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Abstract Background Schmidtea mediterranea (Platyhelminthes, Tricladida, Continenticola) is found in scattered localities on a few islands and in coastal areas of the western Mediterranean. Although S. mediterranea is the object of many regeneration studies, little is known about its evolutionary history. Its present distribution has been proposed to stem from the fragmentation and migration of the Corsica-Sardinia microplate during the formation of the western Mediterranean basin, which implies an ancient origin for the species. To test this hypothesis, we obtained a large number of samples from across its distribution area. Using known and new molecular markers and, for the first time in planarians, a molecular clock, we analysed the genetic variability and demographic parameters within the species and between its sexual and asexual populations to estimate when they diverged. Results A total of 2 kb from three markers (COI, CYB and a nuclear intron N13) was amplified from ~200 specimens. Molecular data clustered the studied populations into three groups that correspond to the west, central and southeastern geographical locations of the current distribution of S. mediterranea. Mitochondrial genes show low haplotype and nucleotide diversity within populations but demonstrate higher values when all individuals are considered. The nuclear marker shows higher values of genetic diversity than the mitochondrial genes at the population level, but asexual populations present lower variability than the sexual ones. Neutrality tests are significant for some populations. Phylogenetic and dating analyses show the three groups to be monophyletic, with the west group being the basal group. The time when the diversification of the species occurred is between ~20 and ~4 mya, although the asexual nature of the western populations could have affected the dating analyses. Conclusions S. mediterranea is an old species that is sparsely distributed in a harsh habitat, which is probably the consequence of the migration of the Corsica-Sardinia block. This species probably adapted to temperate climates in the middle of a changing Mediterranean climate that eventually became dry and hot. These data also suggest that in the mainland localities of Europe and Africa, sexual individuals of S. mediterranea are being replaced by asexual individuals that are either conspecific or are from other species that are better adapted to the Mediterranean climate.

Background: Intimate partner violence (IPV) against women is a global health issue and a breach of human rights. However, the literature lacks understanding of how socioeconomic and geographic disparities influence women's attitudes toward IPV in Guyana over time. This study aimed to assess trends in women's attitudes about IPV in Guyana., Methods: Data from three nationally representative surveys from 2009, 2014 to 2019 were analysed. The prevalence of women's attitudes about IPV was assessed, specifically in response to going out without telling their partners, neglecting their children, arguing with their partner, refusing sex with their partner, or burning food prepared for family meals. A series of stratified subgroup analyses were also completed. We assessed trends in IPV using the slope index of inequality (SII) and the concentration index of inequality (CIX). We used multilevel mixed-effects logistic regression to assess factors associated with women's attitudes justifying IPV., Findings: The prevalence of women's attitudes justifying IPV for any of the five reasons declined from 16.4% (95% CI: 15.1-17.8) in 2009 to 10.8% (95% CI: 9.7-12.0) in 2019. Marked geographic and socioeconomic inequalities were observed among subgroups. The SII for any of the five reasons decreased from -20.02 to -14.28, while the CIX remained constant over time. Key factors associated with women's attitudes about IPV were area of residence, sex of the household head, marital status, respondent's level of education, wealth index quintile, and the frequency of reading newspapers/magazines., Interpretation: From 2009 to 2019, Guyana was able to reduce women's attitudes justifying IPV against women by 34.1% and shortened subgroup inequalities. However, the prevalence remained high in 2019, with persisted inequalities among subgroups. Effective strategies, including the use of media to raise awareness, promotion of community-based approaches, and educational campaigns focusing on geographic and socioeconomic disparities, are essential for continuing to reduce the prevalence of IPV and associated inequalities., Funding: The study was funded in part by the National Institutes of Health, Fogarty International Center grant number D43TW012189., Competing Interests: We declare no competing interests., (© 2024 The Author(s).)

The Catalan Initiative for the Earth BioGenome Project (CBP) is an EBP-affiliated project network aimed at sequencing the genome of the >40 000 eukaryotic species estimated to live in the Catalan-speaking territories (Catalan Linguistic Area, CLA). These territories represent a biodiversity hotspot. While covering less than 1% of Europe, they are home to about one fourth of all known European eukaryotic species. These include a high proportion of endemisms, many of which are threatened. This trend is likely to get worse as the effects of global change are expected to be particularly severe across the Mediterranean Basin, particularly in freshwater ecosystems and mountain areas. Following the EBP model, the CBP is a networked organization that has been able to engage many scientific and non-scientific partners. In the pilot phase, the genomes of 52 species are being sequenced. As a case study in biodiversity conservation, we highlight the genome of the Balearic shearwater Puffinus mauretanicus , sequenced under the CBP umbrella., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Introduction: The main objective of the study will be to evaluate the effects of two widely used standardized mindfulness-based programs [Mindfulness-Based Stress Reduction (MBSR) and Compassion Cultivation Training (CCT)], on epigenetic, neurobiological, psychological, and physiological variables., Methods: The programs will be offered in an intensive retreat format in a general population sample of healthy volunteer adults. During a 7-day retreat, participants will receive MBSR and CCT in a crossover design where participants complete both programs in random order. After finishing their first 3-day training with one of the two programs, participants will be assigned to the second 3-day training with the second program. The effects of the MBSR and CCT programs, and their combination, will be measured by epigenetic changes (i.e., DNA methylation biomarkers), neurobiological and psychophysiological measures (i.e., EEG resting state, EKG, respiration patterns, and diurnal cortisol slopes), self-report questionnaires belonging to different psychological domains (i.e., mindfulness, compassion, well-being, distress, and general functioning), and stress tasks (i.e., an Arithmetic Stress Test and the retrieval of negative autobiographical memories). These measures will be collected from both groups on the mornings of day 1 (pre-program), day 4 (after finishing the first program and before beginning the second program), and day 7 (post-second program). We will conduct a 3-month and a 12-month follow-up using only the set of self-report measures., Discussion: This study aims to shed light on the neurobiological and psychological mechanisms linked to meditation and compassion in the general population. The protocol was registered at clinicaltrials.gov (Identifier: NCT05516355; August 23, 2022)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Diez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The hammerhead worms constitute a land planarian subfamily (Platyhelminthes: Tricladida: Bipaliinae) that is popular among natural historians as well as citizen scientists due to their characteristic semi-lunar-shaped head, the striking colours of many species, and the worldwide presence of introduced species, which has raised much concern about their putative impact on the receiving ecosystems. Despite such current awareness, the majority of Bipaliinae species was described before the second half of the 20th century. Over the past few decades, description of new species has been rather scarce. In the present paper, we describe twelve new species and two new genera from Madagascar, Borneo, and Japan, mainly using specimens that form part of the collections of Naturalis Biodiversity Center, Leiden. Species identification has been based on morphology, inner anatomy, and, when available, molecular information. Our molecular phylogenetic tree suggests that either Malagasy or Bornean representatives form the sister-group to the rest of Bipaliinae. The phylogeny suggests also that the Bipaliinae comprises several new and, as yet, undescribed genera. Although the geographical origin of the subfamily is uncertain and may involve either Madagascar or the Bornean region, a molecular time-calibration of the phylogenetic tree indicated that the origin of the Bipaliinae may date back to about 253 Mya, placing its origin near the Permian-Triassic transition and, thus, on Pangea.

Several studies have focused on the phylogenetic relationships within the Geoplaninae land planarians (Tricladida). In those studies, ancient phylogenetic relationships remained obscure. In this work, the phylogeny of Geoplaninae is assessed through three different datasets, namely morphological, molecular, and both datasets combined, i.e, a total evidence approach (TE). The data matrix consisted of six DNA regions, including a newly developed marker (DOM5), and a morphological matrix with 37 characters. The study produced the best-resolved hypothesis so far for the phylogeny of Geoplaninae, although ancient clades still remain elusive. The effect of the morphological data on the TE tree topology and clade support is seemingly negligible. The phylogenetic tree also suggests that most of the diagnostic morphological characters of the genera are homoplastic, while unambiguous unique synapomorphies can characterize some supra-generic informal groupings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Mediterranean is one of the most biodiverse areas of the Paleartic region. Here, basing on large data sets of single copy orthologs obtained from transcriptomic data, we investigated the evolutionary history of the genus Dugesia in the Western Mediterranean area. The results corroborated that the complex paleogeological history of the region was an important driver of diversification for the genus, speciating as microplates and islands were forming. These processes led to the differentiation of three main biogeographic clades: Iberia-Apennines-Alps, Corsica-Sardinia, and Iberia-Africa. The internal relationships of these major clades were analysed with several representative samples per species. The use of large data sets regarding the number of loci and samples, as well as state-of-the-art phylogenomic inference methods allowed us to answer different unresolved questions about the evolution of particular groups, such as the diversification path of D. subtentaculata in the Iberian Peninsula and its colonization of Africa. Additionally, our results support the differentiation of D. benazzii in two lineages which could represent two species. Finally, we analysed here for the first time a comprehensive number of samples from several asexual Iberian populations whose assignment at the species level has been an enigma through the years. The phylogenies obtained with different inference methods showed a branching topology of asexual individuals at the base of sexual clades. We hypothesize that this unexpected topology is related to long-term asexuality. This work represents the first phylotranscriptomic analysis of Tricladida, laying the first stone of the genomic era in phylogenetic studies on this taxonomic group., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transcriptomic data (obtained from RNA sequencing) has become a very powerful source of information to reconstruct the evolutionary relationships among organisms. Although phylogenetic inference using transcriptomes retains the same core steps as when working with few molecular markers (viz., nucleic acid extraction and sequencing, sequence treatment, and tree inference), all of them show significant differences. First, the needed quantity and quality of the extracted RNA has to be very high. Although this may not represent a challenge when working with certain organisms, it may well be a headache with others, especially for those with small body sizes. Second, the tremendous increase in the quantity of sequences obtained requires a high computational power for both treating the sequences and inferring the subsequent phylogenies. This means that transcriptomic data can no longer be analyzed using personal computers nor local programs with a graphical interface. This, in turn, implies the requirement of an increased set of bioinformatic skills from the researchers. Finally, the genomic peculiarities of each group of organisms, such as the level of heterozygosity or the percentage of base composition, also need to be considered when inferring phylogenies using transcriptomic data., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Switching antipsychotic medication must be done carefully to ensure patient safety and a successful response. Here, we present two major psychotic decompensations that occurred following a switch to aripiprazole in two patients with schizophrenia. Mr. X was treated with paliperidone and experienced residual anxiety. Thus, a switch to aripiprazole was planned with risperidone and a gradual decrease in paliperidone. Initially, an increase in aripiprazole resulted in remission of his residual symptoms. However, two weeks later, he presented an anxiety relapse with persecutory ideas which required hospitalization. Mr. Y, who was treated for many years with risperidone, presented with a treatment resistant psychotic episode. A switch to aripiprazole enhanced his clinical condition. Despite the initial improvement, soon after discharge from the hospital, the patient presented psychotic symptoms requiring home intervention. Ultimately, the patient in the midst of a delusional recrudescence, had killed himself when the health care team arrived. A strong dopamine antagonist may lead to the development of dopaminergic upregulation. The addition of a partial agonist to these hypersensitive neurotransmitter pathways could explain these episodes. We agree with previous reports and recommend careful management when switching from strong dopamine antagonists to aripiprazole., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The sexual strain of the planarian Schmidtea mediterranea, indigenous to Tunisia and several Mediterranean islands, is a hermaphrodite 1,2 . Here we isolate individual chromosomes and use sequencing, Hi-C 3,4 and linkage mapping to assemble a chromosome-scale genome reference. The linkage map reveals an extremely low rate of recombination on chromosome 1. We confirm suppression of recombination on chromosome 1 by genotyping individual sperm cells and oocytes. We show that previously identified genomic regions that maintain heterozygosity even after prolonged inbreeding make up essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicates that this phenomenon has evolved specifically in populations from Sardinia and Corsica. We find that most known master regulators 5-13 of the reproductive system are located on chromosome 1. We used RNA interference 14,15 to knock down a gene with haplotype-biased expression, which led to the formation of a more pronounced female mating organ. On the basis of these observations, we propose that chromosome 1 is a sex-primed autosome primed for evolution into a sex chromosome., (© 2022. The Author(s).)

The Balearic shearwater (Puffinus mauretanicus) is the most threatened seabird in Europe and a member of the most speciose group of pelagic seabirds, the order Procellariiformes, which exhibit extreme adaptations to a pelagic lifestyle. The fossil record suggests that human colonisation of the Balearic Islands resulted in a sharp decrease of the Balearic shearwater population size. Currently, populations of the species continue to be decimated mainly due to predation by introduced mammals and bycatch in longline fisheries, with some studies predicting its extinction by 2070. Here, using a combination of short and long reads, we generate the first high-quality reference genome for the Balearic shearwater, with a completeness amongst the highest across available avian species. We used this reference genome to study critical aspects relevant to the conservation status of the species and to gain insights into the adaptation to a pelagic lifestyle of the order Procellariiformes. We detected relatively high levels of genome-wide heterozygosity in the Balearic shearwater despite its reduced population size. However, the reconstruction of its historical demography uncovered an abrupt population decline potentially linked to a reduction of the neritic zone during the Penultimate Glacial Period (∼194-135 ka). Comparative genomics analyses uncover a set of candidate genes that may have played an important role into the adaptation to a pelagic lifestyle of Procellariiformes, including those for the enhancement of fishing capabilities, night vision, and the development of natriuresis. The reference genome obtained will be the crucial in the future development of genetic tools in conservation efforts for this Critically Endangered species., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The diversification of modern birds has been shaped by a number of radiations. Rapid diversification events make reconstructing the evolutionary relationships among taxa challenging due to the convoluted effects of incomplete lineage sorting (ILS) and introgression. Phylogenomic data sets have the potential to detect patterns of phylogenetic incongruence, and to address their causes. However, the footprints of ILS and introgression on sequence data can vary between different phylogenomic markers at different phylogenetic scales depending on factors such as their evolutionary rates or their selection pressures. We show that combining phylogenomic markers that evolve at different rates, such as paired-end double-digest restriction site-associated DNA (PE-ddRAD) and ultraconserved elements (UCEs), allows a comprehensive exploration of the causes of phylogenetic discordance associated with short internodes at different timescales. We used thousands of UCE and PE-ddRAD markers to produce the first well-resolved phylogeny of shearwaters, a group of medium-sized pelagic seabirds that are among the most phylogenetically controversial and endangered bird groups. We found that phylogenomic conflict was mainly derived from high levels of ILS due to rapid speciation events. We also documented a case of introgression, despite the high philopatry of shearwaters to their breeding sites, which typically limits gene flow. We integrated state-of-the-art concatenated and coalescent-based approaches to expand on previous comparisons of UCE and RAD-Seq data sets for phylogenetics, divergence time estimation, and inference of introgression, and we propose a strategy to optimize RAD-Seq data for phylogenetic analyses. Our results highlight the usefulness of combining phylogenomic markers evolving at different rates to understand the causes of phylogenetic discordance at different timescales. [Aves; incomplete lineage sorting; introgression; PE-ddRAD-Seq; phylogenomics; radiations; shearwaters; UCEs.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

Fructosyltransferase (FTase) catalyzes the transfer of a fructosyl group to a sucrose molecule or a fructooligosaccharide (FOS) when a FOS with a longer chain is formed. Production of FTase by two Aspergillus species and its mixture was exploited using solid-state fermentation (SSF) and employing agave sap as substrate. The maximum FTase activity (1.59 U/mL) by Aspergillus oryzae was obtained after 24 h, using a temperature of 30 °C, with an inoculum of 2 × 10 7 spores/mL. The nucleotide sequence coding for the fructosyltransferase showed 1494 bp and encodes for a protein of 498 amino acids. The hypothetical molecular tertiary structure of Aspergillus oryzae BM-DIA FTase showed the presence of structural domains, such as a five-bladed beta-propeller domain characteristic of GH (glycoside hydrolase) and C terminal, which forms a beta-sandwich module. This study contributes to the knowledge of stability, compatibility, and genetic expression of Aspergillus oryzae BM-DIA under SSF bioprocess conditions for industrial production of fructosyltransferase.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity 1-4 . Sparse taxon sampling has previously been proposed to confound phylogenetic inference 5 , and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.

Terrestrial planarians found in a plant nursery in Spain in 2012 are described as a new species, Caenoplana decolorata . Dorsally they are mahogany brown with a cream median line. Ventrally they are pastel turquoise fading to brown laterally. Molecular data indicate that they are a member of the genus Caenoplana , but that they differ from other Caenoplana species found in Europe. One mature specimen has been partially sectioned, and the musculature and copulatory apparatus is described, confirming the generic placement but distinguishing the species from other members of the genus. It is probable that the species originates from Australia., Competing Interests: Marta Riutort is an Academic Editor for PeerJ., (© 2020 Mateos et al.)

Background: Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study., Methods: To unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features., Results: We discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri . A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area., Competing Interests: Marta Riutort is an Academic Editor for PeerJ., (©2020 Araujo et al.)

The planarian suborder Cavernicola Sluys, 1990 was originally created to house five species of triclad flatworms with special morphological features and a surprisingly discontinuous and broad geographic distribution. These five species could not be accommodated with any degree of certainty in any of the three taxonomic groups existing at that moment, viz., Paludicola Hallez, 1892, Terricola Hallez, 1892, and Maricola Hallez, 1892. The scarce representation of the group and the peculiarities of the morphological features of the species, including several described more recently, have complicated new tests of the monophyly of the Cavernicola, the assessment of its taxonomic status, as well as the resolution of its internal relationships. Here we present the first molecular study including all genera currently known for the group, excepting one. We analysed newly generated 18S and 28S rDNA data for these species, together with a broad representation of other triclad flatworms. The resulting phylogenetic trees supported the monophyly of the Cavernicola, as well as its sister-group relationship to the Maricola. The sister-group relationship to the Maricola and affinities within the Cavernicola falsify the morphology-based phylogeny of the latter that was proposed previously. The relatively high diversity of some cavernicolan genera suggests that the presumed rarity of the group actually may in part be due to a collecting artefact. Ancestral state reconstruction analyses suggest that the ancestral habitat of the group concerned epigean freshwater conditions. Our results point to an evolutionary scenario in which the Cavernicola (a) originated in a freshwater habitat, (b) as the sister clade of the marine triclads, and (c) subsequently radiated and colonized both epigean and hypogean environments. Competition with other planarians, notably members of the Continenticola, or changes in epigean habitat conditions are two possible explanations -still to be tested- for the loss of most epigean diversity of the Cavernicola, which is currently reflected in their highly disjunct distributions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Terrestrial planarians with a dorsal yellow stripe and dark lateral surfaces and up to 15-20 cm long have been found in several countries in Europe, the earliest in 2008. They are similar to two species originally from Australia, Caenoplana variegata (Fletcher Hamilton, 1888) and C. bicolor (Graff, 1899), both described on external characters only, with no anatomical information. Careful reading suggests that there is no significant difference between the original descriptions. Further: observations on live specimens show considerable variation between individuals and in individuals over time and before and after feeding, negating any distinction between descriptions. Examination of three sectioned specimens shows considerable difference in sexual maturity, though one seems almost fully mature and the reproductive system is described. Molecular results show that specimens from the United Kingdom and Spain are of the same species. It is concluded that the planarians should be referred to as C. variegata, C. bicolor being a junior synonym.

The keystone of planarian taxonomy traditionally has been the anatomy of the copulatory apparatus. However, many planarian species comprise asexual fissiparous populations, with the fissiparous animals not developing a copulatory apparatus, thus precluding their morphological identification. Incorporation of molecular data into planarian systematics has been of great value, not only in the identification of fissiparous individuals but also as an additional source of information for determining species boundaries. Nevertheless, the discrepancy between morphological and molecular data has highlighted the need for extra sources of taxonomic information. Moreover, a recent study has pointed out that fissiparous reproduction may lead to high levels of intraindividual genetic diversity in planarians, which may mislead molecular analyses. In the present study we aim to test a new up-to-date integrative taxonomic procedure for planarians, including intraindividual genetic data and additional sources of taxonomic information, besides morphology and DNA, using Dugesia subtentaculata sensu lato as a model organism, a species with an intricate taxonomic history. First, we used three different methods for molecular species delimitation on single locus datasets, both with and without intraindividual information, for formulating Primary Species Hypotheses (PSHs). Subsequently, Secondary Species Hypotheses (SSHs) were formulated on the basis of three types of information: (1) a coalescent-based species delimitation method applied to multilocus data, (2) morphology of the copulatory apparatus, and (3) karyological metrics. This resulted in the delimitation of four morphologically cryptic species within the nominal species D. subtentaculata. Our results provide evidence that the analysis of intraindividual genetic data is essential for properly developing PSHs in planarians. Our study reveals also that karyological differentiation, rather than morphological differentiation, may play an important role in speciation processes in planarians, thus suggesting that the currently known diversity of the group could be highly underestimated., (Copyright © 2019 Elsevier Inc. All rights reserved.)