Accurate Species Delimitation Research Articles

Taxonomic note on the endemic genus Lophopogon (Poaceae: Andropogoneae) from India

Although the genus Lophopogon comprises only two distinct species, their identification has always been bewildering in some of the available grass literature in India. This confusion mainly stems from the tendency of Indian botanists to utilize weak key characters to differentiate the species. We have discussed both the variable as well as distinguishing features of the Lophopogon species and revised the key to make it more efficient. Upon a thorough morphological study of the recently described L. prasannae, we have concluded that it is but a variable form of an obscure species L. kingii. Therefore, we now consider it a new heterotypic synonym of the latter. While re-evaluating the previously misidentified specimens of Lophopogon, we have established two new distributional records of L. kingii for the states of Andhra Pradesh and Karnataka, South India. In L. kingii a characteristic whitish indumentum is observed, which is confined to the base of the plant and is not observed in any of the variable forms of L. tridentatus. Based on this character, we have restructured and simplified the identification key for easy and accurate species delimitation. We have also provided a brief discussion on the phenotypic variability of both the species, along with photo plates to facilitate identification and showcase a variability spectrum in plant habit. Distribution of L. kingii is plotted on the map based on the available specimens at BSI, BSID, CAL and K herbaria.

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.

Integrative taxonomy in a rapid speciation group associated with mating system transition: A case study in the Primula cicutariifolia complex

Accurate species delimitation is the key to biodiversity conservation and is fundamental to most branches of biology. However, species delimitation remains challenging in those evolutionary radiations associated with mating system transition from outcrossing to self-fertilization, which have frequently occurred in angiosperms and are usually accompanied by rapid speciation. Here, using the Primula cicutariifolia complex as a case, we integrated molecular, morphological and reproductive isolation evidence to test and verify whether its outcrossing (distylous) and selfing (homostylous) populations have developed into independent evolutionary lineages. Phylogenetic trees based on whole plastomes and SNPs of the nuclear genome both indicated that the distylous and homostylous populations grouped into two different clades. Multispecies coalescent, gene flow and genetic structure analyses all supported such two clades as two different genetic entities. In morphology, as expected changes in selfing syndrome, homostylous populations have significantly fewer umbel layers and smaller flower and leaf sizes compared to distylous populations, and the variation range of some floral traits, such as corolla diameter and umbel layers, show obvious discontinuity. Furthermore, hand-pollinated hybridization between the two clades produced almost no seeds, indicating that well post-pollination reproductive isolation has been established between them. Therefore, the distylous and homostylous populations in this studied complex are two independent evolutionary lineages, and thus these distylous populations should be treated as a distinct species, here named Primula qiandaoensis W. Zhang & J.W. Shao sp. nov.. Our empirical study of the P. cicutariifolia complex highlights the importance of applying multiple lines of evidence, in particular genomic data, to delimit species in pervasive evolutionary plant radiations associated with mating system transition.

Identification of Acrossocheilus fasciatus (Teleostei: Cyprinidae) from Three Different Phylogeographic Sources using Morphology and COI DNA Barcoding

Background: Accurate species delimitation is essential for phylogenetic, phylogeographic, ecological, conservation and biogeographic investigations. Acrossocheilus species classification has long been contentious, owing to their uncertain evolutionary relationships, high levels of morphological homoplasy and a lack of knowledge of the taxonomic validity of conventionally utilized morphological characters. Methods: A final data set consisting of 102 mitochondrial cytochrome oxidase I (COI) gene sequences was analysed for K2P intraspecific and interspecific divergence in the current study, using reconstruction methods (maximum likelihood) and various models (ABGD, PTP, GMYC) to divide the sequences according to the pattern of genetic variation. We used landmark-based geometric morphometrics to identify specimens. Result: Two taxa were identified according to the phylogenetic tree (maximum likelihood method) and three different models (ABGD, PTP, GMYC). The molecular taxonomy of Acrossocheilus agreed with the morphological identification of specimens from the same collection locality. Therefore, the COI gene and geometric morphometrics were effective in species identification of A. fasciatus.

Mitochondrial introgression and mito-nuclear discordance obscured the closely related species boundaries in Cletus Stål from China (Heteroptera: Coreidae)

Accurate taxonomy and delimitation are of great importance for pest control strategies and management programs. Here, we focus on Cletus (Insecta: Hemiptera: Coreidae), which includes many crop pests. The species boundaries still conflict and only cytochrome c oxidase subunit I (COI) barcoding has been previously used for molecular studies. We generated new mitochondrial genome and nuclear genome-wide SNPs to explore the species boundaries of 46 Cletus samples from China using multiple species delimitation approaches. All results recovered a monophyly with high support, except for two closely related species in clade I - C. punctiger and C. graminis. Mitochondrial data demonstrated admixture in clade I, while genome-wide SNPs unambiguously identified two separate species, which were confirmed by morphological classification. Inconsistent nuclear and mitochondrial data indicated mito-nuclear discordance. Mitochondrial introgression is the most likely explanation, and more extensive sampling and more comprehensive data are needed to ascertain a pattern. Accurate species delimitation will shed light on species status; thus, an accurate taxonomy is of particular concern, as there is a pressing need to implement precise control of agricultural pests and to perform further research on diversification.

Using low-coverage whole genome sequencing (genome skimming) to delineate three introgressed species of buffalofish (Ictiobus)

Consumption of buffalofish has been sporadically associated with Haff disease-like illnesses involving sudden onset muscle pain and weakness due to skeletal muscle rhabdomyolysis, but determination of precisely which species are associated with these illnesses has been impeded by a lack of species-specific DNA-based markers. Here, three closely related species of buffalofish native to the Mississippi River Basin (Ictiobus bubalus, Ictiobus cyprinellus and Ictiobus niger) that have previously proven genetically indistinguishable using both mitochondrial and nuclear single-locus sequencing were reliably discriminated using low-coverage whole genome sequencing (‘genome skimming’). Using 44 specimens representing the three species collected from the mid/upper (Missouri) and lower (Louisiana) regions of the species’ native ranges, the SISRS (Site Identification from Short Read Sequences) bioinformatics pipeline was adapted to (1) identify over 620Mbp of putatively homologous nuclear sequence data and (2) isolate over 140,000 single-nucleotide polymorphisms (SNPs) that supported accurate species delimitation, all without the use of a reference genome or annotation data. These sites were used to classify Ictiobus spp. samples with genome-skim data, along with a larger set (n = 67) where ultraconserved elements (UCEs) were sequenced. Analyses of whole mitochondrial data revealed more limited signal. Nearly all samples matched their purported species based on morphologic identification, but two Missouri samples morphologically identified as I. niger grouped with samples of I. bubalus, albeit with significant enrichment of I. niger SNPs. To our knowledge this is the first report of a DNA-based tool to reliably discriminate these three morphologically distinct species.

Delimiting species in the taxonomically challenging orchid section Pseudophrys: Bayesian analyses of genetic and phenotypic data

Accurate species delimitation is critical for biodiversity conservation. Integrative taxonomy has been advocated for a long time, yet tools allowing true integration of genetic and phenotypic data have been developed quite recently and applied to few models, especially in plants. In this study, we investigated species boundaries within a group of twelve Pseudophrys taxa from France by analyzing genetic, morphometric and chemical (i.e., floral scents) data in a Bayesian framework using the program integrated Bayesian Phylogenetics and Phylogeography (iBPP). We found that these twelve taxa were merged into four species when only genetic data were used, while most formally described species were recognized as such when only phenotypic (either morphometric or chemical) data were used. The result of the iBPP analysis performed on both genetic and phenotypic data supports the proposal to merge Ophrys bilunulata and O. marmorata on the one hand, and O. funerea and O. zonata on the other hand. Our results show that phenotypic data are particularly informative in the section Pseudophrys and that their integration in a model-based method significantly improves the accuracy of species delimitation. We are convinced that the integrative taxonomic approach proposed in this study holds great promise to conduct taxonomic revisions in other orchid groups.

Aneuploidy promotes intraspecific diversification of the endemic East Asian herb Lycoris aurea complex.

Polyploidy has received considerable interest in the past, but aneuploidy and partial rearrangements may also influence genomic divergence. In this study, we reported a comprehensive cytogeographic, morphological and genetic analysis of Lycoris aurea complex throughout its range and attempted to explore the association between aneuploidy and species diversification. The karyotypes of this complex presented aneuploidy variations mainly divided into four cytotypes: I (2n = 10m + 2T), II (2n = 8m + 6T), III (2n = 7m + 8T), and IV (2n = 6m + 10T). Cytotype distributions were highly structured geographically. Two main cytotypes, II and IV, are geographically allopatric. The populations with cytotype II are mainly distributed in central China and the southern islands of Japan. Cytotypes IV is disjunctly distributed in southwestern and southeastern China. The cytotypes with fewer chromosome numbers tend to occur at high latitudes. For analyzing the phylogeographic pattern and genetic structure of this complex, we sequenced four chloroplast DNA fragments (4,748 bp in total) of 241 individuals from 42 populations. Extremely high diversity of cpDNA haplotypes was found, with genetic diversity index (Hd) being 0.932 and 98.61% of the genetic variation occurring among populations, indicating that this complex has undergone strong intraspecific differentiation. The cytotype II had the highest haplotype diversity (Hd = 0.885), while cytotype IV harbored the highest nucleotide diversity (π = 4.09 × 10–3). We detected significant leaf morphological differences not only between cytotype II and IV but also between west lineage and east lineage within cytotype IV. These results illustrated that aneuploidy contributed to extensive morphological and genetic differentiation in L. aurea complex. It was suggested that L. aurea complex should comprise multiple independent evolutionary lineages, and accurate species delimitation needs to be established further in an integrative taxonomic approach.

Phylogenomic structure and speciation in an emerging model: the Sphagnum magellanicum complex (Bryophyta).

Sphagnum magellanicum is one of two Sphagnum species for which a reference-quality genome exists to facilitate research in ecological genomics. Phylogenetic and comparative genomic analyses were conducted based on resequencing data from 48 samples and RADseq analyses based on 187 samples. We report herein that there are four clades/species within the S. magellanicum complex in eastern North America and that the reference genome belongs to Sphagnum divinum. The species exhibit tens of thousands (RADseq) to millions (resequencing) of fixed nucleotide differences. Two species, however, referred to informally as S. diabolicum and S. magni because they have not been formally described, are differentiated by only 100 (RADseq) to 1000 (resequencing) of differences. Introgression among species in the complex is demonstrated using D-statistics and f4 ratios. One ecologically important functional trait, tissue decomposability, which underlies peat (carbon) accumulation, does not differ between segregates in the S. magellanicum complex, although previous research showed that many closely related Sphagnum species have evolved differences in decomposability/carbon sequestration. Phylogenetic resolution and more accurate species delimitation in the S. magellanicum complex substantially increase the value of this group for studying the early evolutionary stages of climate adaptation and ecological evolution more broadly.

Phylogenomics and species delimitation of the economically important Black Basses (Micropterus)

Informed management and conservation efforts are vital to sustainable recreational fishing and biodiversity conservation. Because the taxonomic rank of species is important in conservation and management strategies, success of these efforts depends on accurate species delimitation. The Black Basses (Micropterus) are an iconic lineage of freshwater fishes that include some of the world’s most popular species for recreational fishing and world's most invasive species. Despite their popularity, previous studies to delimit species and lineages in Micropterus suffer from insufficient geographic coverage and uninformative molecular markers. Our phylogenomic analyses of ddRAD data result in the delimitation of 19 species of Micropterus, which includes 14 described species, the undescribed but well-known Altamaha, Bartram’s, and Choctaw basses, and two additional undescribed species currently classified as Smallmouth Bass (M. dolomieu). We provide a revised delimitation of species in the Largemouth Bass complex that necessitates a change in scientific nomenclature: Micropterus salmoides is retained for the Florida Bass and Micropterus nigricans is elevated from synonymy for the Largemouth Bass. The new understanding of diversity, distribution, and systematics of Black Basses will serve as important basis for the management and conservation of this charismatic and economically important clade of fishes.

Towards Large-Scale Integrative Taxonomy (LIT): Resolving the Data Conundrum for Dark Taxa.

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling “dark taxa,” here defined as groups for which n}{}<10n}{}% of all species are described and the estimated diversity exceeds 1,000 species. Species delimitation for these taxa should be based on multiple data sources (“integrative taxonomy”) but collecting multiple types of data risks impeding a discovery process that is already too slow. We here develop large-scale integrative taxonomy (LIT), an explicit method where preliminary species hypotheses are generated based on inexpensive data that can be obtained quickly and cost-effectively. These hypotheses are then evaluated based on a more expensive type of “validation data” that is only obtained for specimens selected based on objective criteria applied to the preliminary species hypotheses. We here use this approach to sort 18,000 scuttle flies (Diptera: Phoridae) into 315 preliminary species hypotheses based on next-generation sequencing barcode (313 bp) clusters (using objective clustering [OC] with a 3n}{}% threshold). These clusters are then evaluated with morphology as the validation data. We develop quantitative indicators for predicting which barcode clusters are likely to be incongruent with morphospecies by randomly selecting 100 clusters for in-depth validation with morphology. A linear model demonstrates that the best predictors for incongruence between barcode clusters and morphology are maximum p-distance within the cluster and a newly proposed index that measures cluster stability across different clustering thresholds. A test of these indicators using the 215 remaining clusters reveals that these predictors correctly identify all clusters that are incongruent with morphology. In our study, all morphospecies are true or disjoint subsets of the initial barcode clusters so that all incongruence can be eliminated by varying clustering thresholds. This leads to a discussion of when a third data source is needed to resolve incongruent grouping statements. The morphological validation step in our study involved 1,039 specimens (5.8n}{}% of the total). The formal LIT protocol we propose would only have required the study of 915 (5.1n}{}%: 2.5 specimens per species), as we show that clusters without signatures of incongruence can be validated by only studying two specimens representing the most divergent haplotypes. To test the generality of our results across different barcode clustering techniques, we establish that the levels of incongruence are similar across OC, Automatic Barcode Gap Discovery (ABGD), Poisson Tree Processes (PTP), and Refined Single Linkage (RESL) (used by Barcode of Life Data System to assign Barcode Index Numbers [BINs]). OC and ABGD achieved a maximum congruence score with the morphology of 89n}{}% while PTP was slightly less effective (84n}{}%). RESL could only be tested for a subset of the specimens because the algorithm is not public. BINs based on 277 of the original 1,714 haplotypes were 86n}{}% congruent with morphology while the values were 89n}{}% for OC, 74n}{}% for PTP, and 72n}{}% for ABGD. [Biodiversity discovery; dark taxa; DNA barcodes; integrative taxonomy.]

Coalescent-based species delimitation in North American pinyon pines using low-copy nuclear genes and plastomes.

PremiseAccurate species delimitation is essential for evolutionary biology, conservation, and biodiversity management. We studied species delimitation in North American pinyon pines, Pinus subsection Cembroides, a natural group with high levels of incomplete lineage sorting.MethodsWe used coalescent‐based methods and multivariate analyses of low‐copy number nuclear genes and nearly complete high‐copy number plastomes generated with the Hyb‐Seq method. The three coalescent‐based species delimitation methods evaluated were the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP), and Trinomial Distribution of Triplets (Tr2). We also measured admixture in populations with possible introgression.ResultsOur results show inconsistencies among GMYC, PTP, and Tr2. The single‐locus based GMYC analysis of plastid DNA recovered a higher number of species (up to 24 entities, including singleton lineages and clusters) than PTP and the multi‐locus coalescent approach. The PTP analysis identified 10 species whereas Tr2 recovered 13, which agreed closely with taxonomic treatments.ConclusionsWe found that PTP and GMYC identified species with low levels of ILS and high morphological divergence (P. maximartinezii, P. pinceana, and P. rzedowskii). However, GMYC method oversplit species by identification of more divergent samples as singletons. Moreover, both PTP and GMYC were incapable of identifying some species that are readily identified morphologically. We suggest that the divergence times between lineages within North American pinyon pines are so disparate that GMYC results are unreliable. Results of the Tr2 method coincided well with previous delimitations based on morphology, DNA, geography, and secondary chemistry.

Two Fish in a Pod. Mislabelling on Board Threatens Sustainability in Mixed Fisheries

Accuracy in reporting captures is a key element to achieve fisheries sustainability. However, identification of the catches might be a challenge when two or more species are morphologically similar and caught jointly, like the mixed fisheries of black hakes in East Atlantic African waters. Black hakes (Merluccius senegalensis and M. polli) are tough to differentiate without previous training due to their high morphological resemblance. The two species are managed as a single stock, although the biological differences between them suggest the need of a separate management. In this study, a total of 806 black hakes were visually identified by fishers on deck of fishing vessels operating in Mauritania and Senegal waters, then assigned to a species by sequencing 450bp of the Mitochondrial Control Region. Comparing the results with visual identification we found 31.4% of the total catch were incorrectly labelled on board by the fishermen. The accuracy of the fishers’ identification depended on the depth of capture and on fish size, larger individuals caught from deeper waters being more correctly assigned to M. polli. Mislabelling biased to M. polli suggests that M. senegalensis, already catalogued as endangered, is being underreported, which could endanger the conservation of this species and threaten the sustainability of black hake fisheries. Our results highlight the need for separate evaluation of the stocks in mixed fisheries for morphologically similar fish. Thus, monitoring through DNA barcoding in the very first step of the seafood chain surveys would improve accurate species delimitation and reduce its impact on the correct assessment of the stocks.

An integrative approach reveals high species diversity in the primitively segmented spider genus

Accurate species delimitation is crucial for our understanding of evolution, biodiversity and conservation. However, morphology-based species delimitation alone appears to be prone to taxonomic errors and ineffective for taxa with high interspecific morphological homogeneity or intraspecific morphological variations, as is the case for mesothele and mygalomorph spiders. Combined molecular–morphology species delimitation has shown great potential to delimit species boundaries in such ancient lineages. In the present study, molecular and morphological evidence were integrated to delimit species of the primitively segmented spider genus Songthela Ono, 2000. The cytochrome c oxidase subunit I gene (COI) was sequenced for 192 novel specimens belonging to 12 putative morphospecies. The evolutionary relationships within Songthela and the 12-morphospecies hypothesis were tested in two steps – species discovery and species validation – using four single-locus species delimitation approaches. All species delimitation analyses supported the 12-species hypothesis. Phylogenetic analyses yielded three major clades in Songthela, which are consistent with morphology. Accordingly, we assigned 19 known and 11 new species (S. aokoulong, sp. nov., S. bispina, sp. nov., S. dapo, sp. nov., S. huayanxi, sp. nov., S. lianhe, sp. nov., S. lingshang, sp. nov., S. multidentata, sp. nov., S. tianmen, sp. nov., S. unispina, sp. nov., S. xiujian, sp. nov., S. zizhu, sp. nov.) of Songthela to three species-groups: the bispina-group, the multidentata-group and the unispina-group. Another new species, S. zimugang, sp. nov., is not included in any species groups, but forms a sister lineage to the bispina- and unispina-groups. These results elucidate a high species diversity of Songthela in a small area and demonstrate that integrating morphology with COI-based species delimitation is fast and cost-effective in delimiting species boundaries. http://zoobank.org/urn:lsid:zoobank.org:pub:AF0F5B31-AFAF-4861-9844-445AE8678B67

The effect of landscape and human settlement on the genetic differentiation and presence of Paragonimus species in Mesoamerica

Foodborne diseases are a neglected research area, and despite the existence of many tools for diagnosis and genetic studies, very little is known about the effect of the landscape on the genetic diversity and presence of parasites. One of these foodborne disease is paragonimiasis, caused by trematodes of the genus Paragonimus, which is responsible for a high number of infections in humans and wild animals. The main Paragonimus sp reported in Mesoamerica is Paragonimus mexicanus, yet there are doubts about its correct identification as a unique species throughout the region. This, together with a lack of detailed knowledge about their ecology, evolution and differentiation, may complicate the implementation of control strategies across the Mesoamerican region. We had the goal of delimiting the species of P. mexicanus found throughout Mesoamerica and determining the effect of landscape and geology on the diversity and presence of the parasite. We found support for the delimitation of five genetic groups. The genetic differentiation among these groups was positively affected by elevation and the isolation of river basins, while the parasite’s presence was affected negatively only by the presence of human settlements. These results suggest that areas with lower elevation, connected rivers basins, and an absence of human settlements have low genetic differentiation and high P. mexicanus presence, which may increase the risk of Paragonimus infection. These demonstrate the importance of accurate species delimitation and consideration of the effect of landscape on Paragonimus in the proposal of adequate control strategies. However, other landscape variables cannot be discarded, including temperature, rainfall regime, and spatial scale (local, landscape and regional). These additional variables were not explored here, and should be considered in future studies.

Toward a global DNA barcode reference library of the intolerant nonbiting midge genus Rheocricotopus Brundin, 1956.

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time‐ and cost‐efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the “Barcode Gap Analysis” and neighbor‐joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well‐separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota.

Deciphering the Taxonomic Delimitation of Ottelia acuminata (Hydrocharitaceae) Using Complete Plastomes as Super-Barcodes

Accurate species delimitation and identification, which is a challenging task in traditional morphology-based taxonomy, is crucial to species conservation. Ottelia acuminata (Hydrocharitaceae) is a severely threatened submerged macrophyte endemic to southwestern China. The taxonomy of O. acuminata, which has long been in dispute, remains unresolved, impeding effective conservation and management practices. Here, we aim to address the long-standing issues concerning species boundary and intraspecific subdivision of O. acuminata using complete plastome sequences as super-barcodes. The taxonomic delimitation of O. acuminata was explored using phylogenetic inference and two independent sequence-based species delimitation schemes: automatic barcode gap discovery (ABGD) and multi-rate Poisson tree processes (mPTP). The reciprocally reinforcing results support the reduction of the closely related congeneric species, O. balansae and O. guanyangensis, as two conspecific varieties of O. acuminata. Within the newly defined O. acuminata, accurate varietal identification can be achieved using plastome super-barcodes. These findings will help inform future decisions regarding conservation, management and restoration of O. acuminata. This case study suggests that the use of plastome super-barcodes can provide a solution for species delimitation and identification in taxonomically difficult plant taxa, thus providing great potential to lessen the challenges of inventorying biodiversity, as well as biologically monitoring and assessing threatened species.

Taxon delimitation, chromosome numbers and genetic diversity of Paspalum polyphyllum and P. bicilium (Poaceae, Paspaleae)

The South American Paspalum polyphyllum and P. bicilium need accurate species delimitation. The aim of this study was to assess morphological variation, through morphometric analysis of specimens from natural populations, chromosome number and genetic diversity of populations based on inter-simple sequence repeat (ISSR) molecular markers, to test taxonomic relationships among these species and other related species. We analyzed 183 individuals of P. polyphyllum and P. bicilium from 31 natural populations from Brazil, Argentina and Bolivia. Multivariate analyses from 23 morphological characters allowed the distinction of P. bicilium from P. polyphyllum, and the most important characters were: rachis width, spikelet and upper floret length. For both species, most of the genetic diversity was shown among the populations (88.36% of the total mean variation in P. polyphyllum and 84.31% in P. bicilium), while the diversity within the populations was low (11, 64% and 15.69%, respectively). Cytogenetic studies revealed a new chromosome number (2n = 36) for P. polyphyllum and P. bicilium, and 2n = 46 for P. burmanii. Results obtained made it possible to recognize P. bicilium as a distinct species from P. polyphyllum. We provide a taxonomic treatment including species descriptions, a distribution map, illustrations, and a comparative table for P. bicilium and P. polyphyllum, besides preliminary data for five related species of Paspalum subgen. Ceresia.

Unveiling cryptic diversity among M\xfcllerian co-mimics: insights from the Western Palaearctic Syntomis moths (Lepidoptera: Erebidae: Arctiinae)

Accurate species delimitation is of primary importance in biodiversity assessments and in reconstructing patterns and processes in the diversification of life. However, the discovery of cryptic species in virtually all taxonomic groups unveiled significant gaps in our knowledge of biodiversity. Mimicry complexes are good candidates to source for cryptic species. Indeed, members of mimicry complexes undergo selective pressures on their habitus, which results in strong resemblance even between distantly related species. In this study, we used a multi-locus genetic approach to investigate the presence of cryptic diversity within a group of mimetic day-flying moths whose systematics has long been controversial, the Euro-Anatolian Syntomis. Results showed incongruence between species boundaries and the currently accepted taxonomy of this group. Both mitochondrial and nuclear markers indicate the presence of four, well-distinct genetic lineages. The genetic distance and time of divergence between the Balkan and Italian populations of S. marjana are the same as those found between S. phegea and S. ragazzii, the last two being well-distinct, broadly sympatrically occurring species. The divergence between the two lineages of S. marjana dates back to the Early Pleistocene, which coincided with substantial changes in climatic conditions and vegetation cover in Southern Europe that have likely induced geographic and ecological vicariance. Syntomis populations belonging to the taxa kruegeri (s. str.), albionica and quercii are now considered a separate species from marjana s. str. and are thus distinguished as Syntomis quercii Verity, 1914, bona sp., stat. nov. Our results show that the species richness of mimicry complexes inhabiting temperate regions might still be severely underestimated.

The first comprehensive DNA barcode reference library of Chinese Tanytarsus (Diptera: Chironomidae) for environmental DNA metabarcoding

AbstractAimReliable DNA barcode reference libraries are critical for biodiversity monitoring based on Environmental DNA (eDNA). DNA barcoding has proven successful in species delimitation and association of life stages in many groups of animals, such as non‐biting midges (Chironomidae). We use the genus Tanytarsus van der Wulp, 1874, second largest genus of the family Chironomidae, to test whether DNA barcodes can be used to identify unknown and cryptic species. A comprehensive DNA barcode library will be built to facilitate further eDNA studies for biodiversity monitoring and conservation in freshwater ecosystems.LocationMainland China.MethodsIn this study, the cytochrome c oxidase subunit 1 (COI) DNA barcodes from 298 individuals, representing 56 morphospecies from China, were analysed, including 136 sequences from previous studies and 162 newly generated DNA barcodes. 298 COI barcodes were applied into the Automatic Barcode Gap Discovery (ABGD) to compare the operational taxonomic units (OTU) number resulting from the Barcode Index Number (BIN) based “Barcode Gap Analysis” and neighbour joining (NJ) tree.ResultsTotal 298 barcode sequences were applied to 66 BINs, including 20 singleton BINs and 46 concordant BINs. ABGD yielded 55 OTUs with a prior intraspecific divergence of Pmax = 3.59%. By Integrating DNA barcodes with morphology, we revealed 21 putative species new to science and two unidentified species from the 56 well‐separated morphospecies. Besides, several cryptic species are uncovered using the NJ tree and TCS haplotype network based on COI DNA barcodes.Main conclusionsOur results demonstrate that DNA barcodes enable the discovery and identification of Tanytarsus non‐biting midges. A comprehensive DNA barcode reference library could speed up accurate species delimitation and discovery by providing a reliable resource for taxonomic studies, and improve the application of eDNA metabarcoding in biomonitoring of freshwater ecosystems.