Species Delimitation Approaches Research Articles

Review of archaic nymphomyiids (Diptera, Nymphomyiidae) of the Russian Far East and bordering territories, with describing of new taxa and DNA barcoding of known species

Nymphomyiidae from the Russian Far East and bordering territories are revised, including data on taxonomy, redescriptions, and results of DNA barcoding. Nymphomyia orientalis sp. nov. with two subspecies N orientalis orientalis subsp. nov. and N. orientalis makcha subsp. nov. are described and information on biology, ecology and distribution are presented. Key of Nymphomyia species for adult males and females of the world is provided. Three species delimitation approaches (ASAP, mPTP, GMYC) using the mitochondrial gene cytochrome c oxidase I (COI) confirm validity of described taxa as well as available nymphomyiids in GenBank. Morphologically indistinguishable specimens of N. orientalis sp. nov. from Makcha and Polovinka rivers belongs to separate mOTUs with the lowest distances in the genus 1.99% that is the lower interspecific threshold.

Cryptic diversity and phylogeographic patterns of Mediodactylus species in the Eastern Mediterranean region

Cryptic diversity poses a great obstacle in our attempts to assess the current biodiversity crisis and may hamper conservation efforts. The gekkonid genus Mediodactylus, a well-known case of hidden species and genetic diversity, has been taxonomically reclassified several times during the last decade. Focusing on the Mediterranean populations, a recent study within the M. kotschyi species complex using classic mtDNA/nuDNA markers suggested the existence of five distinct species, some being endemic and some possibly threatened, yet their relationships have not been fully resolved. Here, we generated genome-wide SNPs (using ddRADseq) and applied molecular species delimitation approaches and population genomic analyses to further disentangle these relationships. Τhe most extensive nuclear dataset, so far, encompassing 2,360 loci and ∼ 699,000 bp from across the genome of Mediodactylus gecko, enabled us to resolve previously obscure phylogenetic relationships among the five, recently elevated, Mediodactylus species and to support the hypothesis that the taxon includes several new, undescribed species. Population genomic analyses within each of the proposed species showed strong genetic structure and high levels of genetic differentiation among populations.

Determination of the species boundaries of genus Dolerus (Hymenoptera: Tenthredinidae) using the COI gene

New generation molecular approaches and methods are being developed to identify species and determine species boundaries. There are many different approaches of species delimitation used to assess the species richness of poorly studied and highly diverse invertebrate taxa. The basis of these approach is DNA barcoding studies. DNA barcoding has been used as a powerful tool for species identification and delimitation. Although DNA barcoding studies have been carried out on the family Tenthredinidae, there are no studies on species delimitation. Herein, we compare species delimitation analyzes belong to Dolerus genus based on cytochrome c oxidase I (COI) region. In this context, it was used five species delimitation approaches (ABGD, ASAP, DNA Taxon, PTP and GMYC). Thirty-six morphotypes were used in the study. These morphotypes separated into six species (Dolerus triplicatus, Dolerus germanicus, Dolerus puncticollis, Dolerus nigratus, Dolerus sp1 and Dolerus sp2) in ABGD, ASAP and DNA Taxon approaches. Two additional species were introduced because of the tree-based PTP and GMYC approaches. These species were named as Dolerus sp3 and Dolerus sp4 which were separated from Dolerus puncticollis clade and Dolerus nigratus clade, respectively. These analyzes were supported by the phylogenetic tree and CBC entities that constitute the ITS2 data.

Identification of fish specimens of the Tocantins River, Brazil, using DNA barcoding.

The fish fauna of the Tocantins River possesses many endemic species; however, it is little studied in molecular terms and is quite threatened by the construction of several hydroelectric dams. Therefore, the objective of this study was to identify the ichthyofauna of the Tocantins River using DNA barcoding. For this, collections were carried out in five points of this river, which resulted in the capture of 725 individuals from which partial sequences of the cytochrome oxidase subunit I (COI) gene were obtained for genetic analysis. A total of 443 haplotypes were recovered with the mean intraspecific K2P genetic distance of 1.82%. Altogether, 138 species were identified based on morphological criteria, which was a quantity that was much lower than that indicated by the four molecular methods (assemble species by automatic partitioning [ASAP], barcode index number [BIN], generalized mixed Yule coalescent (GMYC), and Bayesian Poisson tree processes [bPTP]) through which 152-157 molecular entities were identified. In all, 41 unique BINs were obtained based on the data generated in the BOLDSystems platform. According to the result indicated by ASAP (species delimitation approach considered the most appropriate in the present study), there was an increase of 17 molecular entities (12.32%), when compared to the number of species identified through their morphological criteria, as it can show cryptic diversity, candidates for new species, and misidentifications. There were 21 incongruities indicated between the different identification approaches for species. Therefore, it is suggested that these taxonomic problems be cautiously evaluated by experts to solve such taxonomic issues.

Multi-Locus Sequence Analysis Indicates Potential Cryptic Speciation in the Chigger Mite Neoschoengastia gallinarum (Hatori, 1920) Parasitising Birds in Asia.

Neoschoengastia gallinarum is widely distributed in Asia, preferentially parasitising birds, and heavy infestations have clinical impacts on domestic fowl. In common with other trombiculid mites, the genetic diversity and potential variation in host preferences or pathology induced by N. gallinarum are poorly understood. This study aimed to unravel the geographical variation and population structure of N. gallinarum collected from galliform birds in Peninsular Malaysia and Thailand by inference from concatenated mitochondrial-encoded cytochrome c oxidase subunit I (COI), and nuclear-encoded internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA gene sequences, including a comparison with previously published data from southeastern China. Our multi-locus sequence analysis revealed three monophyletic clades comprising (A) specimens from Peninsular Malaysia, (B) the samples from Thailand together with a minority of Chinese sequences, and (C) the majority of sequences from China. Similarly, most species delimitation approaches divided the specimens into three operational taxonomic units. Analysis of molecular variance revealed 96.41% genetic divergence between Malaysian and Thai populations, further supported by the absence of gene flow (Nm = 0.01). In conclusion, despite the two countries sharing a land border, populations of N. gallinarum from Peninsular Malaysia and Thailand appear to be genetically segregated and may represent distinct cryptic species.

Molecular Evidence Reveals Taxonomic Uncertainties and Cryptic Diversity in the Neotropical Catfish of the Genus Pimelodus (Siluriformes: Pimelodidae).

Pimelodus is the most speciose genus of the family Pimelodidae, and is amply distributed in the Neotropical region. The species-level taxonomy and phylogenetic relationships within this genus are still poorly resolved, however. These taxonomic problems and the general lack of data have generated major uncertainties with regard to the identification of specimens from different localities. In the present study, we applied a single-locus species delimitation approach to identify the MOTUs found within the genus Pimelodus and provide sound evidence for the evaluation of the species richness of this genus in the different river basins of the Neotropical region. The study was based on the analysis of sequences of the mitochondrial COI gene of 13 nominal species, which resulted in the identification of 24 consensus MOTUs. Only six nominal species were recovered as well-defined molecular entities by both the traditional barcoding analysis and the molecular delimitation methods, while the other seven presented cryptic diversity or persistent taxonomic uncertainties. The lineages identified from the Parnaíba ecoregions, Amazonas Estuary and Coastal Drainages may represent a much greater diversity of Pimelodus species than that recognized currently, although a more detailed study of this diversity will be necessary to provide a more definitive classification of the genus.

Species richness in Myotis (Chiroptera: Vespertilionidae): species delimitation and expanded geographic sampling reveal high Neotropical diversity

Abstract With up to 137 species worldwide, Myotis,— a genus of small, insectivorous bats are an example for understanding biogeographic and evolutionary processes such as intercontinental colonization, diversification, speciation, and convergent adaptive evolution. Species limits and associated distributions, however, remain poorly delineated across significant portions of this diverse group and encompassing a vast geographic range. Here, we explore how diversity is partitioned across this radiation with a focus on Neotropical species of Myotis, using a barcoding approach (706 bp of the mitochondrial DNA gene cytochrome-b) that includes 544 individual sequences (116 newly generated sequences and 428 sequences from GenBank, including outgroups). Single-locus molecular barcoding is useful for identifying cryptic diversity and establishing hypotheses that can guide investigations of species boundaries and phylogenetic relationships, an especially important initial step in assessing poorly characterized, diverse clades. The global phylogeny produced from these data is consistent with the hypothesized monophyly of the New World clade and places the evolutionary relationships of Neotropical species of Myotis within the context of the Palearctic and Nearctic clades. We then explore species boundaries for 25 of the 36 nominal species of Neotropical Myotis, using 3 alternative species delimitation approaches (1 distance-based ABGD [Automatic Barcode Gap Discovery]; and 2 coalescent-based mPTP [multi-rate Poisson Tree Processes], GMYC [Generalized Mixed Yule Coalescent] model). The mPTP (n = 29 species) and ABGD (n = 26) approaches inferred different numbers of species than that recognized by current Neotropical Myotis taxonomy, whereas GMYC (n = 25) inferred the same number of species. In addition to the original 36 Neotropical nominal species currently recognized, we suggest that taxonomic revisions are needed to assess whether M. oxyotus gardneri and M. nigricans osculatii should be elevated to the species level and whether M. dinellii is distinct from M. levis. With the 7 potentially cryptic species identified herein that require further study, the number of Neotropical Myotis may approach 45 species. Taken together, our results suggest that rather than a classic example of a temperate radiation, there may be more species of Myotis in the Neotropics than Nearctic, Afrotropic, Indomalayan, and Oceanian Realms, with only the Palearctic region showing higher species richness.

DNA taxonomy reveals high species diversity among the stygobiont genus Metastenasellus (Crustacea, Isopoda) in African groundwater

This study aimed to explore the species diversity within the isopod genus Metastenasellus in Benin and Cameroon. Compared to other parts of the world, the described diversity of stygobiotic crustaceans in Africa is low due to a dearth of studies and taxonomic expertise. However, recent research activities in Benin and Cameroon suggest higher groundwater diversity than previously envisioned. Recent sampling campaigns in these countries have shown that Metastenasellus is a major group in the underground aquatic environment. The accumulation of biological material provided an opportunity to explore species diversity within the genus using a DNA taxonomy approach based on the cytochrome c oxidase subunit I (COI) gene fragment. Despite the limitations of using a single-locus approach for species delimitation, an overview of the diversity within the genus Metastenasellus was obtained, revealing the presence of 23 distinct lineages. Several elements suggest that most, if not all, of these lineages represent valid species. These include high genetic distances between lineages, morphologically distinct species separated by genetic distances of the same order of magnitude as between other described lineages, and the coexistence of different lineages at the same stations. Despite a limited sampling effort, these first results indicate a high level of species diversity and endemism within Metastenasellus in the studied regions. The narrow geographic distribution of the lineages suggests strong isolation and limited dispersal abilities. This study highlights the potential for discovering a significant number of new species within this genus and emphasizes the need for further research to uncover the extent of diversity in African stygobiotic isopods.

High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science.

Applying molecular methods to fungi establishing lichenized associations with green algae or cyanobacteria has repeatedly revealed the existence of numerous phylogenetic taxa overlooked by classical taxonomic approaches. Here, we report taxonomical conclusions based on multiple species delimitation and validation analyses performed on an eight-locus dataset that includes world-wide representatives of the dolichorhizoid and scabrosoid clades in section Polydactylon of the genus Peltigera. Following the recommendations resulting from a consensus species delimitation approach and additional species validation analysis (BPP) performed in this study, we present a total of 25 species in the dolichorhizoid clade and nine in the scabrosoid clade, including respectively 18 and six species that are new to science and formally described. Additionally, one combination and three varieties (including two new to science) are proposed in the dolichorhizoid clade. The following 24 new species are described: P. appalachiensis, P. asiatica, P. borealis, P. borinquensis, P. chabanenkoae, P. clathrata, P. elixii, P. esslingeri, P. flabellae, P. gallowayi, P. hawaiiensis, P. holtanhartwigii, P. itatiaiae, P. hokkaidoensis, P. kukwae, P. massonii, P. mikado, P. nigriventris, P. orientalis, P. rangiferina, P. sipmanii, P. stanleyensis, P. vitikainenii and P. willdenowii; the following new varieties are introduced: P. kukwae var. phyllidiata and P. truculenta var. austroscabrosa; and the following new combination is introduced: P. hymenina var. dissecta. Each species from the dolichorhizoid and scabrosoid clades is morphologically and chemically described, illustrated, and characterised with ITS sequences. Identification keys are provided for the main biogeographic regions where species from the two clades occur. Morphological and chemical characters that are commonly used for species identification in the genus Peltigera cannot be applied to unambiguously recognise most molecularly circumscribed species, due to high variation of thalli formed by individuals within a fungal species, including the presence of distinct morphs in some cases, or low interspecific variation in others. The four commonly recognised morphospecies: P. dolichorhiza, P. neopolydactyla, P. pulverulenta and P. scabrosa in the dolichorhizoid and scabrosoid clades represent species complexes spread across multiple and often phylogenetically distantly related lineages. Geographic origin of specimens is often helpful for species recognition; however, ITS sequences are frequently required for a reliable identification. Citation: Magain N, Miadlikowska J, Goffinet B, et al. 2023. High species richness in the lichen genus Peltigera (Ascomycota, Lecanoromycetes): 34 species in the dolichorhizoid and scabrosoid clades of section Polydactylon, including 24 new to science. Persoonia 51: 1-88. doi: 10.3767/persoonia.2023.51.01.

Metazoa-level USCOs as markers in species delimitation and classification.

Metazoa-level universal single-copy orthologs (mzl-USCOs) are universally applicable markers for DNA taxonomy in animals that can replace or supplement single-gene barcodes. Previously, mzl-USCOs from target enrichment data were shown to reliably distinguish species. Here, we tested whether USCOs are an evenly distributed, representative sample of a given metazoan genome and therefore able to cope with past hybridization events and incomplete lineage sorting. This is relevant for coalescent-based species delimitation approaches, which critically depend on the assumption that the investigated loci do not exhibit autocorrelation due to physical linkage. Based on 239 chromosome-level assembled genomes, we confirmed that mzl-USCOs are genetically unlinked for practical purposes and a representative sample of a genome in terms of reciprocal distances between USCOs on a chromosome and of distribution across chromosomes. We tested the suitability of mzl-USCOs extracted from genomes for species delimitation and phylogeny in four case studies: Anopheles mosquitos, Drosophila fruit flies, Heliconius butterflies and Darwin's finches. In almost all instances, USCOs allowed delineating species and yielded phylogenies that corresponded to those generated from whole genome data. Our phylogenetic analyses demonstrate that USCOs may complement single-gene DNA barcodes and provide more accurate taxonomic inferences. Combining USCOs from sources that used different versions of ortholog reference libraries to infer marker orthology may be challenging and, at times, impact taxonomic conclusions. However, we expect this problem to become less severe as the rapidly growing number of reference genomes provides a better representation of the number and diversity of organismal lineages.

Integrative species delimitation uncovers hidden diversity within the Pithecopus hypochondrialis species complex (Hylidae, Phyllomedusinae) and its phylogeography reveals Plio-Pleistocene connectivity among Neotropical savannas

Despite their limited vagility and pronounced habitat heterogeneity in the tropics, many anuran species have unexpectedly extensive geographic ranges. One prominent example of this phenomenon is Pithecopus hypochondrialis, which is found in the Cerrado, Guianan savanna, and Llanos domains, as well as isolated tracts of savanna and open habitat within the Amazon Forest. The present study employs an integrative species delimitation approach to test the hypothesis that P. hypochondrialis is in fact a species complex. We also reconstruct the relationships among the lineages delimited here and other Pithecopus species. In this study, we employ Ecological Niche Modelling (ENM) and spatiotemporal phylogeographic reconstruction approaches to evaluate a multitude of scenarios of connectivity across the Neotropical savannas. We identified three divergent lineages, two of which have been described previously. The lineages were allocated to a lowland Pithecopus clade, although the relationships among these lineages are weakly supported. Both the ENM and the phylogeographic reconstruction highlight the occurrence of periods of connectivity among the Neotropical savannas over the course of the Pliocene and Pleistocene epochs. These processes extended from eastern Amazonia to the northern coast of Brazil. The findings of the present study highlight the presence of hidden diversity within P. hypochondrialis, and reinforce the need for a comprehensive taxonomic review. These findings also indicate intricate and highly dynamic patterns of connectivity across the Neotropical savannas that date back to the Pliocene.

Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

DNA barcodes provide insights into the diversity and biogeography of the non-biting midge Polypedilum (Diptera, Chironomidae) in South America.

South America, particularly within its tropical belt, is renowned for its unparalleled high levels of species richness, surpassing other major biomes. Certain neotropical areas harbor fragmented knowledge of insect diversity and face imminent threats from biodiversity loss and climate change. Hence, there is an urgent need for rapid estimation methods to complement slower traditional taxonomic approaches. A variety of algorithms for delimiting species through single-locus DNA barcodes have been developed and applied for rapid species diversity estimates across diverse taxa. However, tree-based and distance-based methods may yield different group assignments, leading to potential overestimation or underestimation of putative species. Here, we investigate the performance of different DNA-based species delimitation approaches to rapidly estimate the diversity of Polypedilum (Chironomidae, Diptera) in South America. Additionally, we test the hypothesis that significant differences exist in the community structure of Polypedilum fauna between South America and its neighboring regions, particularly the Nearctic. Our analysis encompasses a dataset of 1492 specimens from 598 locations worldwide, with a specific focus on South America. Within this region, we analyzed a subset of 247 specimens reported from 37 locations. Using various methods including the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), multi-rate Poisson tree processes (mPTP), single-rate Poisson tree processes (sPTP), and generalized mixed Yule coalescent (sGMYC), we identify molecular operational taxonomic units (MOTUs) ranging from 267 to 520. Our results indicate that the sGMYC method is the most suitable for estimating putative species in our dataset, resulting in the identification of 75 species in the Neotropical region, particularly in South America. Notably, this region exhibited higher species richness in comparison to the Palearctic and Oriental realms. Additionally, our findings suggest potential differences in species composition of Polypedilum fauna between the Neotropical and the adjacent Nearctic realms, highlighting high levels of endemism and species richness in the first. These results support our hypothesis that there are substantial differences exist in species composition between the Polypedilum fauna in South America and the neighboring regions.

SPdel: A pipeline to compare and visualize species delimitation methods for single-locus datasets.

An accurate species delimitation is critical for biological studies. In this context, the use of molecular techniques along with species delimitation methods would help to a rapid and accurate biodiversity assessment. The species delimitation methods cluster data sets of orthologous sequences in molecular operational taxonomic units (MOTU). In particular, the methods based on a single gene are easily integrated with the widely used DNA barcoding approach. We developed SPdel a user-friendly pipeline to integrate different single-gene species delimitation methods. SPdel is designed to calculate and compare MOTUs obtained by different species delimitation approaches. SPdel also outputs diverse ready-to-publish quality figures, that facilitate the interpretation of results. SPdel aims to help researchers use species delimitation methods that would improve biodiversity studies.

Phylogenetic insight into the Lecidea atrobrunnea complex – evidence of narrow geographic endemics and the pressing need for integrative taxonomic revisions

AbstractSpecies of lichen-forming fungi (LFF) display an array of geographical distribution patterns. Among the broadly distributed lichen-forming fungal species, the degree of reproductive isolation and genetic substructure among populations varies widely, in some cases masking unrecognized diversity or meaningful biogeographical patterns. Lecidea atrobrunnea (Raymond ex Lam. & DC.) Schaer. s. lat. (Lecideaceae) is a widespread species complex that has been studied for over two centuries since its initial description. The diversity of the L. atrobrunnea group is highest in western North America, where a dizzying array of morphologies and chemistry can occur at local scales. Here we investigate whether the assumed cosmopolitan distribution of L. atrobrunnea s. lat. is an artifact of taxonomic limitations and masks biogeographical patterns in this species complex. To address these questions, we compiled sequence data from the standard fungal barcoding marker (ITS) for over 100 specimens within this complex, in addition to genome-scale data from a subset of these representing over 1600 single-copy nuclear genes spanning over 3 Mb of the genome. Our study corroborates the perspective that the morphologically and chemically variable Lecidea atrobrunnea group reflects a complex of distinct species-level lineages, with 42–83 candidate species inferred from the ITS region and high levels of diversity inferred from a subset of specimens using genome-scale data. However, both phenotype- and molecular-based species boundaries remained unsettled, with the most common nominal taxa recovered as highly polyphyletic and with conflict among different molecular species delimitation approaches. Our study also highlights the potential for geographically restricted species, with fascinating biogeographical patterns, challenging, in part, the assumed cosmopolitan distribution of L. atrobrunnea s. lat. This study provides valuable direction for future research that will be crucial in understanding diversification and establishing a robust taxonomy for this well-known species complex.

Historical Climate Change Dynamics Facilitated Speciation and Hybridization Between Highland and Lowland Species of Baripus Ground Beetles From Patagonia

One of the largest beetles of Patagonia, Baripus (Cardiophthalmus), includes 20 currently described species. Its distribution ranges from the tip of Patagonia, on Tierra del Fuego Island, to isolated patches along the Andes and extra-Andes mountains in northern Patagonia on the Payunia at >3000 m elevation. Here, using RADseq data, evidence is found of mixed ancestry in different lineages. Phylogenetic network reconstruction shows two hybridizing edges between lowland and highland species. Using environmental niche modeling, we show changes in geographic distribution of potential niches of species during the last glacial maximum compared to their present distribution. Increasing potential niche overlap among different species pairs possibly explains how lineages came into secondary contact, supporting the hypothesis of hybridization. In addition, morphological evolution is studied using geometric morphometrics on the network, and evidence of transgressive evolution has been found involving the pronotum shape, as well as highland/lowland habitat preferences. Finally, based on genomics and morphological data and using an integrative coalescent-based species delimitation approach, the separate evolution of two lineages in early stages of speciation is found. Taken together, dynamics of diversification of Baripus beetles in both space and time are discussed.

Unravelling the diversity of the genus Afronurus Lestage, 1924 (Ephemeroptera, Heptageniidae) in Thailand.

The genus Afronurus in Thailand is investigated using an integrative approach (morphology based, ootaxonomy and molecular data) for species delimitation. A total of four species of Afronurus was identified; A.cervina (Braasch & Soldán, 1984), A.gilliesiana (Braasch, 1990), A.rainulfiana (Braasch, 1990), and A.rubromaculata (You et al., 1981). The subimago of A.gilliesiana is described for the first time based on reared specimens. The egg structure of all four species is also described for the first time. Morphological and molecular data strongly support their species delimitation. The egg chorionic structure of the genus Afronurus, together with other morphological characters, is useful for species identification. A key to mature nymphs of the known species is provided.

Contrasting results of multiple species delimitation approaches cause uncertainty in synecological studies: A case study on Sri Lankan chafers

Abstract Biodiversity patterns are the sum of multiple overlapping species distributions. Their analysis therefore requires proper species inference. DNA‐based species delimitation has become increasingly popular for such assessments, but their robustness is often problematic due to incongruence between multiple delimitation approaches. Here, we explored how contrasting results of different species’ delimitations translate into conclusions of synecological studies, exemplified by assemblages of phytophagous scarab beetles in Sri Lanka from different elevations and forest types. Particularly, we compared estimates based on complete assemblages and on cumulated species inventories inferred from individually analysed subclades. The patterns of assemblage similarity were analysed across different spatial scales with reference to morphospecies, molecular operational taxonomic units (MOTUs) and haplotypes. Method‐related ambiguity of species (MOTUs) estimates, which also included subclade inferences, severely affected the certainty of apparent biodiversity patterns at most spatial scales. Even more contrasting patterns resulted from individual clade‐wise analyses of faunal similarity or even from cumulated species inventories from individual clade‐wise species delimitation analysis. In this case study of tropical chafer beetles, haplotypes provided only very little explanatory information, since genetically highly diverse populations widely lacked shared haplotypes. Therefore, searching proper species boundaries should be the ultimate goal of biodiversity assessments to lend an enduring meaning to biodiversity research and its sustainable application. Our study underlines the need for awareness when synecological observations from different studies are integrated, which use different species delimitation methods for their biodiversity assessment, and its potential impact on conservation management.

A global phylogeny of Elysia Risso, 1818 (Gastropoda: Heterobranchia): molecular systematic insights focusing on European taxa and description of a new species

Abstract The genus Elysia comprises about one-third of the species richness in Sacoglossa. However, the species diversity in the genus remains poorly characterized in some areas like the north-eastern Atlantic and Mediterranean waters. To clarify the systematics of this genus and to characterize the species diversity in undersampled regions, we performed an integrative study based on a thorough literature review, molecular and morphological analyses, and species delimitation approaches. We conducted phylogenetic analyses of partial sequences of two mitochondrial genes (COI and 16S) and two nuclear genes (H3, 28S) using Bayesian inference and maximum likelihood methods, which confirmed the presence of five of the recognized European Elysia species: Elysia viridis, E. timida, E. flava, E. margaritae, and E. rubeni. Moreover, a new species (Elysia azorica sp. nov.) was identified in the Azores, and E. gordanae, currently considered a junior synonym of E. margaritae, was recovered as a distinct species. In addition, we consider E. hetta as a junior synonym of E. gordanae, and E. translucens as a taxon inquirendum. Finally, the tropical E. evelinae is recorded along European coasts for the first time. Our results demonstrate the value of integrative approaches in resolving taxonomic uncertainty surrounding polymorphism and unravelling potential cases of cryptic or pseudocryptic species complexes.

DNA Barcoding of Cardinalfish (Apogonidae) in Gilimanuk Bay, Bali, Indonesia

Highlight Research Eight species of cardinalfish in Gilimanuk Bay were delineated using the barcoding method. This study provides the first nucleotide sequence for Ostorhinchus hartzfeldii in GenBank online database. The phylogenetic tree showed that each sample resided in a distinct cluster, indicating that the barcoding method efficiently differentiated at the species level. The mean genetic distance between genera within the family was 31.8-fold higher than the mean genetic distance within species. Abstract The Apogonidae is estimated to consist of nearly 300 fish species, most of which inhabit coral reef areas. The lack of distinctive body markings and overlapping species distribution makes species assignment challenging. Therefore, this study aimed to delineate species and establish barcoding reference databases of Apogonidae in Gilimanuk Bay (Bali, Indonesia) using the Cytochrome Oxidase I (COI) gene of the mitochondrial DNA. A total of 22 fish tissue samples were extracted with 10% Chelex solution. BLAST analysis was performed and genetic differentiation between species was calculated. The phylogenetic tree was constructed using the Maximum Likelihood method and tree visualization was generated using iTOL V5. The morphology and genetic identification results based on the mitochondrial COI gene revealed eight species of seven genera, and one species was new to GenBank online database. This study was the first-ever addition of COI sequence for Ostorhinchus hartzfeldii into the GenBank database. The average K2P genetic distance within species and K2P distance between genera within the family were 0.60% and 19.10%, respectively. The mean genetic distance between genera within the family was 31.8-fold higher than the mean genetic distance within species. The phylogenetic tree showed that each sample resided in a distinct cluster, which indicates that DNA barcoding is a reliable and effective approach for species delimitation in Apogonidae fishes.