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DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity

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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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... 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. ...
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The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.
... In Europe, apart from taxon-specific projects, large national barcoding initiatives are all in north and central Europe, for example, Germany (Gemeinholzer et al., 2011), the Netherlands (Beentjes, Speksnijder, Van der Hoorn, & Van Tol, 2015), Norway (Ekrem et al., 2015), and Finland (Huemer, Mutanen, Sefc, & Hebert, 2014;Pentinsaari, Hebert, & Mutanen, 2014), far from the southern Peninsulas (Iberia, Italy, and the Balkans) that host higher levels of biodiversity, endemism, and genetic diversity (Geiger et al., 2014;Hewitt, 1996;Murienne & Giribet, 2009;Pinto, Muñoz, Chávez-Galarza, & De la Rúa, 2012). In fact, when a few smaller-scale barcoding initiatives have been carried out in southern Europe for specific groups (like butterflies in the Iberian Peninsula of freshwater fish around the Mediterranean Basin), the results have revealed a high genetic richness and distinctiveness and the existence of a number of potential cryptic species (Dincă et al., 2015;Geiger et al., 2014). ...
... The peninsulas of southern Europe are hot spots of species and genetic diversity (Geiger et al., 2014;Murienne & Giribet, 2009;Pinto et al., 2012); thus, when they are undersampled, intraspecific genetic divergence is underestimated more than expected by the mere reduction of the geographical scale. The low availability of DNA barcodes or their reduced geographic distribution is a main concern in DNA barcoding (Bergsten et al., 2012;Dincă et al., 2015;Geiger et al., 2014;Savolainen et al., 2005). Our results show that, to capture as much intraspecific genetic variability as possible, sequencing efforts should be concentrated in southern F I G U R E 5 Relationship between geographic distance (x-axis, km) and intraspecific genetic divergence (y-axis, K2P%) (Mean ± SD) in pairwise contrasts between Iberian populations (IBIB, green line) and only European populations (EUEU, red line) ...
... Subdivided taxa and number of operational taxonomic units defined by GMYC (single and multiple threshold), jMOTU (at a cutoff value of 1.1%) and ABGD (at a 0.01 prior on maximum intraspecific divergence and gap width (X) as default) See Table 6 and Figure 2 for an exploration of the effect of different parameter values in ABGD and jMOTU analyses European genetic diversity hot spots (Dincă et al., 2015;Murienne & Giribet, 2009;Pinto et al., 2012) where, paradoxically, the number of available DNA barcodes is lower. ...
Article
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DNA barcoding identification needs a good characterization of intraspecific genetic divergence to establish the limits between species. Yet, the number of barcodes per species is many times low and geographically restricted. A poor coverage of the species distribution range may hamper identification, especially when undersampled areas host genetically distinct lineages. If so, the genetic distance between some query sequences and reference barcodes may exceed the maximum intraspecific threshold for unequivocal species assignation. Taking a group of Quercus herbivores (moths) in Europe as model system, we found that the number of DNA barcodes from southern Europe is proportionally very low in the Barcoding of Life Data Systems. This geographical bias complicates the identification of southern query sequences, due to their high intraspecific genetic distance with respect to barcodes from higher latitudes. Pairwise intraspecific genetic divergence increased along with spatial distance, but was higher when at least one of the sampling sites was in southern Europe. Accordingly, GMYC (General Mixed Yule Coalescent) single-threshold model retrieved clusters constituted exclusively by Iberian haplotypes, some of which could correspond to cryptic species. The number of putative species retrieved was more reliable than that of multiple-threshold GMYC but very similar to results from ABGD and jMOTU. Our results support GMYC as a key resource for species delimitation within poorly inventoried biogeographic regions in Europe, where historical factors (e.g., glaciations) have promoted genetic diversity and singularity. Future European DNA barcoding initiatives should be preferentially performed along latitudinal gradients , with special focus on southern peninsulas.
... 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;Dinca et al., 2011;Hausmann et al., 2011;Dinca et al., 2015;Litman et al., 2018;Dinca et al., 2021). In this well-known group, species delineation methods revealed a portion of polyphyletic, paraphyletic or barcode-sharing species. ...
... For example, Dinca 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;Dinca 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 Dinca et al. (2021). ...
Article
Roughly 18,000 species are described annually as new to science, while estimated extinction rates are comparable to or even exceeding these new discoveries. Considering the estimates of up to 15 million extant eukaryotic species on Earth, of which only about 2 million have been described so far, there has been a recent 'boom' of new potential approaches to more quickly discover and describe the millions of unknown species. This deficit is particularly noted in hyperdiverse taxa, as the current rate of species discovery is considered too slow. Recently, a 'minimalist' alpha taxonomic approach was proposed, relying solely on DNA barcoding and a habitus photograph, in a claimed effort to expedite the naming of new species to combat the so-called taxonomic impediment. In this paper, we point to limitations of minimalist taxonomy, present arguments in favour of the integrative approach, and finally explore a number of potential solutions to combat the taxonomic impediment in hyperdiverse taxa without sacrificing utility and quality for apparent speed and quantity.
... 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. ...
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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.
... Compared to other groups of insects, the phylogenetic relationships of butterflies are reasonably well-known, with robust backbone molecular phylogenies at the subfamily (Wahlberg et al. 2005a;Heikkilä et al. 2012;Espeland et al. 2018) and genuslevel (Chazot et al. 2019). In addition, molecular phylogenies also exist for most butterfly families (Campbell et al. 2000;Caterino et al. 2001;Wahlberg et al. 2003;Braby et al. 2006;Warren et al. 2008;Wahlberg et al. 2009;Wahlberg et al. 2014;Espeland et al. 2015;Sahoo et al. 2016;Seraphim et al. 2018;Toussaint et al. 2018;Allio et al. 2020) as well as major subgroups (Wahlberg et al. 2005b;Peña et al. 2006;Nylin and Wahlberg 2008;Peña and Wahlberg 2008;Wiemers et al. 2010;Talavera et al. 2013;Peña et al. 2015;Condamine et al. 2018) and comprehensive COI data at the species level are available from DNA barcoding studies (Wiemers and Fiedler 2007;Dincă et al. 2011;Hausmann et al. 2011;Dincă et al. 2015;Huemer and Wiesmair 2017;Litman et al. 2018). Some ecological studies on butterflies have already incorporated phylogenetic information, e.g., on the impact of climate change on abundance trends (Bowler et al. 2015;Bowler et al. 2017), the sensitivity of butterflies to invasive species (Gallien et al. 2017;Schleuning et al. 2016) or the ecological determinants of butterfly vulnerability (Essens et al. 2017). ...
... Species identities of the chosen sequences for the dataset were validated by blasting the DNA barcode sequences against the Barcode Of Life Database (http://www.boldsystems.org/), which has a good representation of European butterfly species due to a number of barcoding projects implemented in different countries (e.g., Wiemers and Fiedler 2007;Dincă et al. 2011;Hausmann et al. 2011;Dincă et al. 2015;Huemer and Wiesmair 2017;Litman et al. 2018). In almost all cases, the sequences came from the same voucher specimen itself. ...
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With the aim of supporting ecological analyses in butterflies, the third most species-rich superfamily of Lepidoptera, this paper presents the first time-calibrated phylogeny of all 496 extant butterfly species in Europe, including 18 very localised endemics for which no public DNA sequences had been available previously. It is based on a concatenated alignment of the mitochondrial gene COI and up to eleven nuclear gene fragments, using Bayesian inferences of phylogeny. To avoid analytical biases that could result from our region-focussed sampling, our European tree was grafted upon a global genus-level backbone butterfly phylogeny for analyses. In addition to a consensus tree, the posterior distribution of trees and the fully concatenated alignment are provided for future analyses. Altogether a complete phylogenetic framework of European butterflies for use by the ecological and evolutionary communities is presented.
... 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 ...
Article
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.
... For a 5th data set, "Congolese fishes", the authors also mention identification problems due to the lack of identification tools (Decru et al. 2016). However, none of the these problems are likely to plague a data set like "Iberian butterflies", where the authors consider it likely that introgression and cryptic species are responsible for the lack of congruence (Dincȃ et al. 2015). Note, however, that these problems do not appear to affect the remaining Lepidoptera data sets for which we observe high levels of congruence (five data sets). ...
Article
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New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared to full-length barcodes. This is here tested based on 20 empirical datasets covering ca. 30,000 specimens (5,500 species) and six clade-specific datasets from GenBank covering ca. 98,000 specimens (>20,000 species). All specimens in these datasets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (3 windows: 100-bp, 200-bp, 300-bp) and by excising nine mini-barcodes with established primers (length: 94 - 407-bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular Operational Taxonomic Units (mOTUs) that were obtained using three different species delimitation techniques (PTP, ABGD, objective clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length (>200-bp). Only very short mini-barcodes (<200-bp) perform poorly, especially when they are located near the 5' end of the Folmer region. The mean congruence between morphospecies and mOTUs was ca. 75% for barcodes >200-bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes. https://academic.oup.com/sysbio/article-abstract/69/5/999/5739457
... 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). ...
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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).
... 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. ...
Article
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.
... Alternatively, we used a qualitative approach generating a list of endemics for regionalisation and comparison of species traits. Our evaluation, based on an "and/or" approach, allowed to include several species that were lumped in a COIbased GMYC analysis, likely due to events of post-speciation mitochondrial introgression (Dincă et al., 2015). In particular, 18 endemic entities, widely recognised as good species by butterfly specialists also based on nuclear markers (Wiemers et al., 2018), were recovered as LE and LME. ...
... 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. ...
Chapter
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.
... DNA barcoding has been applied in the biodiversity studies and species identification of diverse fauna, including fishes, amphibians, reptiles, mammals, birds, and invertebrates [20,[29][30][31][32][33][34][35][36]. Also, past studies have confirmed the relevance of this approach for lepidopterans with a variety of applications in taxonomy, biodiversity documentation, and ecology [35][36][37][38][39][40][41][42][43][44][45]. ...
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Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require both morphological diagnostics and molecular tools, such as DNA barcoding, for correct identification. In particular, the use of the subunit I of the mitochondrial cytochrome c oxidase (COI) gene for DNA barcoding has proven useful in species identification for insects. However, to date, no studies have been carried out on the DNA barcoding of Nigerian butterflies. We evaluated the utility of DNA barcoding applied for the first time to 735 butterfly specimens from southern Nigeria. In total, 699 DNA barcodes, resulting in a record of 116 species belonging to 57 genera, were generated. Our study sample comprised 807 DNA barcodes based on sequences generated from our current study and 108 others retrieved from BOLD. Different molecular analyses, including genetic distance-based evaluation (Neighbor-Joining, Maximum Likelihood and Bayesian trees) and species delimitation tests (TaxonDNA, Automated Barcode Gap Discovery, General Mixed Yule-Coalescent, and Bayesian Poisson Tree Processes) were performed to accurately identify and delineate species. The genetic distance-based analyses resulted in 163 well-separated clusters consisting of 147 described and 16 unidentified species. Our findings indicate that about 90.20% of the butterfly species were explicitly discriminated using DNA barcodes. Also, our field collections reported the first country records of ten butterfly species—Acraea serena, Amauris cf. dannfelti, Aterica galena extensa, Axiocerces tjoane rubescens, Charaxes galleyanus, Papilio lormieri lormieri, Pentila alba, Precis actia, Precis tugela, and Tagiades flesus. Further, DNA barcodes revealed a high mitochondrial intraspecific divergence of more than 3% in Bicyclus vulgaris vulgaris and Colotis evagore. Furthermore, our result revealed an overall high haplotype (gene) diversity (0.9764), suggesting that DNA barcoding can provide information at a population level for Nigerian butterflies. The present study confirms the efficiency of DNA barcoding for identifying butterflies from Nigeria. To gain a better understanding of regional variation in DNA barcodes of this biogeographically complex area, future work should expand the DNA barcode reference library to include all butterfly species from Nigeria as well as surrounding countries. Also, further studies, involving relevant genetic and eco-morphological datasets, are required to understand processes governing mitochondrial intraspecific divergences reported in some species complexes.
... These approaches facilitate genotyping, species identifications and phylogenetic studies [23]. Specifically, DNA barcoding is usually considered to be a reliable, commercially available and simple molecular identification technique with widespread applicability across metazoan taxa [24][25][26][27][28][29][30] owing to large data sets at the Barcode of Life Database (BOLD) and National Centre for Biotechnology Information (NCBI) [31,32]. DNA barcoding even identifies species from a trace number of sample individuals at all insect life stages (eggs, larvae, nymphs and pupae), which are frequently impossible to identify otherwise [33]. ...
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Morphometric and molecular divergence among four butterfy species of the families Nymphalidae and Pieridae from the western Himalaya region were investigated using molecular tools, traditional morphometric measures and a truss network system. The considered species were Danaus chrysippus, Vanessa cardui, Pieris brassicae and Pieris canidia. Traditional taxonomy is sometimes unable to discriminate cryptic species or species that have close morphological features. Although taxonomists carefully examine external body features to diferentiate the species; however, there is a risk for misidentifca- tion during a visual assessment of cryptic species. Therefore, we aimed to use the truss network system of 14 morphological landmarks interconnected to yield 90 variables about molecular taxonomy. Principal component analysis (PCA), discriminant function analysis (DFA) and cluster analysis (CA) were employed to determine morphometric variations. In the traditional analysis, 79, 68, 16 and 5 characters out of 90 were found signifcant (p<0.05) for D. chrysippus, V. cardui, P. brassicae and P. canidia, respectively. One to seven principal components were extracted through PCA; they explained 87.5–100% of the total variance in samples. Notably, DFA correctly classifed 100% of the original grouped cases and 100% of the cross- validated grouped cases. However, the variations were not the same for the two diferent methods (truss and traditional) employed for the analysis. We correctly identifed all the species; the interspecies sequence divergence was between 0.1034 and 0.1398, and the intra-species sequence divergence range was 0.0001 to 0.0128 using the Cytochrome c oxidase subunit-I (COI) gene. The present study provides useful information about the application and complementary role of traditional with truss morphometric analysis for the precise identifcation and classifcation of the selected species.
... 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. ...
Article
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.
... 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). ...
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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.
... Moreover, reduced costs, coupled with technical refinements such as multi-marker approaches (da Silva et al., 2019) and the availability of ever more comprehensive barcode databases (e.g. Dincă 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. ...
Article
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 fibre. However, this would require identifying key service providers, which may be challenging where 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 use DNA metabarcoding to provide detailed data on pest species predation by diverse predator communities. Then, these data feed into an extensive network analysis, where 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 consume 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, though additional information is needed to evaluate their actual effectiveness in pest regulation.
... 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). ...
Article
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.
... Alternatively, we used a qualitative approach generating a list of endemics for regionalisation and comparison of species traits. Our evaluation, based on an "and/or" approach, allowed to include several species that were lumped in a COIbased GMYC analysis, likely due to events of post-speciation mitochondrial introgression (Dincă et al., 2015). In particular, 18 endemic entities, widely recognised as good species by butterfly specialists also based on nuclear markers (Wiemers et al., 2018), were recovered as LE and LME. ...
Article
Endemics co‐occur because they evolved in situ and persist regionally or because they evolved ex situ and later dispersed to shared habitats, generating evolutionary or ecological endemicity centres, respectively. We investigate whether different endemicity centres can intertwine in the region ranging from Alps to Sicily, by studying their butterfly fauna. We gathered an extensive occurrence dataset for butterflies of the study area (27,123 records, 269 species, in cells of 0.5x0.5 degrees of latitude‐longitude). We applied molecular‐based delimitation methods (GMYC model) to 26,557 COI sequences of Western Palearctic butterflies. We identified entities based on molecular delimitations and/or the checklist of European butterflies and objectively attributed occurrences to their most probable entity. We obtained a zoogeographic regionalisation based on the 69 endemics of the area. Using phylogenetic ANOVA we tested if endemics from different centres differ from each other and from non‐endemics for key ecological traits and divergence time. Endemicity showed high incidence in the Alps and Southern Italy. The regionalisation separated the Alps from the Italian Peninsula and Sicily. The endemics of different centres showed a high turnover and differed in phylogenetic distances, phenology and distribution traits. Endemics are on average younger than non‐endemics and the Peninsula‐Sicily endemics also have lower variance in divergence than those from the Alps. The observed variation identifies Alpine endemics as paleoendemics, now occupying an ecological centre, and the Peninsula‐Sicily ones as neoendemics, that diverged in the region since the Pleistocene. The results challenge the common view of the Alpine‐Apennine area as a single “Italian refugium”.
... Mitochondrial DNA is particularly susceptible to introgression (i.e., where the mitochondrial genome of one species is incorporated into the gene pool of a different species as a result of hybridization and backcrossing) because of the lack of recombination for this genome (Funk & Omland, 2003;Patten et al., 2015). In addition, hybridization and mitochondrial introgression is common among butterfly species (Zakharov et al., 2009;Dinc a et al., 2011;Brower, 2013;Dinc a et al., 2015). Since nuclear DNA (nDNA) is recombinant and therefore less susceptible to this phenomenon, the DNA sequence data for two nDNA regions, elongation factor 1-α (EF1-α) and wingless (Wgl), were also collected in addition to COI sequence data. ...
Article
1. The Pelopidas are a widely distributed genus of hesperiids ranging from the Afrotropical to Australasia. Based on traditional classification, four species are recorded from Sri Lanka: Pelopidas agna, P. mathias, P. subochracea and P. conjuncta. 2. The aim is to test the taxonomic hypotheses of the Sri Lankan Pelopidas as they are currently recognised based on morphology. We achieve this by analysing the phyloge-netic relationships among the Pelopidas as well as between the Pelopidas and tribe Baor-ini both inside and outside of Sri Lanka. 3. We used DNA sequence data from the cytochrome oxidase c subunit I (COI) "bar-code" region and two nuclear DNA regions, elongation factor 1-α (EF1-α) and wingless (Wgl) collected for Sri Lankan Pelopidas and combined with all available Pelopidas sequence data from public databases. 4. The reconstructed phylogeny showed that P. mathias, P. agna and P. conjuncta were not monophyletic. Additionally, P. agna in Sri Lanka may now be a hybrid population that retains the P. agna morphology but possesses the mitochondrial DNA of P. mathias (termed a 'ghost' population). Finally, two lineages of P. conjuncta were identified in Sri Lanka, appearing more closely related to other species of Pelopidas than to each other. 5. This study demonstrates the phylogenetic complexity within the Pelopidas and the uniqueness of the Sri Lankan Pelopidas that have not been previously appreciated. This new knowledge is important in planning conservation management and may also guide future work on the potential mechanisms of evolutionary diversification of island butterflies.
... 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). ...
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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.
... 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. ...
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We studied the evolutionary relationship of two widely distributed parapatric butterfly species, Melitaea athalia and Melitaea celadussa, using the ddRAD sequencing approach, as well as genital morphology and mtDNA data. M. athalia was retrieved as paraphyletic with respect to M. celadussa. Several cases of mito-nuclear discordance and morpho-genetic mismatch were found in the contact zone. A strongly diverged and marginally sympatric clade of M. athalia from the Balkans was revealed. An in-depth analysis of genomic structure detected high levels of admixture between M. athalia and M. celadussa at the contact zone, though not reaching the Balkan clade. The demographic modelling of populations supported the intermediate genetic make-up of European M. athalia populations with regards to M. celadussa and the Balkan clade. However, the dissimilarity matrix of genotype data (PCoA) suggested the Balkan lineage having a genetic component that is unrelated to the athalia-celadussa group. Although narrowly sympatric, almost no signs of gene flow were found between the main M. athalia group and the Balkan clade. We propose two possible scenarios on the historical evolution of our model taxa and the role of the last glacial maximum in shaping their current distribution. Finally, we discuss the complexities regarding the taxonomic delimitation of parapatric taxa.
... 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. ...
Article
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.
... However, recent studies on several genera of European Gelechiidae , Huemer and Mutanen 2012, Huemer and Karsholt 2014, Landry et al. 2017) revealed the power of this approach to aid species delimitation in taxonomically difficult groups, even those with a high level of unrecorded species and cryptic diversity. Similar patterns have been analyzed in several other Lepidoptera in different parts of the world, e.g., in another gelechioid group , in Iberian butterflies (Dincă et al. 2015), in North American Noctuoidea (Zahiri et al. 2017), or in the Lepidoptera fauna of Costa Rica (Janzen and Hallwachs 2016). These results motivated the present effort to compile a comprehensive DNA barcode library for the European Gelechiidae fauna, with the aim of simplifying future revisionary studies while also improving their quality. ...
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For the first time, a nearly complete barcode library for European Gelechiidae is provided. DNA barcode sequences (COI gene-cytochrome c oxidase 1) from 751 out of 865 nominal species, belonging to 105 genera, were successfully recovered. A total of 741 species represented by specimens with sequences ≥ 500bp and an additional ten species represented by specimens with shorter sequences were used to produce 53 NJ trees. Intraspecific barcode divergence averaged only 0.54% whereas distance to the Nearest Neighbour species averaged 5.58%. Of these, 710 species possessed unique DNA barcodes, but 31 species could not be reliably discriminated because of barcode sharing or partial barcode overlap. Species A peer-reviewed open-access journal Peter Huemer et al. / ZooKeys 921: 141-157 (2020) 142 discrimination based on the Barcode Index System (BIN) was successful for 668 out of 723 species which clustered from minimum one to maximum 22 unique BINs. Fifty-five species shared a BIN with up to four species and identification from DNA barcode data is uncertain. Finally, 65 clusters with a unique BIN remained unidentified to species level. These putative taxa, as well as 114 nominal species with more than one BIN, suggest the presence of considerable cryptic diversity, cases which should be examined in future revisionary studies.
... 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. ...
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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)]. Our results confirm E. bazae as a well‐differentiated species and show 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 and complementary data gathered from other studies, we propose to consider it ‘Endangered’ [EN B1ab(i,ii,iii,iv); B2ab(i,ii,iii,iv)]. Tot i que s'han atribuït tant efectes positius (expansió d'espècies termòfiles) com negatius (contracció d'espècies adaptades al fred) a l'escalfament global, canvis en les distribucions poden estar condicionats pels trets ecològics de les espècies. Això pot ser especialment cert per a organismes altament especialitzats com la grogueta del desert (Euchloe bazae), un endemisme ibèric especialista d'estepes semiàrides amb un estatus taxonòmic i de conservació debatut. En aquest estudi, primer cerquem esclarir l'estatus taxonòmic d'aquesta papallona i de les seves poblacions mitjançant la inferència filogenètica multilocus. També actualitzem la seva distribució i fem servir la modelització de nínxols ecològics combinada amb altres dades per reavaluar el seu estatus de conservació. Els nostres resultats confirmen que E. bazae és una espècie ben diferenciada i amb una de les distribucions més restringides entre les papallones europees. Demostrem que els seus dos nuclis de població, cadascun considerat una subespècie, són diferents genèticament i s'han de tractar com a unitats de gestió independents. Els models climàtics elaborats amb dos escenaris d'emissions futurs suggereixen un augment de l'àrea climàticament adient. No obstant això, s'estima que la idoneïtat de l'àrea actualment ocupada disminuirà, el que podria requerir canvis ràpids en la seva distribució per garantir‐ne la supervivència. Tenint en compte que E. bazae disposaria d'una capacitat de dispersió baixa, la distribució actual reduïda, l'alt grau de fragmentació (dividida en dues poblacions llunyanes), la baixa variabilitat genètica intrapoblacional, la disminució de l'extensió i l'abundància, les baixes densitats, les elevades fluctuacions anuals i les nombroses amenaces al seu hàbitat, considerem que les poblacions d'aquesta papallona són extremament fràgils i proposem considerar‐la “En perill” [EN B1ab(i,ii,iii,iv); B2ab(i,ii,iii,iv)].
... Zahiri et al., 2014) and butterflies in particular (e.g. Huemer et al., 2014;Dincǎ et al., 2015) has been a strong focus of this large endeavour. The University of Alaska Museum has been collaborating with the Kenai National Wildlife Refuge to build a DNA barcode library of the state's non-marine arthropods (Sikes et al., 2017). ...
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Ongoing efforts to build a DNA barcode library of the non-marine arthropod fauna of Alaska have, combined with work done in Canada, so far yielded an estimated coverage of~48.5% of the species in the state. Among these are 71 of the 80 species of butterflies that are known residents of Alaska. This study compares the state's butterfly fauna using the DNA barcode BIN system, which is a proxy for species based on DNA barcodes, and the BOLD identification engine, to the count of species based on traditional taxonomy. The 71 traditional species for which DNA barcodes were obtained correspond to 56 unique BINs. Eleven of these BINs are shared among species of Alaskan butterflies (i.e. more than one Alaskan butterfly species occurs in two or more of these 11 BINs). The greatest BIN sharing was seen in the genus Colias which includes 8 traditional species that share two BINs. All the remaining BIN sharing was seen in species of the family Nymphalidae with members of Boloria, Speyeria, Polygonia, Erebia, and Oeneis sharing BINs. Currently 45 Alaskan butterfly species are in BINs not shared with other Alaskan butterflies, thus allowing a direct match to an Alaskan butterfly species name using the DNA barcode BIN system. Only three species had samples split into more than one BIN (suggestive of high within-species diversity). The test of the BOLD identification engine resulted in 82 of the 108 sequences (76%) being correctly identified. However, sampling of Alaskan butterflies has been sparse, with many species represented by only one or two DNA barcoded specimens, so these conclusions may change as sampling is increased.
... This approach has become increasingly popular as an efficient tool for studying and categorizing Afrotropical moth species to prioritize conservation efforts [19,20]. In lepidopteran studies, DNA barcoding has proven effective in investigating biodiversity [21][22][23][24][25][26][27][28], resolving taxonomic problems [29,30], and unraveling cryptic diversities [27,[30][31][32][33][34]. Apart from this, the largescale availability of reliable COI sequence data in the global databases has proven useful for quick and accurate identification of lepidopterans [1,24,25,29]. ...
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Comprehensive biodiversity assessment of moths in Nigeria rely greatly on accurate species identification. While most of the Nigerian moths are identified effortlessly using their morphological traits, some taxa are morphologically indistinguishable, which makes it difficult for taxon diagnosis. We investigated the efficiency of the DNA barcode, a fragment of the mitochondrial Cytochrome C oxidase subunit I, as a tool for the identification of Nigerian moths. We barcoded 152 individuals comprising 18 morphospecies collected from one of the remaining and threatened rainforest blocks of Nigeria – the Cross River National Park. Phenetic neighbor-joining tree and phylogenetic Maximum Likelihood approach were employed for the molecular-based species identification. Results showed that DNA barcodes enabled species-level identification of most of the individuals collected from the Park. Additionally, DNA barcoding unraveled the presence of at least six potential new and yet undescribed species – Amnemopsyche sp., Arctia sp., Deinypena sp., Hodebertia sp., Otroeda sp., and Palpita sp. The phylogenetic Maximum Likelihood using the combined dataset of all the newly assembled sequences from Nigeria showed that all species formed unique clades. The phylogenetic analyses provided evidence of population divergence in Euchromia lethe, Nyctemera leuconoe, and Deinypena lacista. This study thus illustrates the efficacy of DNA barcoding for species identification and discovery of potential new species, which demonstrates its relevance in biodiversity documentation of Nigerian moths. Future work should, therefore, extend to the creation of an exhaustive DNA barcode reference library comprising all species of moths from Nigeria to have a comprehensive insight on the diversity of moths in the country. Finally, we propose integrated taxonomic methods that would combine morphological, ecological, and molecular data in the identification and diversity studies of moths in Nigeria.
Article
Aim Occurrence data are fundamental to macroecology, but accuracy is often compromised when multiple units are lumped together (e.g., in recently separated cryptic species or in citizen science records). Using amalgamated data leads to inaccuracy in species mapping, to biased beta‐diversity assessments and to potentially erroneously predicted responses to climate change. We provide a set of R functions (biodecrypt) to objectively attribute unidentified occurrences to the most probable taxon based on a subset of identified records. Innovation Biodecrypt assumes that unidentified occurrences can only be attributed at certain distances from areas of sympatry. The function draws concave hulls based on the subset of identified records; subsequently, based on hull geometry, it attributes (or not) unidientified records to a given taxon. Concavity can be imposed with an alpha value and sea or land areas can be excluded. A cross‐validation function tests attribution reliability and another function optimises the parameters (alpha, buffer, distance ratio between hulls). We applied the procedure to 16 European butterfly complexes recently separated into 33 cryptic species for which most records were amalgamated. We compared niche similarity and divergence between cryptic taxa, and re‐calculated and contributed updated climatic niche characteristics of the butterflies in Europe (CLIMBER). Main conclusions Biodecrypt showed a cross‐validated correct attribution of known records always ≥ 98% and attributed more than 80% of unidientified records to the most likely taxon in parapatric species. The functions determined where records can be assigned even for largely sympatric species, and highlighted areas where further sampling is required. All the cryptic taxa showed significantly diverging climatic niches, reflected in different values of mean temperature and precipitation compared to the values originally provided in the CLIMBER database. The substantial fraction of cryptic taxa existing across different taxonomic groups and their divergence in climatic niches highlight the importance of using reliably assigned occurrence data in macroecology.
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Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction‐site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies.
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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.
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The Melitaea phoebe group is constituted by six species distributed throughout the Palearctic. One of the most widespread species is Melitaea ornata Christoph, 1893, present from France (Provence) to Central Asia. Recently, populations of M. ornata were discovered in a mountainous region of southeastern Iberia, although doubts about their taxonomy existed. To clarify the taxonomic status of these populations and to revise the distribution of this taxon in Iberia, we have sequenced mitochondrial (COI barcode region) and nuclear (wg, RPS5, MDH, and EF-1α) markers, and analyzed the male genitalia for 72 Iberian individuals and for all the species of the M. phoebe group. This information was complemented with phenological and ecological data. Our results unveiled that the Iberian M. ornata-like taxon is in fact distributed through most of the Iberian Peninsula, except for the southwest and northeast. In contrast to the univoltine M. ornata, the Iberian taxon can be bivoltine in the wild. The Iberian taxon was retrieved to be related to M. ornata, but the differences in the genetic markers and genitalia were comparable to those found between species in the group. Based on the evidence here presented and according to species delimitation results, we propose to consider the Iberian taxon as a novel species , tentatively named Melitaea pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.
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Gracillariidae is the most species-rich leaf-mining moth family with over 2,000 described species worldwide. In Europe, there are 263 valid named species recognized, many of which are difficult to identify using morphology only. Here we explore the use of DNA barcodes as a tool for identification and species discovery in European gracillariids. We present a barcode library including 6,791 COI sequences representing 242 of the 263 (92%) resident species. Our results indicate high congruence between morphology and barcodes with 91.3% (221/242) of European species forming monophyletic clades that can be identified accurately using barcodes alone. The remaining 8.7% represent cases of non-monophyly making their identification uncertain using barcodes. Species discrimination based on the Barcode Index Number system (BIN) was successful for 93% of species with 7% of species sharing BINs. We discovered as many as 21 undescribed candidate species, of which six were confirmed from an integrative approach; the other 15 require additional material and study to confirm preliminary evidence. Most of these new candidate species are found in mountainous regions of Mediterranean countries, the South-Eastern Alps and the Balkans, with nine candidate species found only on islands. In addition, 13 species were classified as deep conspecific lineages, comprising a total of 27 BINs with no intraspecific morphological differences found, and no known ecological differentiation. Double-digest restriction-site associated DNA sequencing (ddRAD) analysis showed strong mitonuclear discrepancy in four out of five species studied. This discordance is not explained by Wolbachia-mediated genetic sweeps. Finally, 26 species were classified as “unassessed species splits” containing 71 BINs and some involving geographical isolation or ecological specialization that will require further study to test whether they represent new cryptic species.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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&apos;s human population and our ecological demands on biodiversity.
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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.
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