Building Phylogenies from Transcriptomic Data.

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.

Phylogenetic relationships of the Geoplaninae land planarians (Platyhelminthes, Tricladida) assessed with a total evidence approach, with the description of a new species of Gigantea.

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.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds.

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.

Phylotranscriptomics interrogation uncovers a complex evolutionary history for the planarian genus Dugesia (Platyhelminthes, Tricladida) in the Western Mediterranean.

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.

Molecular phylogenetics facilitates the first historical biogeographic analysis of the hammerhead worms (Platyhelminthes: Tricladida: Bipaliinae), with the description of twelve new species and two new genera.

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.

Island-specific evolution of a sex-primed autosome in a sexual planarian.

The sexual strain of the planarian Schmidtea mediterranea, indigenous to Tunisia and several Mediterranean islands, is a hermaphrodite1,2. Here we isolate individual chromosomes and use sequencing, Hi-C3,4 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 regulators5-13 of the reproductive system are located on chromosome 1. We used RNA interference14,15 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.

The First Genome of the Balearic Shearwater (Puffinus mauretanicus) Provides a Valuable Resource for Conservation Genomics and Sheds Light on Adaptation to a Pelagic lifestyle.

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.

Fructosyltransferase production by Aspergillus oryzae BM-DIA using solid-state fermentation and the properties of its nucleotide and protein sequences.

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 × 107 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.

Integrating Sequence Capture and Restriction Site-Associated DNA Sequencing to Resolve Recent Radiations of Pelagic Seabirds.

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.].

A new species of alien terrestrial planarian in Spain: Caenoplana decolorata.

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.

Five new pseudocryptic land planarian species of Cratera (Platyhelminthes: Tricladida) unveiled through integrative taxonomy.

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.

The identity of the invasive yellow-striped terrestrial planarian found recently in Europe: Caenoplana variegata (Fletcher amp; Hamilton, 1888) or Caenoplana bicolor (Graff, 1899)?

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.

Phylogeny and biogeography of the Cavernicola (Platyhelminthes: Tricladida): Relicts of an epigean group sheltering in caves?

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.

Cryptic species delineation in freshwater planarians of the genus Dugesia (Platyhelminthes, Tricladida): Extreme intraindividual genetic diversity, morphological stasis, and karyological variability.

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.

First record of freshwater planarian Girardia tigrina (Platyhelminthes, Tricladida, Continenticola) in Eastern Europe.

The genus Girardia Ball, 1974 houses freshwater planarian species and is the second after Dugesia Girard, 1850 within Dugesiidae regarding the number of species. It contains 45 species (Tyler et al. 2006-2019) including three recently described: one from China-Girardia sinensis Chen Wang 2015 (Chen et al. 2015), and two cave-dwelling planarians from Brasil-Girardia desiderensis Souza Leal-Zanchet 2016 and Girardia pierremartini Souza Leal-Zanchet 2016 (Souza et al. 2016).

Diet assessment of two land planarian species using high-throughput sequencing data.

Geoplanidae (Platyhelminthes: Tricladida) feed on soil invertebrates. Observations of their predatory behavior in nature are scarce, and most of the information has been obtained from food preference experiments. Although these experiments are based on a wide variety of prey, this catalog is often far from being representative of the fauna present in the natural habitat of planarians. As some geoplanid species have recently become invasive, obtaining accurate knowledge about their feeding habits is crucial for the development of plans to control and prevent their expansion. Using high throughput sequencing data, we perform a metagenomic analysis to identify the in situ diet of two endemic and codistributed species of geoplanids from the Brazilian Atlantic Forest: Imbira marcusi and Cephaloflexa bergi. We have tested four different methods of taxonomic assignment and find that phylogenetic-based assignment methods outperform those based on similarity. The results show that the diet of I. marcusi is restricted to earthworms, whereas C. bergi preys on spiders, harvestmen, woodlice, grasshoppers, Hymenoptera, Lepidoptera and possibly other geoplanids. Furthermore, both species change their feeding habits among the different sample locations. In conclusion, the integration of metagenomics with phylogenetics should be considered when establishing studies on the feeding habits of invertebrates.

Outstanding intraindividual genetic diversity in fissiparous planarians (Dugesia, Platyhelminthes) with facultative sex.

BACKGROUND: Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller's Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller's Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction. RESULTS: In the present study, we generate and analyze intraindividual cloned data of a nuclear and a mitochondrial gene of sexual, fissiparous and facultative wild populations of the species Dugesia subtentaculata. We find that most individuals, independently of their reproductive strategy, are mosaics. However, the intraindividual haplotype and nucleotide diversity of fissiparous and facultative individuals is significantly higher than in sexual individuals, with no signs of Muller's Ratchet. Finally, we also find that this high intraindividual genetic diversity of fissiparous and facultative individuals is composed by different combinations of ancestral and derived haplotypes of the species. CONCLUSIONS: The intraindividual analyses of genetic diversity point out that fissiparous reproduction leaves a very special genetic footprint in individuals, characterized by mosaicism combined with the Meselson effect (named in the present study as the mosaic Meselson effect). Interestingly, the different intraindividual combinations of ancestral and derivate genetic diversity indicate that haplotypes generated during periods of fissiparous reproduction can be also transmitted to the progeny through sexual events, resulting in offspring showing a wide range of genetic diversity and putatively allowing purifying selection to act at both intraindividual and individual level. Further investigations, using Dugesia planarians as model organisms, would be of great value to delve into this new model of genetic evolution by the combination of fission and sex.

Hidden diversity in forest soils: Characterization and comparison of terrestrial flatworm's communities in two national parks in Spain.

Terrestrial flatworms (Platyhelminthes, Tricladida, and Geoplanidae) belong to what is known as cryptic soil fauna of humid forests and are animals not easily found or captured in traps. Nonetheless, they have been demonstrated to be good indicators of the conservation status of their habitat as well as a good model to reconstruct the recent and old events affecting biodiversity. This is mainly due to their delicate constitution, their dependence on the integrity of their habitat, and their very low dispersal capacity. At present, little is known about their communities, except for some studies performed in Brazil. In this work, we analyze for the first time in Europe terrestrial flatworm communities. We have selected two protected areas belonging to the Red Española de Parques Nacionales. Our aims include performing a first study of the species richness and community structure for European terrestrial planarian species at regional and local scale. We evaluate the effect of type of forests in the community composition and flatworms' abundance, but also have into account the phylogenetic framework (never considered in previous studies) analyzed based on molecular data. We find differences in the species composition among parks, with an astonishingly high diversity of endemic species in the Parque Nacional de Picos de Europa and an extremely low diversity of species in the Parque Nacional de Ordesa y Monte Perdido. These divergent patterns cannot be attributed to differences in physical variables, and in addition, the analyses of their phylogenetic relationships and, for a few species, their genetic structure, point to a more probable historical explanation.