Tool For Species Delimitation Research Articles

A Geometric Morphometrics Approach to the Study of Natural Variations and Hybrid Detection in Populations of Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák.

Landmark-based geometric morphometrics (GM) was used to examine, for the first time, spontaneous hybridization between Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák, and to assess inter- and intrapopulation variability in leaf shape, leaf size and venation in natural populations in Serbia (Western Balkans). Two geographically distant (30 km) and two close (1.2 km) populations were selected to examine hybridization. The variability in leaf shapes was assessed by canonical variate analysis and linear discriminant analysis performed on the symmetric component of variation. Covariation between the symmetric component of shape variation and the number of pairs of secondary leaf veins was investigated with partial least squares analysis. Static allometry was examined for the first time in the genus Alnus Mill. A higher proportion of A. incana leaves was classified as A. rohlenae in geographically close populations, which is in accordance with the hypothesis about spontaneous hybridization. No single leaf of A. rohlenae was classified as A. incana, indicating that putative hybrids can only be found in grey alder populations. This study demonstrates that GM is a powerful tool for species delimitation and hybrid detection in the genus Alnus and it can be used for preliminary screening in hybrid zones.

Limits of mitochondrial genes in delimiting species within a Carbula species complex (Hemiptera: Pentatomidae).

Molecular data has become a powerful tool for species delimitation, particularly among those that present limited morphological differences; while the mitochondrial genome, with its moderate length, low cost of sequencing and fast lineage sorting, has emerged as a practical data set. Due to the limited morphological differences among the closely related species of Carbula Stål 1865, the species boundaries between Carbula abbreviata (Motschulsky, 1866), Carbula humerigera (Uhler, 1860), and Carbula putoni (Jakovlev, 1876) have remained particularly unclear. In this study, we applied two phylogenetic reconstruction methods to two data sets (mitogenome and COI) to assess the phylogeny of Carbula distributed in Asia, and five species delimitation methods to determine the boundaries between East Asian Carbula species. Our phylogenetic analyses showed Carbula to be paraphyletic; the seven known species distributed within East Asia to form a single monophyletic group, and within this, C. abbreviata, C. humerigera, C. putoni and middle-type to comprise a C. humerigera species complex. Our results show that mitogenome data alone, while effective in the differentiation of more distantly related Carbula species, is not sufficient to accurately delimit the species within this newly described complex.

New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Evaluating species richness using proteomic fingerprinting and DNA barcoding—a case study on meiobenthic copepods from the Clarion Clipperton Fracture Zone

The Clarion Clipperton Fracture Zone (CCZ) is a vast deep-sea region harboring a highly diverse benthic fauna, which will be affected by potential future deep-sea mining of metal-rich polymetallic nodules. Despite the need for conservation plans and monitoring strategies in this context, the majority of taxonomic groups remain scientifically undescribed. However, molecular rapid assessment methods such as DNA barcoding and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) provide the potential to accelerate specimen identification and biodiversity assessment significantly in the deep-sea areas. In this study, we successfully applied both methods to investigate the diversity of meiobenthic copepods in the eastern CCZ, including the first application of MALDI-TOF MS for the identification of these deep-sea organisms. Comparing several different species delimitation tools for both datasets, we found that biodiversity values were very similar, with Pielou’s evenness varying between 0.97 and 0.99 in all datasets. Still, direct comparisons of species clusters revealed differences between all techniques and methods, which are likely caused by the high number of rare species being represented by only one specimen, despite our extensive dataset of more than 2000 specimens. Hence, we regard our study as a first approach toward setting up a reference library for mass spectrometry data of the CCZ in combination with DNA barcodes. We conclude that proteome fingerprinting, as well as the more established DNA barcoding, can be seen as a valuable tool for rapid biodiversity assessments in the future, even when no reference information is available.

The impact of species complexes on tree abundance patterns in Amazonia.

The impact of species complexes on tree abundance patterns in Amazonia.

Integrative taxonomy based on morphometric and molecular data supports recognition of the three cryptic species within the Encyrtus sasakii complex (Hymenoptera, Encyrtidae)

Morphometrics has established itself as one of the most powerful tools for species delimitation, particularly for morphologically-conserved groups of insects. An interesting example is the parasitoid Encyrtus sasakii Ishii (Hymenoptera: Chalcidoidea: Encyrtidae), which was recently subdivided into three cryptic species that are seemingly well-delimited with the available DNA data but nearly indistinguishable morphologically. Here, we performed linear morphometric analyses of the antenna as well as shape analyses of the ovipositor and hypopygium (the last two are key structures associated with host location and selection) to shed further light on the taxonomic status of the E. sasakii complex. Principal component analyses were carried out to visualize the amount and direction of shape variation in the ovipositor and hypopygium. Complementarily, we constructed phylogenetic trees using a Bayesian approach based on two markers (28S and COI). We found statistically-significant differences in the relative size of the funicle and of the two proximal claval antennomeres among the three species. Our analyses also indicated that the outer plates of the ovipositor show remarkable allometric changes and that both the stylus and shield of the ovipositor are relatively well conserved among species. We nonetheless found consistent interspecific differences in the shape of the 2nd outer plate of the ovipositor and hypopygium. Also, both our COI and combined trees recovered three strongly-supported major clades, each corresponding to one of the three cryptic species. We discuss that changes in the shape of the ovipositor may have played an important role in host shift and speciation within the E. sasakii complex. Even though the recent descriptions of both E. eulecaniumiae Wang & Zhang, 2016 and E. rhodococcusiae Wang & Zhang, 2016 appear not to fully satisfy the International Code of Zoological Nomenclature, a simple resolution for the sake of taxonomic stability is proposed herein.

SPEDE-sampler: An R Shiny application to assess how methodological choices and taxon sampling can affect Generalized Mixed Yule Coalescent output and interpretation.

Species delimitation tools are vital to taxonomy and the discovery of new species. These tools can make use of genetic data to estimate species boundaries, where one of the most widely used methods is the Generalized Mixed Yule Coalescent (GMYC) model. Despite its popularity, a number of factors are known to influence the performance and resulting inferences of the GMYC. Moreover, the few studies that have assessed model performance to date have been predominantly based on simulated data sets, where model assumptions are not violated. Here, we present a user‐friendly R Shiny application, ‘SPEDE‐sampler’ (SPEcies DElimitation sampler), that assesses the effect of computational and methodological choices, in combination with sampling effects, on the GMYC model. Output phylogenies are used to test the effect that (1) sample size, (2) BEAST and GMYC parameters (e.g. prior settings, single vs multiple threshold, clock model), and (3) singletons have on GMYC output. Optional predefined grouping information (e.g. morphospecies/ecotypes) can be uploaded in order to compare it with GMYC species and estimate percentage match scores. Additionally, predefined groups that contribute to inflated species richness estimates are identified by SPEDE‐sampler, allowing for the further investigation of potential cryptic species or geographical substructuring in those groups. Merging by the GMYC is also recorded to identify where traditional taxonomy has overestimated species numbers. Four worked examples are provided to illustrate the functionality of the program's workflow, and the variation that can arise when applying the GMYC model to empirical data sets. The R Shiny program is available for download at https://github.com/clarkevansteenderen/spede_sampler_R.

Description of three new species of Geometridae (Lepidoptera) using species delimitation in an integrative taxonomy approach for a cryptic species complex.

The genus Eois Hbner (Geometridae: Larentiinae) comprises 254 valid species, 217 of which were described from the Neotropics and 31 of those having their type locality in Brazil. Since this species rich genus has never been revised, and may potentially include many cryptic undescribed species, Eois embodies a problematic taxonomic scenario. The actual diversity of Eois is greatly underestimated and the Brazilian fauna is poorly known, both because of inadequate sampling and because of the potential existence of cryptic species "hidden" within some nominal taxa. In this study we investigated the diversity within a cryptic species complexes associated to the E. pallidicosta and E. odatis clades. We describe three new species Eois oya Moraes & Montebello sp. nov., Eois ewa Moraes & Stanton sp. nov., and Eois oxum Moraes & Freitas sp. nov., in an integrative taxonomy approach, using morphology, host plant use and species delimitation tools.

How often do they do it? An in-depth analysis of the hybrid zone of two grass snake species (Natrix astreptophoraandNatrix helvetica)

AbstractWe examined the contact zone of two parapatric species of grass snake (Natrix astreptophora and Natrix helvetica) in southern France. To this end, we used comprehensive sampling, analysed mtDNA sequences and microsatellite loci, and built Species Distribution Models for current and past climatic conditions. The contact zone had established by the mid-Holocene during range expansions from glacial refuges in the Iberian Peninsula (N. astreptophora) and southern or western France (N. helvetica). The contact zone represents a narrow bimodal hybrid zone, with steep genetic transition from one taxon to the other and rare hybridization, supporting species status for N. astreptophora and N. helvetica. Our results suggest that the steepness of the clines is a more robust tool for species delimitation than cline width. In addition, we discovered in western France, beyond the hybrid zone, a remote population of N. helvetica with genetic signatures of hybridization with N. astreptophora, most likely the result of human-mediated long-distance dispersal. For N. helvetica, we identified a southern and a northern population cluster, connected by broad-scale gene flow in a unimodal hybrid zone running across France. This pattern either reflects genetic divergence caused by allopatry in two microrefuges and subsequent secondary contact or introgression of foreign alleles into the southern cluster.

One or two species? Floral characteristics and pollination biology aid in Sinningia (Gesneriaceae) species circumscription

Plant species are mostly delimited solely by morphological characteristics, but this method has drawbacks and may result in identification errors. Pollination and ecological speciation studies may serve as additional tools for species delimitation. Sinningia lutea and S. allagophylla are species hitherto considered synonyms, and recent accounts suggest that the taxa present overlapping morphological features. Here we seek to improve the taxonomic circumscription of both taxa through the comparative study of floral traits, pollination and breeding systems. Our results indicate significant differences in morphological characters and confirm that the two species are pollinated by different animals. Therefore, both species are ecologically isolated and can be considered independent species. Our results highlight the potential of supplementing morphological analyses with ecological data to establish correct taxonomic relationships.

Morphological description and DNA barcoding of some Diamesinae (Diptera, Chironomidae) from the Severnaya Zemlya Archipelago and the Vaigach Island (Russian Arctic).

Chironomids of the Diamesinae subfamily from the Russian Arctic were studied using both morphological characters and molecular data. Adult males of Diamesa urvantsevi sp. nov., D. amplexivirilia Hansen, Arctodiamesa appendiculata (Lundström) from Severnaya Zemlya Archipelago and D. arctica (Boheman), Pseudokiefferiella sp. from Vaigach Island were described, redescribed, annotated and figured. A reference 658 bp barcode sequence from a fragment of the mitochondrial gene cytochrome oxidase I (COI) was used as a tool for species delimitation. For D. arctica (Boheman) and Pseudokiefferiella sp. close DNA barcodes from Norway were performed, which allowed to relate these specimens to the described species. Comparisons with corresponding regions of COI between each described species and close related congeneric species produced K2P genetic distances of 0.11-0.16, values well associated with interspecific variation. Phylogenetic relationships for genera Arctodiamesa Makarchenko and Pseudokiefferiella Zavřel were reconstructed for the first time.

BÚSQUEDA DE BIOMARCADORES PARA LA DISCRIMINACIÓN DE LAS VARIEDADES DE KAÑIWA (Chenopodium pallidicaule Aellen) “CHILLIWA” Y “RAMIS” POR MEDIO DE METABOLÓMICA NON-TARGETED

La “kañiwa” (Chenopodium pallidicaule Aellen) es un grano andino muy nutritivo caracterizado por su alto contenido de proteínas y buen perfil de aminoácidos. La diversidad de esta especie viene siendo caracterizada por estudios agro-morfológicos y genómicos, los cuales no son completamente eficientes para discriminar entre las accesiones y variedades. Actualmente se utiliza la metabolómica como una herramienta adicional para la delimitación de especies. En tal sentido, en el presente trabajo se llevó a cabo el análisis metabolómico “non-targeted” de dos variedades de kañiwa (“Ramis” y “Chilliwa”) provenientes del departamento de Puno, Perú. Para el análisis se utilizó la cromatografía líquida de ultra-alta performance acoplada a la espectrometría de masas de alta resolución con analizador Orbitrap (UHPLC-HRMS). Para la identificación de los metabolitos se utilizaron los softwares MSDIAL y MSFINDER. Los perfiles químicos obtenidos fueron resumidos en una sola tabla para su análisis estadístico con el software SIMCA P. El modelo estadístico OPLS-DA permitió la clasificación de ambas variedades de kañiwa y el gráfico S-plot puso en evidencia los principales biomarcadores putativos para la discriminación de ambas variedades. El metabolito delfinidina-O-(6''-O-α-ramnopiranosil-β-glucopiranósido) fue identificado como uno de los principales biomarcadores para la kañiwa “Ramis”.

Museomics reveals extensive cryptic diversity of Australian prionine longhorn beetles with implications for their classification and conservation

AbstractWe combined museum collection genomics and morphology to identify new genera and species, and to otherwise revise the taxonomy of the poorly understood Australian longhorn beetles in the subfamily Prioninae. Moreover, we produced a comprehensive DNA reference library for molecular species identification and a first comprehensive molecular phylogeny for all Australian Prioninae, using mitogenomic data. The Prioninae include some of the world's largest beetles, and their long‐lived larvae are major recyclers of solid dead timber, yet these longhorn beetles are rarely seen in nature due to their concealed habits, short adult lifespans and nocturnal habits. Furthermore, many species are difficult to identify using morphological characters. We applied Whole Genome Shotgun (WGS) sequencing to 213 historical museum specimens, representing all morphological types of Australian Prioninae. Complete mitochondrial genomes were obtained for all genera, and at least one mitochondrial protein‐coding gene (PCG) was acquired for an additional 141 specimens of Australian Prioninae. Using molecular species delimitation tools, we identified 48 new species of Australian Prioninae, nearly doubling the number of currently recognized Australian species of Prioninae. We assign generic status to Catypnes Pascoe, 1864 stat.r. and Hermerius Newman, 1844 stat.r., propose a new synonymy for Cnemoplites Newman, 1842 (= Paroplites Lameere, 1903 syn.n.), describe six new genera, including Paulhutchinsonia Jin, de Keyzer & Ślipiński gen.n., Papunya Jin, de Keyzer & Ślipiński gen.n., Dunmorium Jin, de Keyzer & Ślipiński gen.n., Hagrides Jin, de Keyzer & Ślipiński gen.n., Cryptipus Jin, de Keyzer & Ślipiński gen.n., Geoffmonteithia Jin, de Keyzer & Ślipiński gen.n., and revise the tribal composition of the Australian Macrotomini. Moreover, our phylogeny of Prioninae provides valuable new insights into the evolutionary origins and nearest relatives of several Australian endemic taxa. Our results emphasize the importance of natural history museums as a repository for information about biodiversity, and highlight the opportunities associated with using museum specimens as a source for molecular data in phylogenetic studies.

Concise review of genus Chondracanthus (Rhodophyta: Gigartinales)

Members of the genus Chondracanthus (Rhodophyta, Gigartinales) inhabit the shores of both hemispheres and have been used for decades in some countries as raw material for carrageenan extraction and in some cases destined for human consumption. This has created potential business opportunities, which has drawn the attention of scientists and producers and increased the number of scientific and applied research studies in the last few years. The broad morphological variation of Chondracanthus species makes necessary the use of molecular tools for species delimitation, which has led to the re-grouping and geographic re-distribution of some entities. The present study is a review of the available literature on members of the genus Chondracanthus, with the exception of Chondracanthus teedei, regarding their identity, distribution, ecology, life history, population structure, chemical traits, handling, and culture.

Morphological description and DNA barcoding of Hydrobaenus laticaudus Sæther, 1976 (Diptera: Chironomidae: Orthocladiinae) from Amur River basin (Russian Far East).

Illustrated redescription of adult male and description for the first time of pupa and fourth instar larva, as well as DNA barcoding, of Hydrobaenus laticaudus Sæther in comparison with close related known species of Hydrobaenus Fries, and an updated part of a key to species of Hydrobaenus from the Russian Far East are provided. A reference 658 bp barcode sequence from a fragment of the mitochondrial gene cytochrome oxidase I (COI) was used as a tool for species delimitation. For the estimation of interspecific distances and constructing detailed Bayesian tree within Hydrobaenus, we have obtained DNA barcoding for the two other species, H. distinctus and Hydrobaenus sp.1. The intraspecific sequence divergence of H. laticaudus based on the Kimura-2-parameter (K2P) distance ranged from 0.0-0.031, whereas the interspecific sequence divergence based on the K2P distance ranged from 0.106-0.197% between H. laticaudus and other species of genus Hydrobaenus. The well-supported monophyly as well as results of an ABGD analysis confirms the validity of H. laticaudus.

Molecular species delimitation of reef-building coral genera, Porites and Turbinaria (Anthozoa: Scleractinia), from the intertidal fringing reefs of Gulf of Kutch, India reveals unrecognized diversity

Knowledge of extant biodiversity with accurate species boundaries is one of the crucial prerequisites for guiding conservation priorities in the face of global climate change. DNA taxonomy promises to provide a rapid and reliable tool for species delimitation of morphologically challenging taxa, such as reef-building corals. Despite harbouring diverse coral assemblages, molecular studies of hard corals occurring in the seas around India are lacking. The Gulf of Kutch (GoK), located in the north-western part of India, is one such region. Information on coral species inhabiting this unique ecosystem is limited to morphology, which is often highly variable and phenotypically plastic, obscuring species boundaries. Here, we present the first exploration of molecular divergence of hard corals of the GoK in order to identify evolutionarily distinct lineages. Nuclear ribosomal ITS and mitochondrial COI markers were sequenced for samples of dominant genera, Porites and Turbinaria for phylogenetic comparisons with available data from other ecosystems of the world. Five molecular species delimitation methods were applied to the datasets and their performance was evaluated. Our analyses clearly suggest the occurrence of unique genotypes of both these genera in the GoK, unequivocally discerned by all the five methods as well as microskeletal features. This first integrative assessment provides implications for the phylogeny of these genera and identifies previously unrecognized scleractinian species hidden in the GoK ecoregion.

Mitochondrial divergence suggests unexpected high species diversity in the opsariichthine fishes (Teleostei: Cyprinidae) and the revalidation of Opsariichthys macrolepis.

Opsariichthine (sensu Oceanologi Et Limnologia Sinica, 1982, 13, 293–298) is a cyprinid group consisting of five genera and endemic to East Asia. Previous studies suggested that there may be many possible cryptic species in this group, but this has not been confirmed. In this study, using mitochondrial cyt b sequences on 1,388 samples and 739 haplotypes, we showed very high species diversity within this group. The results showed that phylogenetic relationships of the opsariichthine group were as ([Nipponocypris‐Parazacco‐Candidia] + [Zacco + Opsariichthys]), and there were multiple deep lineages within several species, flagging putative cryptic species. When a 3% genetic distance was used as a threshold for species delimitation, 35 haplogroups were found, nine haplogroups in Candidia‐Parazacco‐Nipponocypris group, six haplogroups in the Zacco group, and 20 haplogroups in the Opsariichthys group. We consider all of them to be putative until determination of distinct species based on the tree topology, geographic distributions, or a combination of both. In addition, two kinds of species delimitation tools, ABGD and PTP, were applied to construct molecular operational taxonomic units (MOTUs). The ABGD method revealed nine MOTUs in Candidia‐Parazacco‐Nipponocypris group, two MOTUs in the Zacco group, and 17 MOTUs in the Opsariichthys group. And the PTP method revealed 10 MOTUs in Candidia‐Parazacco‐Nipponocypris group, 10 MOTUs in the Zacco group, and 29 MOTUs in the Opsariichthys group. Therefore, there should be more species in the opsariichthine group than presently described. Based on the molecular data and morphological characteristics, we proposed Opsariichthys macrolepis as a valid species and described its morphological diagnostic characters.

DNA-based approaches uncover cryptic diversity in the European Lepidocyrtus lanuginosus species group (Collembola: Entomobryidae)

DNA sequence data and phylogenies are useful tools for species delimitation, especially in taxa comprising cryptic species. The Lepidocyrtus lanuginosus species group (Collembola: Entomobryidae) comprises three morphospecies and distinct cryptic species. We applied three DNA-based methods to delimit species boundaries in the L. lanuginosus species group across central and southern Europe. Using cytochrome c oxidase subunit I and II, we identified gaps of genetic distances that indicate species boundaries and found 10 and 9 distinct genetic lineages in L. cyaneus and L. lanuginosus, respectively. The nuclear gene elongation factor 1-α delimited 89% of the lineages but 28S rDNA (D1–2 domain) was too conserved for this purpose. The phylogenetic trees showed that L. cyaneus and L. lanuginosus are polyphyletic, suggesting that body colour is insufficient for delimiting species in the L. lanuginosus species group. Our study challenges the current morphology-based species delimitation in the L. lanuginosus species group and suggests that molecular approaches are needed for fast and accurate determination of Collembola species in both taxonomic and ecological studies. Overall, the results suggest that wide geographic sampling combined with molecular phylogenetic approaches is needed to delimit species and to understand the full range of cryptic diversity in Collembola.

Morphological and molecular characterization of entomopathogenic nematode, Heterorhabditis baujardi (Rhabditida, Heterorhabditidae) from Mizoram, northeastern India.

Isolation of entomopathogenic nematodes from Mizoram, northeastern part of India includes a Heterorhabditis species never been recorded in India. The morphological and multigene (ITS rRNA, 28S rRNA and COI) sequences analysis had revealed that the isolated Heterorhabditis belongs to Heterorhabditis baujardi, originally described from Vietnam. The phylogenetic tree (both MP and ML) revealed that the H. baujardi belongs to H. indica clade, and further, forms a monophyletic clade (99/98% and 94/92% bootstrap support for ITS and 28S respectively) with H. amazonensis, H. floridensis, H. mexicana and H. taysearae forming H. baujardi sub clade. The multigene characterization revealed that both the ITS and 28S rRNA showed similar result in resolving the phylogenetic relationship of the genus Heterorhabditis, with the ITS rRNA being superior based on the strong bootstrap support, whereas, the cytochrome c oxidase I (M1-M6 partition) can be a good supportive tool for species delimitation. This is the first report of H. baujardi from India envisaging its future use as a biological control agent, and further incorporated into the IPM.

Morphological description and DNA barcoding of Chaetocladius (Chaetocladius) elisabethae sp. nov. (Diptera: Chironomidae: Orthocladiinae) from the Moscow Region

Illustrated descriptions of the adult male, pupa and fourth instar larva, as well as DNA barcoding results of Chaetocladius (Chaetocladius) elisabethae sp. nov. in comparison with closely related species of Chaetocladius s. str. from the Moscow Region are provided. A reference 658 bp barcode sequence from a fragment of the mitochondrial gene cytochrome oxidase I (COI) was used as a tool for species delimitation. Comparisons with corresponding regions of COI between C. (s. str.) elisabethae sp. nov. and other species of the subgenus produced K2P genetic distances of 0.11-0.16, values well associated with interspecific variation. The barcodes of the new species were identical to the Chaetocladius sp. 2ES in BOLD systems. Molecular data were also used for the reconstruction of the phylogenetic relationships within the subgenus Chaetocladius s. str.