptic diversity as evidenced by the presence of lineages with over 2% maximum barcode divergence in Europe, in North America or between the two continents. Our study also reveals cases where taxonomic names have been used inconsistently between regions and exposes misidentifications. Overall, DNA barcodes have great potential to both increase taxonomic resolution and to make decisions concerning the taxonomic status of allopatric populations more objective.
Cryptic diversity, defined as two or more distinct species that were classified as a single one due to morphological similarity, is believed to be a potentially important factor influencing future conservation decisions. A re