What do Biologists Make of the Species Problem?

The concept of species is one of the core concepts in biology and one of the cornerstones of evolutionary biology, yet it is rife with conceptual problems. Philosophers of biology have been discussing the concept of species for decades, and in doing so they sometimes appeal to the views of biologists. However, their statements as to what biologists think are seldom supported by empirical data. In order to investigate what biologists actually think about the key issues related to the problem of species, we have conducted a survey on the sample of 193 biologists from the population of biologists from over 150 biology departments at universities in the US and the EU. This article presents and discusses the results of the survey. Some results confirm and others falsify the reiterated statements of philosophers of biology as to what biologists think, but all results we obtained should be informative and relevant for future discussions of the problem of species.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.

Algal endosymbionts in European Hydra strains reflect multiple origins of the zoochlorella symbiosis.

Symbiotic associations are of broad significance in evolution and biodiversity. Green Hydra is a classic example of endosymbiosis. In its gastrodermal myoepithelial cells it harbors endosymbiotic unicellular green algae, most commonly from the genus Chlorella. We reconstructed the phylogeny of cultured algal endosymbionts isolated and maintained in laboratory conditions for years from green Hydra strains collected from four different geographical sites within Croatia, one from Germany and one from Israel. Nuclear (18S rDNA, ITS region) and chloroplast markers (16S, rbcL) for maximum likelihood phylogenetic analyses were used. We focused on investigating the positions of these algal endosymbiotic strains within the chlorophyte lineage. Molecular analyses established that different genera and species of unicellular green algae are present as endosymbionts in green Hydra, showing that endosymbiotic algae growing within green Hydra sampled from four Croatian localities are not monophyletic. Our results indicate that the intracellular algal endosymbionts of green Hydra have become established several times independently in evolution.

Genetic identification of a rare record of Ommastrephes Bartramii (Cephalopoda: Ommastrephidae) from the Eastern Adriatic Sea.

The neon flying squid Ommastrephes bartramii Lesueur, 1821 belongs to the Ommastrephidae, Cephalopoda family. The family Ommastrephidae (Mollusca: Cephalopoda) includes many commercially important species, dispersed around the world. The Ommastrephidae family is conventionally divided into three subfamilies (Illicinae, Todarodinae, and Ommastrephinae). We report a specimen of neon flying squid caught in the winter 2013 at Luka Sipanska, Island of Sipan, Croatia and identified at the genetic level using the standard mitochondrial COI barcode region. This record represents the first genetic identification of a neon flying squid from the Adriatic Sea.

Protein phosphorylation in bacterial signal transduction.

BACKGROUND: Protein phosphorylation has emerged as one of the major post translational modifications in bacteria, involved in regulating a myriad of physiological processes. In a complex and dynamic system such as the bacterial cell, connectivity of its components accounts for a number of emergent properties. This article is part of a Special Issue entitled: Systems Biology of Microorganisms. SCOPE OF REVIEW: This review focuses on the implications of bacterial protein phosphorylation in cell signaling and regulation and highlights the connections and cross talk between various signaling pathways: bacterial two-component systems and serine/threonine kinases, but also the interference between phosphorylation and other post-translational modifications (methylation and acetylation). MAJOR CONCLUSIONS: Recent technical developments in high accuracy mass spectrometry have profoundly transformed proteomics, and today exhaustive site-specific phosphoproteomes are available for a number of bacterial species. Nevertheless, prediction of phosphorylation sites remains the main guide for many researchers, so we discuss the characteristics, limits and advantages of available phosphorylation predictors. GENERAL SIGNIFICANCE: The advent of quantitative phosphoproteomics has brought the field on the doorstep of systems biology, but a number of challenges remain before the bacterial phosphorylation networks can be efficiently modeled and their physiological role understood. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Lepocranus and Valalyllum gen. nov. (Orthoptera, Tetrigidae, Cladonotinae), endangered Malagasy dead-leaf-like grasshoppers.

Only two leaf-like pygmy grasshopper species and specimens are known from Madagascar: the Leatherback Pygmy Grasshopper (Lepocranusfuscus Devriese, 1991) -which has a relatively low median carina of the pronotum; and the Malagasy Litterhopper (Valalyllumfolium gen. et. sp. nov.), herein described - which has a high median carina. Lepocranusfuscus is known from the rainforests around Tampolo, Manakambahiny, and Mahavelona (Foulpointe). The new taxon, Valalyllumfolium gen. et. sp. nov. is known only from the Belanono forest. Both species inhabit northeastern Madagascar. The new species could be rare or not-easy-to-spot in the rainforest leaf litter, where it most probably lives. A new tribe, Valalyllinitrib. nov., is described for the two mentioned genera because its members are different from the Caribbean leaf-like Choriphyllini Cadena-Castañeda & Silva, 2019, from the African leaf-like Xerophyllini Günther, 1979, and from the Asian leaf-like Cladonotini Bolívar, 1887. A tabular key to the tribes of Cladonotinae with leaf-like representatives is provided, together with photographs of type specimens of both species belonging to the newly described tribe. The holotype of the new species belongs to the Muséum national d'Histoire naturelle Orthoptera collection, Paris.

Genomic characterization and assessment of pathogenic potential of Legionella spp. isolates from environmental monitoring.

Several species in the genus Legionella are known to cause an acute pneumonia when the aerosols containing the bacteria from man-made water systems are inhaled. The disease is usually caused by Legionella pneumophila, but other species have been implicated in the infection. The disease is frequently manifested as an outbreak, which means several people are affected when exposed to the common source of Legionella contamination. Therefor environmental surveillance which includes isolation and identification of Legionella is performed routinely. However, usually no molecular or genome-based methods are employed in further characterization of the isolates during routine environmental monitoring. During several years of such monitoring, isolates from different geographical locations were collected and 39 of them were sequenced by hybrid de novo approach utilizing short and long sequencing reads. In addition, the isolates were typed by standard culture and MALDI-TOF method. The sequencing reads were assembled and annotated to produce high-quality genomes. By employing discriminatory genome typing, four potential new species in the Legionella genus were identified, which are yet to be biochemically and morphologically characterized. Moreover, functional annotations concerning virulence and antimicrobial resistance were performed on the sequenced genomes. The study contributes to the knowledge on little-known non-pneumophila species present in man-made water systems and establishes support for future genetic relatedness studies as well as understanding of their pathogenic potential.

Evidence for a Syncytial Origin of Eukaryotes from Ancestral State Reconstruction.

Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA's multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other's lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized.

Isolation and cultivation of endosymbiotic algae from green hydra and phylogenetic analysis of 18S rDNA sequences.

Symbiotic associations are of wide significance in evolution and biodiversity. The green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells it harbors the individuals of a unicellular green algae. Endosymbiotic algae from green hydra have been successfully isolated and permanently maintained in a stable clean lab culture for the first time. We reconstructed the phylogeny of isolated endosymbiotic algae using the 18S rRNA gene to clarify its current status and to validate the traditional inclusion of these endosymbiotic algae within the Chlorella genus. Molecular analyses established that different genera and species of unicellular green algae could be present as symbionts in green hydra, depending on the natural habitat of a particular strain of green hydra.

The story of the Malagasy devils (Orthoptera, Tetrigidae): Holocerus lucifer in the north and H. devriesei sp. nov. in the south?

Madagascar is home to some of the largest and most colorful pygmy grasshoppers (Tetrigidae) in the world, known as 'Malagasy Metrodorinae'. Among them, Devil's pygmy grasshoppers (genus Holocerus Bolívar, 1887) are unique in having two long spines on the back, which are modified internal lateral pronotal carinae. The genus Holocerus was composed of two species - H. lucifer (Serville, 1838) and H. taurus Rehn, 1929 syn. nov., but here it is evidenced that the latter represents a junior synonym of the former. Simultaneously, H. devriesei sp. nov. is described as a species new to science. Holocerus lucifer is a northern species of paler coloration and longer spines (distributed from Marojejy and Maroantsetra in the north to Zahamena in the south), whereas H. devriesei sp. nov. represents the southern and darker species (distributed from Vohimana and Andasibe-Mantadia in the south to the Antongil Bay in the north). There are potential overlaps in the distribution of the two species, but without more georeferenced localities, it is impossible to discriminate whether they occur only sympatrically or also syntopically.

Wide-nosed pygmy grasshoppers (Cladonotinae: Cladonotini, Xerophyllini) of India and Sri Lanka: catalogue with an identification key and description of a new species of the genus Tettilobus.

Wide nosed pygmy grasshoppers (Tetrigidae: Cladonotinae) are grasshoppers in which the frontal costa bifurcates into strongly divergent facial carinae, between which there is wide scutellum. Altogether seven genera and thirteen species inhabit India and Sri Lanka. Tribe Cladonotini is represented in this region by five genera and nine species (Cladonotus-4 spp., Deltonotus-2 spp., Gignotettix-1 sp., Hancockella-1 sp., and Yunnantettix-1 sp.), while the tribe Xerophyllini by two genera, Tettilobus (3 sp.) and Potua (1 sp.). For this Potua species it is not clear if it belongs to this genus. A simple identification key to Cladonotinae of India and Sri Lanka is provided. New synonyms are: Deltonotus humilis syn. nov., Epitettix tamilus syn. nov. and Potua aptera syn. nov., synonymous with Deltonotus gibbiceps. Deltonotus cristatus should be regarded nomen nudum. A new species, assigned to the genus Tettilobus is described from the Western Ghats of Kerala, Tettilobus trishula Skejo, Bhaskar et Stermsek sp. n. Furthermore, we present the first records of D. subcucullatus from India (Kerala).

Online social media tells a story of Anaselina, Paraselina, and Selivinga (Orthoptera, Tetrigidae), rare Australian pygmy grasshoppers.

Knowledge on the pygmy grasshoppers of Australia is, despite the numerous endemics being described from this unique continent, still scarce. Of interest is the Vingselina genus group, including genera Anaselina Storozhenko, 2019, Paraselina Storozhenko, 2019, Selivinga Storozhenko, 2019 and Vingselina Sjöstedt, 1921. The systematic position of this group, currently assigned to Batrachideinae (Bufonidini), is probably not correct. In this study new records are presented of Anaselina minor (Sjöstedt, 1921), Paraselina brunneri (Bolívar, 1887), P. trituberculata (Sjöstedt, 1932), and Selivinga tribulata Storozhenko, 2019, all except A. minor the first records of the species since their original descriptions. The first photographs of living specimens of A. minor, P. brunneri, P. trituberculata and S. tribulata are provided and their habitats described. All the records were compiled by citizen scientists who use online social media, such as iNaturalist. Lastly, P. multifora (Rehn, 1952) syn. nov. represents a junior synonym of P. brunneri.

The limits of cryptic diversity in groundwater: phylogeography of the cave shrimp Troglocaris anophthalmus (Crustacea: Decapoda: Atyidae).

Recent studies have revealed high local diversity and endemism in groundwaters, and showed that species with large ranges are extremely rare. One of such species is the cave shrimp Troglocaris anophthalmus from the Dinaric Karst on the western Balkan Peninsula, apparently uniform across a range of more than 500 kilometers. As such it contradicts the paradigm that subterranean organisms form localized, long-term stable populations that cannot disperse over long distances. We tested it for possible cryptic diversity and/or unexpected evolutionary processes, analysing mitochondrial (COI, 16S rRNA) and nuclear (ITS2) genes of 232 specimens from the entire range. The results of an array of phylogeographical procedures congruently suggested that the picture of a widespread, continuously distributed and homogenous T. anophthalmus was wrong. The taxon is composed of four or possibly five monophyletic, geographically defined phylogroups that meet several species delimitation criteria, two of them showing evidence of biological reproductive isolation in sympatry. COI genetic distances between phylogroups turned out to be a poor predictor, as they were much lower than the sometimes suggested crustacean threshold value of 0.16 substitutions per site. Most results confirmed the nondispersal hypothesis of subterranean fauna, but the southern Adriatic phylogroup displayed a paradoxical pattern of recent dispersal across 300 kilometers of hydrographically fragmented karst terrain. We suggest a model of migration under extreme water-level conditions, when flooded poljes could act as stepping-stones. In the north of the range (Slovenia), the results confirmed the existence of a zone of unique biogeographical conflict, where surface fauna is concordant with the current watershed, and subterranean fauna is not.

Oriental macropterous leaf-mimic pygmy grasshoppers-genera Oxyphyllum and Paraphyllum (Orthoptera: Tetrigidae) and their taxonomic assignment.

There are numerous pygmy grasshoppers (Tetrigidae) that exhibit leaf-like appearance. Leaf-mimic species can be found mainly in the subfamily Cladonotinae (tribes Cladonotini, Xerophyllini). Two leaf-mimic pygmy grasshopper species found in India, Pakistan (Oxyphyllum pennatum Hancock, 1909) and Borneo (Paraphyllum antennatum Hancock, 1913) are the only macropterous leaf-like species of Asia and were traditionally assigned to subfamily Cladonotinae. Our study present new records of these species, as well as updated descriptions. Oxyphyllum pennatum is recorded for the first time from Chhattisgarh (Central India), and furthermore, records from Pakistan were reviewed and confirmed. Paraphyllum antennatum is reported from a few localities in the mountains of Borneo (East Malaysia). New records found in online social media (Flickr, iNaturalist) were implemented. Brachypronotal specimens of P. antennatum are reported. After comparison with Cladonotini and Xerophyllini members, we conclude that Oxyphyllum and Paraphyllum should not be regarded Cladonotinae members. Traditional assignment was based on superficial resemblance to leaf-like Cladonotinae genera (e.g. Phyllotettix, Hymenotes, Holoarcus, Xerophyllum). However, comparison of morphological characters reveals that Oxyphyllum shares morphology with Tetriginae genera, while Paraphyllum shares morphology with Asian Metrodorinae. Here, we assign the genus Oxyphyllum to Tetriginae and the genus Paraphyllum to Metrodorinae.

Limited Genetic Structure of Gypsy Moth Populations Reflecting a Recent History in Europe.

The gypsy moth, Lymantria dispar, a prominent polyphagous species native to Eurasia, causes severe impacts in deciduous forests during irregular periodical outbreaks. This study aimed to describe the genetic structure and diversity among European gypsy moth populations. Analysis of about 500 individuals using a partial region of the mitochondrial COI gene, L. dispar was characterized by low genetic diversity, limited population structure, and strong evidence that all extant haplogroups arose via a single Holocene population expansion event. Overall 60 haplotypes connected to a single parsimony network were detected and genetic diversity was highest for the coastal populations Croatia, Italy, and France, while lowest in continental populations. Phylogenetic reconstruction resulted in three groups that were geographically located in Central Europe, Dinaric Alps, and the Balkan Peninsula. In addition to recent events, the genetic structure reflects strong gene flow and the ability of gypsy moth to feed on about 400 deciduous and conifer species. Distinct genetic groups were detected in populations from Georgia. This remote population exhibited haplotypes intermediate to the European L. dispar dispar, Asian L. dispar asiatica, and L. dispar japonica clusters, highlighting this area as a possible hybridization zone of this species for future studies applying genomic approaches.

Serine/Threonine Protein Kinases from Bacteria, Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life.

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988 (Science, 241, 42-52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term "eukaryotic-type kinases" propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family.

Conversion of Glycerol to 3-Hydroxypropanoic Acid by Genetically Engineered Bacillus subtilis.

3-Hydroxypropanoic acid (3-HP) is an important biomass-derivable platform chemical that can be converted into a number of industrially relevant compounds. There have been several attempts to produce 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from K. pneumoniae. Genetic engineering, driven by in silico optimization, and optimization of cultivation conditions resulted in a 3-HP titer of 10 g/L, in a standard batch cultivation. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis. With this relatively high titer in batch, and the robustness of B. subtilis in high density fermentation conditions, we expect that our production strains may constitute a solid basis for commercial production of 3-HP.

Resistance of two planarian species to UV-irradiation.

The aim of this work was to determine the effects of 20, 25 and 30 minute UV-irradiation periods lambda = 253.5 nm to two planarian species Dugesia tigrina (Gir.) and Polycelis felina (Daly.). In vivo, UV light effects have been reported to affect intracellular receptors and disrupt simple behaviour. The effects of UV-rays on mortality and behavior as well as morphological, cytological and histological changes in the two planarian species were assessed, and the course and the dynamics of regenerative processes were compared between them. Experimental populations of Dugesia tigrina and Polycelis felina species were maintained in laboratory conditions at room temperature. Mortality, behavioral and morphological changes were monitored daily by means of a light stereomicroscope. For cytological and histopathological analysis, planarians were fixed in Bouine fixative on the first, second, third, fifth and seventh day after exposure to UV-irradiation, respectively. They were embedded in paraffin, cut on a microtome, stained with toluidin blue and embedded in Canada-balsam. UV-rays caused mortality, behavioral, morphological, cytological and histological changes in each planarian species. In regeneration of damaged body parts reticular cells and neoblasts played the main role. Neoblasts as totipotent cells extremely increased in number in the area of damaged tissue, immediately after UV-exposure. Dugesia tigrina was more sensitive to UV-rays than Polycelis felina due to possession of less pigmented cells. The course of regeneration in both species was similar. Most individuals of both species regenerated in 5 to 12 days after UV-irradiation.

Freshwater and brackish bryozoan species of Croatia (Bryozoa: Gymnolaemata, Phylactolaemata) and their genetic identification.

Freshwater and brackish species of bryozoans belong to the Phylactolaemata and Gymnolaemata class. Twelve species of bryozoans were recorded and morphologically determined at eight locations in the Black Sea and the Adriatic basin in Croatia. Twelve species of Bryozoa have been listed in the taxonomic index for Croatia (Conopeum seurati, Lophopus crystallinus Paludicella articulata, Cristatella mucedo, Fredericella sultana, Hyalinella punctata, Plumatella casmiana, Plumatella emarginata, Plumatella fruticosa, Plumatella fungosa, Plumatella geimermassardi and Plumatella repens). For the purposes of gene identification of recorded species, molecular markers for nuclear 18S and 28S genes, ITS2 region and mitochondrial COI gene were amplified. Genetic identifications of morphologically determined bryozoan species were confirmed using highly similar sequences local alignment analysis. Proliferation of freshwater bryozoan species over long distances with the help of the vector animals was confirmed by defining haplotypes on the base of 18S, 28S and ITS2 sequences associated with the Black Sea-Mediterranean waterfowl flyway.