Species Delimitation Methods Research Articles

The Species Diversity Assessment of Azygia Looss, 1899 (Digenea: Azygiidae) from the Volga, Ob, and Artyomovka Rivers Basins (Russia), with Description of A. sibirica n. sp.

This study is devoted to the investigation of Azygia (Digenea: Azygiidae) species diversity using classical morphological, recent molecular tools (28S rRNA and cox1 mtDNA for genetic-based inference) and robust statistical techniques (Principal component analysis, PCA). The analysis revealed that the genus Azygia included four valid species: A. lucii, A. longa, A. hwangtsiyui, and A. sibirica n. sp. The distribution of the type species A. lucii was confirmed in the largest Russian rivers: the Volga and the Ob. The worms isolated from Perccottus glenii were determined as the Chinese species A. hwangtsiyui, according to the genetic data for the cox1 mtDNA gene, at 1.32–1.56%. The new species, Azygia sibirica n. sp, was described from Esox lucius in the Ob River and differentiated from the type species A. lucii by the smaller ovary, testes and prostatic sac, wider body, very narrow pharyngeal lumen and form of anterior margin of ovary. In addition, multivariate analysis and three methods for species delimitation (ABGD, GMYC, bPTP) showed the subdivision of A. lucii and A. sibirica n. sp. into two separate groups, one from the Volga River and another from the Ob River, respectively. To conclude, A. lucii infects Esox lucius in the western (European part of Russia, the Volga River basin), and northern (Western Siberia, the Ob River basin) parts of Russia; A. sibirica n. sp. has also been found to infect Esox lucius in the Ob River, while A. hwangtsiyui infects Perccottus glenii in the South of the Russian Far East (the Artymovka River basin).

Pseudophilautus pardus, a junior synonym of P. viridis (Amphibia: Anura: Rhacophoridae).

Pseudophilautus pardus, was first described in 2007, based on a single specimen collected in Sri Lanka during the 19th century. Its absence in recent surveys suggested that the species was extinct. The distinctive spotted dorsal coloration, together with other morphological features, was used to distinguish it from congeners. In 2013, we discovered a specimen resembling the holotype of P. pardus in Samanala Strict Nature Reserve. A DNA analysis based on the 16S rRNA mt-DNA locus recovered a low uncorrected pairwise genetic distance of 0.77-0.96% between the recent example of 'P. pardus' and a congener, P. viridis. Molecular species delimitation methods suggest P. pardus and P. virids to be a single operational taxonomic unit. Morphological analyses using the freshly collected specimen, together with numerous photographic records show P. viridis to be a highly polychromatic species within which the coloration observed in P. pardus too occurs, though rarely. We conclude that P. pardus is a junior synonym of P. viridis.

Morphological and molecular characterization of Udotea species (Bryopsidales, Chlorophyta) from northeastern Brazil with a new record of U. dotyi from the western south Atlantic

Udotea is an exclusively marine green macroalgal genus with wide distribution in both tropical and subtropical regions. Due to the difficulty in properly delimiting infrageneric taxa based on morpho-anatomical characteristics by the presence of cryptic species and phenotypic plasticity, the use of techniques of DNA barcode and phylogenetic analysis have been fundamental to assess the Udotea species diversity. Information concerning this genus in Brazil is currently restricted to a few publications in checklists and/or regional floristic surveys along the tropical coast. This study aimed to evaluate the diversity of Udotea in northeastern Brazil through morphological-based taxonomy allied with plastid molecular markers, tufA and rbcL. We generated phylogenies of Udotea sensu lato, and we used different species delimitation methods to assess the diversity among sequences of selected tufA clades within Udotea sensu stricto. None of the eight infrageneric taxa reported for Brazil based only on morphology were confirmed by using molecular data. Udotea dotyi was revealed by tufA and rbcL sequences and represents its first occurrence for the western south Atlantic Ocean. A taxon named here as ‘Udotea cyathiformis’ requires further studies for its definition. This first molecular study on Udotea from the Brazilian coast shows that a broader sampling will be necessary to assess the true diversity of the genus in the western south Atlantic, which may be overestimated due to its high morphological plasticity.

Multi-gene phylogeny reveals a new genus and species of Hapalidiales (Rhodophyta) from Antarctica: Thalassolithon adeliense gen. & sp. nov.

ABSTRACT Non-geniculate coralline algal specimens were collected in 2013 during the XXVIII Italian Expedition to Antarctica in Adélie Cove (Terra Nova Bay; Ross Sea) and deposited in the collections of the Italian National Antarctic Museum (MNAIT, Section of Genoa). Specimens were characterized through a polyphasic approach combining DNA sequence data obtained for four genes (psbA, rbcL, 18S rDNA and cox1) with morpho-anatomical observations. DNA sequences revealed that all specimens belonged to the same species. Phylogenetic reconstructions unambiguously recovered this alga as a member of the order Hapalidiales, but without any close relationship to a genus of this order currently recognized on a molecular phylogenetic basis. Instead, it formed a well-supported lineage with specimens named ‘Hapalidiales sp. ZH-Twist-2019’, collected in New Zealand, for which no formal assignment at genus level has been proposed. Species delimitation methods (ABGD, PTP, GMYC) applied to the psbA dataset indicated that the Adélie Cove coralline alga is a distinct species from all other known hapalidialean species for which such sequences are available. A new genus, Thalassolithon gen. nov., is proposed for T. adeliense sp. nov.

Cryptic, sibling or neither of the two? Integrative species delimitation ofPsylliodesflea beetles with overlapping ranges

AbstractSpecies identification and delimitation are particularly challenging for morphologically similar and geographically overlapping species, such as in the case of Western Palaearctic flea beetle speciesPsylliodes kiesenwetteriKutschera, 1864 andPsylliodes ruffoiLeonardi, 1975. In this study, we implemented an integrative taxonomic approach based on a comprehensive geographic assessment of morphological and genetic variation, including 142 adult specimens from 15 sympatric and allopatric populations. Results of species delimitation methods using the barcode marker COI show that molecular identification and delimitation betweenP. kiesenwetteriandP. ruffoiare straightforward. Single‐locus and multi‐locus phylogenetic analyses indicate these two species have a large genetic divergence and belong to two distinct clades of thePsylliodes gibbosaspecies group. Morphological identification based on qualitative characters shows great differences in identification success depending on the character used. Characters of male and female genitalia perform very well, but can only be assessed on fully sclerified individuals, whereas the colour of antennae was discovered as a new reliable diagnostic character both for teneral and fully sclerified individuals. Morphological identification is particularly sharp when multiple characters are used in combination or when a morphometric approach is performed. In conclusion, despite overall morphological similarity,P. kiesenwetteriandP. ruffoiare not cryptic species neither they are sibling species, as they belong to distinct clades within thegibbosaspecies group, and they can be reliably distinguished by morphological characters. This study substantiates the relevance of a range‐wide assessment of morphological and genetic variation, including individuals from the type locality, and both sympatric and allopatric populations, for taxonomic assessment of morphologically similar species with overlapping ranges.

Molecular delimitation methods validate morphologically similar species of red snappers (Perciformes: Lutjanidae).

In this study, we tested the taxonomic validation of red snappers species (Southern red snapper Lutjanus purpureus; Silk snapper L. vivanus; Blackfin snapper L. buccanella; and Pacific red snapper L. peru) based on comparative analysis, using four methods for species delimitation. These methods were based on either genetic similarity or phylogenetic trees inferred from two mitochondrial (Cytochrome b and D-loop) and two nuclear (Myostatin and S7 introns) markers. On one hand, the genetic results corroborated the presence of four red snapper species, confirming their taxonomic validation despite their remarkable morphological similarity. On the other hand, few incongruencies in the species delimitation methods were observed according to the phylogenetic reconstruction method (maximum likelihood or Bayesian inference) when using. Based on the phylogenetic results, L. buccanella should represent a more ancient lineage in relation to the clade that encompasses L. purpureus, L. peru and L. vivanus. The single-locus phylogenetic analysis based on Cytb recovered each the red snapper species as a well-supported clade. Overall, this study provided a DNA-based validation of the traditional morphological taxonomy of red snappers.

We don’t know the half of it: morphological and molecular evidence reveal dramatic underestimation of diversity in a key pollinator group (Nemestrinidae)

Nemestrinidae (tangle-veined flies) are important pollinators of numerous southern African plant species. Despite their known ecological importance, the family has received little taxonomic attention in recent years and the systematics of the group is poorly understood. In this study we aimed to assess the phylogenetic relationships and species diversity among three southern African nemestrinid genera from the Nemestrininae subfamily: Prosoeca, Moegistorhynchus and Stenobasipteron, with a specific focus on the largest among these, Prosoeca. We reconstructed a molecular phylogeny using both mitochondrial and nuclear (COI, 16S rRNA, 28S rRNA and CAD) DNA sequence data. Both morphology and molecular species delimitation methods (Automatic Barcode Gap Discovery and the Bayesian Poisson Tree Process) were used to estimate species diversity. The topology from the combined analysis places a monophyletic Moegistorhynchus as the sister group to a paraphyletic Prosoeca with Stenobasipteron nested inside Prosoeca. In all three genera, almost half of the putative species sampled did not match the concept of described species based on morphology. Analysis of phylogenetic diversity showed that undescribed putative species make a substantial contribution to the overall phylogenetic diversity among the sampled species. Comparisons among biogeographic regions suggested that diversity is concentrated in multiple biodiversity hotspots and biomes, particularly in Fynbos and Grassland biomes. The numerous undescribed species and paraphyly of Prosoeca both emphasise the need for increased taxonomic attention for this ecologically important group of flies in particular, and for southern African insect taxa in general.

Integrative taxonomy of the stalk‐eyed bug genus Chauliops (Heteroptera: Malcidae: Chauliopinae) reveals orogeny‐driven speciation

AbstractEconomically significant bean pests of the genus Chauliops are species rich in the areas surrounding the Qinghai–Tibet Plateau and provide an excellent system for speciation studies. Here, an integrative taxonomic approach, employing morphological analyses, population genetic methods, and multiple molecular species delimitation methods, was used to clarify the taxonomy of Chauliops in East and Southeast Asia. Four new species (Chauliops parahorizontalis Li & Bu, sp. nov., Chauliops albida Li & Bu, sp. nov., Chauliops bicoloripes Li & Bu, sp. nov., and Chauliops paraconica Li & Bu, sp. nov.) were described, which increases the number of Chauliops species in this area from six to 10; a key for Chauliops species is also provided. Phylogenetic analysis and divergence time estimation revealed that Chauliops was divided into four clades: Clade A (Chauliops bisontula + [Chauliops horizontalis + C. parahorizontalis sp. nov.]), Clade B (C. albida sp. nov. and C. bicoloripes sp. nov.), Clade C (Chauliops quaternaria and Chauliops zhengi), and Clade D (Chauliops fallax + [Chauliops conica + C. paraconica sp. nov.]). Two species diversification events of Chauliops estimated to have occurred 7–1 million years ago (Ma) and 25–13 Ma were detected. These speciation events were consistent with the two historical uplift events of the Qinghai–Tibet Plateau, suggesting that orogeny might have provided opportunities for the diversification of Chauliops species on the southeastern margin of the Qinghai–Tibet Plateau. Our findings show that population genetic analyses can be used to delimit related species and that orogeny is a key driver of species diversification on the southeastern margin of the Qinghai–Tibet Plateau.

Environmentally driven phenotypic convergence and niche conservatism accompany speciation in hoary bats

Species that are geographically widespread may exist across environmentally heterogeneous landscapes that could influence patterns of occupation and phylogeographic structure. Previous studies have suggested that geographic range size should be positively correlated with niche breadth, allowing widespread species to sustain viable populations over diverse environmental gradients. We examined the congruence of phenotypic and phylogenetic divergence with the environmental factors that help maintain species level diversity in the geographically widespread hoary bats (Lasiurus cinereus sensu lato) across their distribution. Genetic sequences were analyzed using multiple phylogenetic and species delimitation methods, and phenotypic data were analyzed using supervised and unsupervised machine learning approaches. Spatial data from environmental, geographic, and topographic features were analyzed in a multiple regression analysis to determine their relative effect on phenotypic diversity. Ecological niches of each hoary bat species were examined in environmental space to quantify niche overlap, equivalency, and the magnitude of niche differentiation. Phylogenetic and species delimitation analyses support existence of three geographically structured species of hoary bat, each of which is phenotypically distinct. However, the Hawaiian hoary bat is morphologically more similar to the South American species than to the North American species despite a closer phylogenetic relationship to the latter. Multiple regression and niche analyses revealed higher environmental similarities between the South American and Hawaiian species. Hoary bats thus exhibit a pattern of phenotypic variation that disagrees with well-supported genetic divergences, instead indicating phenotypic convergence driven by similar environmental features and relatively conserved niches occupied in tropical latitudes.

Consolidation of Chloridium: new classification into eight sections with 37 species and reinstatement of the genera Gongromeriza and Psilobotrys.

Chloridium is a little-studied group of soil- and wood-inhabiting dematiaceous hyphomycetes that share a rare mode of phialidic conidiogenesis on multiple loci. The genus has historically been divided into three morphological sections, i.e. Chloridium, Gongromeriza, and Psilobotrys. Sexual morphs have been placed in the widely perceived genus Chaetosphaeria, but unlike their asexual counterparts, they show little or no morphological variation. Recent molecular studies have expanded the generic concept to include species defined by a new set of morphological characters, such as the collar-like hyphae, setae, discrete phialides, and penicillately branched conidiophores. The study is based on the consilience of molecular species delimitation methods, phylogenetic analyses, ancestral state reconstruction, morphological hypotheses, and global biogeographic analyses. The multilocus phylogeny demonstrated that the classic concept of Chloridium is polyphyletic, and the original sections are not congeneric. Therefore, we abolish the existing classification and propose to restore the generic status of Gongromeriza and Psilobotrys. We present a new generic concept and define Chloridium as a monophyletic, polythetic genus comprising 37 species distributed in eight sections. In addition, of the taxa earlier referred to Gongromeriza, two have been redisposed to the new genus Gongromerizella. Analysis of published metabarcoding data showed that Chloridium is a common soil fungus representing a significant (0.3 %) proportion of sequence reads in environmental samples deposited in the GlobalFungi database. The analysis also showed that they are typically associated with forest habitats, and their distribution is strongly influenced by climate, which is confirmed by our data on their ability to grow at different temperatures. We demonstrated that Chloridium forms species-specific ranges of distribution, which is rarely documented for microscopic soil fungi. Our study shows the feasibility of using the GlobalFungi database to study the biogeography and ecology of fungi. Taxonomic novelties: New genus: Gongromerizella Réblová; New sections: Chloridium section Cryptogonytrichum Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium section Gonytrichopsis Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium section Metachloridium Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium section Volubilia Réblová, Hern.-Restr., M. Kolařík & F. Sklenar; New species: Chloridium bellum Réblová & Hern.-Restr., Chloridium biforme Réblová & Hern.-Restr., Chloridium detriticola Réblová & Hern.-Restr., Chloridium gamsii Réblová & Hern.-Restr., Chloridium guttiferum Réblová & Hern.-Restr., Chloridium moratum Réblová & Hern.-Restr., Chloridium peruense Réblová & Hern.-Restr., Chloridium novae-zelandiae Réblová & Hern.-Restr., Chloridium elongatum Réblová & Hern.-Restr., Chloridium volubile Réblová & Hern.-Restr.; New varieties: Chloridium bellum var. luteum Réblová & Hern.-Restr., Chloridium detriticola var. effusum Réblová & Hern.-Restr., Chloridium chloridioides var. convolutum Réblová & Hern.-Restr.; New combinations: Chloridium section Gonytrichum (Nees & T. Nees) Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium section Mesobotrys (Sacc.) Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium section Pseudophialocephala (M.S. Calabon et al.) Réblová, Hern.-Restr., M. Kolařík & F. Sklenar, Chloridium simile (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium chloridioides (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium subglobosum (W. Gams & Hol.-Jech.) Réblová & Hern.-Restr., Chloridium fuscum (Corda) Réblová & Hern.-Restr., Chloridium ypsilosporum (Hol.-Jech.) Réblová & Hern.-Restr., Chloridium costaricense (G. Weber et al.) Réblová & Hern.-Restr., Chloridium cuneatum (N.G. Liu et al.) Réblová & Hern.-Restr., Fusichloridium cylindrosporum (W. Gams & Hol.-Jech.) Réblová, Gongromeriza myriocarpa (Fr.) Réblová, Gongromeriza pygmaea (P. Karst.) Réblová, Gongromerizella lignicola (F. Mangenot) Réblová, Gongromerizella pachytrachela (W. Gams & Hol.-Jech) Réblová, Gongromerizella pini (Crous & Akulov) Réblová; New name: Chloridium pellucidum Réblová & Hern.-Restr.; Epitypifications (basionyms): Chaetopsis fusca Corda, Gonytrichum caesium var. subglobosum W. Gams & Hol.-Jech.; Lectotypification (basionym): Gonytrichum caesium Nees & T. Nees. Citation: Réblová M, Hernández-Restrepo M, Sklenář F, Nekvindová J, Réblová K, Kolařík M (2022). Consolidation of Chloridium: new classification into eight sections with 37 species and reinstatement of the genera Gongromeriza and Psilobotrys. Studies in Mycology 103: 87-212. doi: 10.3114/sim.2022.103.04.

DNA barcoding of Scomberomorus (Scombridae, Actinopterygii) reveals cryptic diversity and misidentifications.

The genus Scomberomorus is economically important; however, the taxonomic status and phylogenetic relationships in this genus are not clearly resolved, making it difficult to effectively protect and exploit fish resources. To clarify the taxonomic status of Scomberomorus species, mitochondrial cytochrome c oxidase I (COI) gene sequences of 150 samples were analyzed. The average genetic distance among 14 species was approximately 11 times greater than the distances within species, in accordance with the '10× rule' of species identification. Five of the 14 species did not form monophyletic clades based on a Bayesian inference gene tree. The application of four DNA-based species delimitation methods (automatic barcode gap discovery, barcode index numbers, Poisson tree process, and the K/θ method) yielded several key results. (1) Cryptic species were detected within Scomberomoruscommerson. (2) A Scomberomorusqueenslandicus sample from Australia was misidentified as S.commerson in the Barcode of Life Data System (BOLD). (3) Specimens originally identified as Scomberomorusguttatus was differentiated into four OTUs or species, two in the Yellow, South China, and Java seas, and two in geographically distant areas, one each in the Arabian Sea and the Bay of Bengal. (4) Six specimens from South Africa originally identified as S.plurilineatus most likely do not belong to the species. (5) Specimens identified as S.maculatus and S.regalis were conspecific; however, introgression cannot be ruled out. Our findings revealed cryptic diversity and difficulties in morphological identification of species in the genus Scomberomorus. This study provides scientifically based support for the conservation of germplasm resources of the genus Scomberomorus.

Diversity of interstitial nemerteans of the genus Ototyphlonemertes (Nemertea: Monostilifera: Ototyphlonemertidae) in the South China Sea, with a comment on the distribution pattern of the genus

The genus Ototyphlonemertes Diesing, 1863, consisting of 33 named species and numerous unnamed morphospecies/molecular entities, is a unique group of nemerteans that possess cerebral statocysts and specifically live in coarse-grained sands. Only eight named species of this genus have yet been recorded from the Indo-Polynesian biogeographic province, which harbors the highest marine biodiversity in the world. In recent years, Ototyphlonemertes were collected from eight sites along the South China Sea coasts. Nine species/entities were revealed by four phylogenetic markers (COI, 16S, 18S, 28S) analyzed by three species delimitation methods: Automatic Barcode Gap Discovery (ABGD), Poisson Tree Process (PTP), and Generalized Mixed Yule Coalescent model (GMYC). Six entities are described as new species based on integration of morphological and molecular species delimitations: Ototyphlonemertes conicobasis sp. nov., Ototyphlonemertes coralli sp. nov., Ototyphlonemertes similis sp. nov., Ototyphlonemertes sinica sp. nov., Ototyphlonemertes subrubra sp. nov., and Ototyphlonemertes yingge sp. nov. No morphological differences were detected between two entities and Ototyphlonemertes chernyshevi Kajihara et al., 2018, despite large genetic differences, so are treated as candidate species. Ototyphlonemertes ani Chernyshev, 2007 is first recorded in China. Based mostly on results of phylogenetic analyses, two previously established subgenera are re-defined, and a new subgenus, Procso subgen. nov., is established. Through reviewing the existing studies, we recognize 101 species/entities of Ototyphlonemertes, which are distributed in 18 marine biogeographic provinces. Most (88.1%) of them are endemic to a single biogeographic province, and evolutionary lineages endemic to a geographic area are not uncommon. Maximum diversity has been recorded in the Indo-Polynesian Province (22 species), though sampling to date has covered only a small part of the biogeographic province.

Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Turcogammarus aralensis (Uljanin, 1875), a relict pontogammarid amphipod crustacean from the Aralo-Caspian paleobasin: redescription, phylogenetic position and biogeography

The Ponto-Caspian region harbours the world's second most diverse lacustrine radiation of amphipod crustaceans. The taxonomic position of some of these species is unclear, due to incorrectly defined species limits, poor descriptions, and in some cases, poorly understood characters, such as armament of dorsal integuments. A pertinent example is Turcogammarus aralensis (Uljanin, 1875), which inhabits the Aral Sea and adjacent water bodies like the brackish lake Shalkar (Charkhal), lower reaches of the Syr Darya and Volga, and extends ca. 2500 km westwards in the Black Sea basin. This species has never been properly described, which has led to frequent taxonomic reshuffling. Herein, we provide a full re-description, ascertain its phylogenetic position using the cytochrome c oxidase subunit I (COI) marker, and review its geographical distribution. Our molecular analyses reveal T. aralensis as a sister species to the widespread Obesogammarus crassus (Sars, 1894), and its species status was further confirmed by three molecular species delimitation methods. Turcogammarus aralensis and O. crassus share many similarities such as the setation of the antennae and pereopods 3-4, and poorly setose posterior margin of pereopod 7 basis. Further study is needed to ascertain the validity of the genus Turcogammarus.

Morphological Description and Phylogenetic Analyses of a New Species of Callistoctopus (Cephalopoda, Octopodidae) from China

A new octopus species, Callistoctopus tenuipes sp. nov., was formally described from the southeastern coastal waters of China using morphological description and molecular analysis methods. C. tenuipes sp. nov. is a small- to moderate-sized octopus, which is characterized by very narrow and long arms. Although it was previously misidentified as the juvenile of Octopus minor (Sasaki, 1920), it can be recognised by spots, gill lamellae count, funnel organ shape, enlarged suckers, and ligula shape. C. tenuipes sp. nov. differs from the small-sized octopus Callistoctopus xiaohongxu, mainly in the gill lamellae count, funnel organ shape, and arm-length index. In the molecular analysis, sequences obtained from the cytochrome c-oxidase subunit I (COI) gene of eight specimens were 590 bp in length. The pairwise Kimura 2-parameter (K2P) genetic distances between Octopodidae species ranged from 8.58 to 23.79% based on the COI gene. The phylogenetic analyses suggested that C. tenuipes sp. nov. belonged to the Callistoctopus clade and may have a close affinity with C. xiaohongxu and O. minor. Moreover, three species delimitation methods all strongly supported C. tenuipes as a separate species.

Comparing the Efficiency of Single-Locus Species Delimitation Methods within Trochoidea (Gastropoda: Vetigastropoda)

In the context of diminishing global biodiversity, the validity and practicality of species delimitation methods for the identification of many neglected and undescribed biodiverse species have been paid increasing attention. DNA sequence-based species delimitation methods are mainly classified into two categories, namely, distance-based and tree-based methods, and have been widely adopted in many studies. In the present study, we performed three distance-based (ad hoc threshold, ABGD, and ASAP) and four tree-based (sGMYC, mGMYC, PTP, and mPTP) analyses based on Trochoidea COI data and analyzed the discordance between them. Moreover, we also observed the performance of these methods at different taxonomic ranks (the genus, subfamily, and family ranks). The results suggested that the distance-based approach is generally superior to the tree-based approach, with the ASAP method being the most efficient. In terms of phylogenetic methods, the single threshold version performed better than the multiple threshold version of GMYC, and PTP showed higher efficiency than mPTP in delimiting species. Additionally, GMYC was found to be significantly influenced by taxonomic rank, showing poorer efficiency in datasets at the genus level than at higher levels. Finally, our results highlighted that cryptic diversity within Trochoidea (Mollusca: Vetigastropoda) might be underestimated, which provides quantitative evidence for excavating the cryptic lineages of these species.

Morphology, molecular phylogeny, and species delimitation within microalgal genera Eubrownia, Spongiococcum, and Chlorococcum (Chlorophyceae, Chlorophyta)

• Our knowledge of their taxonomic and phylogenetic relationships is still deficient. • Some genus and species from Moewusinia clade differ specific feature combinations. • The ITS2 of Eubrownia and Chlorococcum genera is unique within the chlorophytes. Despite that the diversity of chlorophycean microalgae has been studied for a long time, our knowledge of their taxonomic and phylogenetic relationships is still deficient. This study represents one of the first efforts to examine the congruence of the morphological identification of microalgal species from genera Eubrownia, Spongiococcum , and Chlorococcum with DNA-based species delimitation methods. We found unique combinations of morphological, molecular, and ecological features, distinguishing some members of the Moewusinia clade at the genus and species levels. Most genetic groups recovered by GMYC, ABGD, and PTP analyses of 18S rRNA and ITS2 were consistent with their morphological identification. The ITS2 region of genera Eubrownia and Chlorococcum is among the longest within the chlorophytes theretofore studied and has a unique secondary structure with branched helix III and uncommonly long helix IV. Moreover, the molecular signatures were found in the basal stem region of helix I, distinguishing two genera. The CBC analysis is able to effectively discriminate between E. isobilateralis, E. aggregata , and all Chlorococcum species, except for C. infusionum and C. echinozygotum . Thus, we confirmed tentative hypotheses of species, obtained by DNA-based delimitation methods, with morphological data, molecular signatures, ITS2 secondary structure, and CBC criterion.

Phylogenetic Relationships in Earthworm Megascolex Species (Oligochaeta: Megascolecidae) with Addition of Two New Species

Megascolex (Oligochaeta: Megascolecidae) are endemic species to India and Sri Lanka, however, to date their molecular taxonomy and phylogenetic relationships have not been reported. We applied the first integrative approach using morpho-anatomical features and a COI dataset to unveil species delimitation (SD), molecular taxonomy, and phylogenetic relationships in Megascolex species. Our morpho-anatomical results revealed nine Megascolex species, namely, M. auriculata, M. cochinensis cochinensis, M. filiciseta, M. ratus, M. travancorensis travancorensis, M. triangularis, M. konkanensis konkanensis, M. polytheca polytheca, and M. polytheca zonatus. We also reported the occurrence of two new species, namely, M. papparensis sp. nov, and M. vazhichlensis sp. nov. Such findings were also supported by the analysed COI dataset, in which these new species appeared distinct on the phylogenetic trees with strong support. The studied Megascolex species appeared paraphyletic and formed three subclades on Bayesian inference (BI) and Maximum Likelihood (ML) phylogenetic trees. The first clade consisted of six species: M. cochinensis cochinensis, M. polytheca polytheca, M. polytheca zonatus, M. konkanensis konkanensis, M. filiciseta, and M. auriculata with strong posterior probability support. The second clade consisted of M. travancorensis travancorensis, M. papparensis sp. nov, and M. vazhichlensis sp. nov with strong support. The third clade consisted of M. ratus and M. triangularis with good support. In addition, the validation of species was confirmed by SD methods, in which the congruence among OTUs was observed with the clear barcode gap of 12–14% suggested by ABGD analysis. However, the species M. ratus and M. travancorensis travancorensis show deep intraspecific divergence and, therefore, require more sampling data. Such findings are essential to study the phylogenetics and evolution of the genus and, nonetheless, demand larger COI datasets to make concrete conclusions.

Diversity of Siphonaria Sowerby I, 1823 (Gastropoda, Siphonariidae) in the Seychelles Bank and beyond

AbstractMolecular phylogenetic studies have shown that the characters of the reduced shell of the false limpets of the genus Siphonaria Sowerby I, 1823 are highly variable and often insufficient for species delimitation. The taxonomy and distribution of Siphonaria in the Indian Ocean are poorly known. We sampled Siphonaria in the Seychelles Bank to check the occurrence of recorded species using DNA sequences and to study the paths through which Siphonaria species have colonised the Seychelles Bank by reconstructing their phylogenetic relationships. Analyses of a dataset comprising 16 S rRNA gene sequences of 33 specimens from the Seychelles Bank and 300 additional Siphonaria sequences from other regions from GenBank with various methods for species delimitation resulted in 19–102 primary species hypotheses. Assemble Species by Automatic Partitioning provided a conservative estimate of the species number (42) in which several indisputable species were lumped. The results of Automatic Barcode Gap Discovery depended strongly on the assumed prior maximum intraspecific divergence, whereas the tree‐based methods Generalised Mixed Yule Coalescent and Poisson Tree Processes resulted in high overestimates. The specimens from the Seychelles Bank represent three clades, belonging to the Siphonaria ‘atra’ group, the Siphonaria ‘normalis’ group and a possibly undescribed species recorded previously only from Hainan. At least two of the three species recorded from the Seychelles Bank came from the east, i.e., from the Coral Triangle in the Indo‐Australian Archipelago, the region with the highest marine biodiversity worldwide. A major transport mechanism across the Indian Ocean was probably the South Equatorial Current.

Cryptic diversity impacts model selection and macroevolutionary inferences in diversification analyses.

Species persist in landscapes through ecological dynamics but proliferate at wider spatial scales through evolutionary mechanisms. Disentangling the contribution of each dynamic is challenging, but the increasing use of dated molecular phylogenies opened new perspectives. First, the increasing use of DNA sequences in biodiversity inventory shed light on a substantial amount of cryptic diversity in species-rich ecosystems. Second, explicit diversification models accounting for various eco-evolutionary models are now available. Integrating both advances, we explored diversification trajectories among 10 lineages of freshwater fishes in Sundaland, for which time-calibrated and taxonomically rich phylogenies are available. By fitting diversification models to dated phylogenies and incorporating DNA-based species delimitation methods, the impact of cryptic diversity on diversification model selection and related inferences is explored. Eight clades display constant speciation rate model as the most likely if cryptic diversity is accounted, but nine display a signature of diversification slowdowns when cryptic diversity is ignored. Cryptic diversification occurs during the last 5 Myr for most groups, and palaeoecological models received little support. Most cryptic lineages display restricted range distribution, supporting geographical isolation across homogeneous landscapes as the main driver of diversification. These patterns question the persistence of cryptic diversity and its role during species proliferation.