Development of molecular and morphological markers to improve species-specific monitoring and systematics of Northeast Atlantic and Mediterranean skates (Rajiformes)

The Northeast Atlantic and Mediterranean skates (Rajidae) showed remarkable species diversity but with high morphological and ecological conservatism. Since skates are particularly vulnerable to the bottom trawl fishery, species-specific demographic surveys as well as studies defining life history and evolutionary traits are important in prioritising conservation programs. However, the identification of juveniles and adults of some species may be difficult using referenced guidelines and identification keys. Therefore, we attempt to develop markers for species identification through the parallel analysis of a 16S rDNA gene sequence and of several morphological characters on 135 individuals collected by trawl surveys in the Adriatic Sea and putatively assigned to six taxa. Species-specific haplotypes were defined for Raja miraletus, Raja montagui, Dipturus oxyrinchus, since a solid accordance between species boundaries and well-differentiated haplotypes was observed. Comparative analysis of 16S rDNA sequences allowed the identification of three juvenile specimens of Leucoraja circularis, a species that rarely occurs in the Adriatic Sea. On the contrary, morphological traits and haplotype distribution were largely discordant in Raja asterias and R. clavata. While all putative R. clavata individuals showed a unique haplotype (H-CLA), only 8 of 30 putative R. asterias individuals possessed a second weakly divergent haplotype (H-AST). The remaining 22 R. asterias carried the H-CLA. The multivariate analyses of morphometric and meristic characters in putative R. clavata and R. asterias revealed the clustering of individuals regardless of haplotypes. However, a bimodal distribution of R. asterias and R. clavata samples would suggest that two separated taxa might exist, both sharing the two 16S rDNA haplotypes. The haplotype distribution appeared to be significantly correlated only to the standardised disc length/total length (DL/TL) variation. Three alternative explanations may support this scenario: (i) an incomplete lineage sorting process in two morphologically yet distinct taxa; (ii) a recent hybridisation between the two taxa; (iii) the two taxa are morphologically plastic species and all considered morphological characters may be misleading in discriminating between them at all maturity stages, except for the DL/TL. However, further analyses on larger data sets and using molecular key markers (i.e. nuclear genes) will be needed to definitely resolve the status of these taxa. Molecular relationships among rajid taxa are largely consistent with systematics based on internal and external anatomical features. This multidisciplinary study contributed to defining the pattern of species diversity and abundance of rajids in the Adriatic Sea.     

Resolving taxonomic uncertainty in vulnerable elasmobranchs: are the Madeira skate (<Emphasis Type="Italic">Raja maderensis</Emphasis>) and the thornback ray (<Emphasis Type="Italic">Raja clavata</Emphasis>) distinct species?

Skates and rays constitute the most speciose group of chondrichthyan fishes, yet are characterised by remarkable levels of morphological and ecological conservatism. They can be challenging to identify, which makes monitoring species compositions for fisheries management purposes problematic. Owing to their slow growth and low fecundity, skates are vulnerable to exploitation and species exhibiting endemism or limited ranges are considered to be the most at risk. The Madeira skate Raja maderensis is endemic and classified as ‘Data Deficient’ by the IUCN, yet its taxonomic distinctiveness from the morphologically similar and more wide-ranging thornback ray Raja clavata is unresolved. This study evaluated the sequence divergence of both the variable control region and cytochrome oxidase I ‘DNA barcode’ gene of the mitochondrial genome to elucidate the genetic differentiation of specimens identified as R. maderensis and R. clavata collected across much of their geographic ranges. Genetic evidence was insufficient to support the different species designations. However regardless of putative species identification, individuals occupying waters around the Azores and North African Seamounts represent an evolutionarily significant unit worthy of special consideration for conservation management.     

Molecular barcoding of skates (Chondrichthyes: Rajidae) from the southern Northeast Atlantic

Serra‐Pereira, B., Moura, T., Griffiths, A. M., Gordo, L. S. & Figueiredo, I. (2010). Molecular barcoding of skates (Chondrichthyes: Rajidae) from the southern Northeast Atlantic. —Zoologica Scripta, 40, 76–84. Due to their vulnerability to fishing pressure, many species of skate (Rajidae) in the Northeast Atlantic are undergoing declines in abundance. The assessment of stock status and subsequent proposal of management measures are often complicated by high levels of species diversity and endemism, coupled with morphological and ecological conservatism, which makes distinguishing between species difficult. To improve the identification of skates and investigate the phylogenetic position of endemic species the cytochrome c oxidase subunit I (COI) was sequenced in 12 species (Dipturus oxyrinchus, Leucoraja naevus, Leucoraja circularis, Neoraja iberica, Raja brachyura, Raja clavata, Raja maderensis, Raja microocellata, Raja miraletus, Raja montagui, Raja undulata, Rostroraja alba) inhabiting the Portuguese waters. Based on sequence divergence R. maderensis and R. clavata only differ by 1% of the 652 bp COI sequence, questioning the recognition of R. maderensis (considered to be endemic to Madeira and the Azores), as a reproductively isolated species. Otherwise, there was clear phylogenetic support for the different genera and all the remaining species, although the genetic divergence was low compared to other chordates. In particular, COI analysis allowed clear identification of the morphologically similar species R. brachyura and R. montagui.     

Dietary habits of the polkadot skate <Emphasis Type="Italic">Dipturus chinensis</Emphasis> in the East China Sea

We investigated the dietary habits of the polkadot skate, Dipturus chinensis (Japanese name: Gangi-ei), in the East China Sea by analyzing the stomach contents of 529 individuals. Of these, 18 (3.40%) had empty stomachs. The mean and maximum fullness indices (FIs) were 0.67% and 6.12%, respectively. The mean FI of female skates > 500 mm in total length (TL) was higher than that of males and smaller females. The most dominant prey category was crustaceans, which included shrimps, crabs, and anomurans, followed by fishes. Cephalopods were additionally observed. Dipturus chinensis fed on a wide variety of crustacean taxa, and among them, Caridea and Galatheidae were particularly common. Fishes that could be identified were mostly demersal fishes, such as Callionymidae, Triglidae, and Rajidae. Some juvenile D. chinensis were also observed, indicating that cannibalism occurs in this species. Even though dental sexual dimorphism was observed in large size classes (mainly > 500 mm TL), the diet of males and females significantly overlapped. Size-related dietary shifts from small crustaceans, such as shrimps and anomurans, to relatively fast-swimming prey, such as fishes, crabs, and cephalopods, were observed in all seasons. The composition of these prey categories varied among seasons. Shannon–Wiener diversity indices calculated for each size class ranged from 2.01 to 2.51, indicating D. chinensis are generalist predators. As size increased, trophic level increased from 3.58 to 3.85, indicating that larger specimens are at a slightly higher trophic level than smaller specimens. These results suggest that D. chinensis are middle-trophic-level generalist predators that utilize diverse prey depending on their size and the season, and thus play an influential role in the demersal ecosystem of the East China Sea.     

Heat resistance of the skeletal muscle in Western Palearctic green frogs (<Emphasis Type="Italic">Rana esculenta</Emphasis> complex)

Heat resistance of the gastrocnemius muscle was studied in five species of the Rana esculenta complex. It was similar in R. bedriagae, R. lessonae, and in the European form of R. ridibunda; while North African R. saharica demonstrated a lower heat resistance. No heterosis was expressed in R. esculenta, a clonal hybrid of R. lessonae and R. ridibunda, for the heat resistance of the muscle. Moreover, this species demonstrated low heat resistance at the highest test temperature (42°C). Comparison of diploid and triploid R. esculenta syntopically occurring in the same water bodies demonstrated no differences between them, thus, suggesting that polyploidy has no effect on this parameter at least in this case.     

Mechanisms of influence of invasive grass litter on germination and growth of coexisting species in California

In grasslands, litter has been recognized as an important factor promoting grass persistence and the suppression of forbs. The invasive European annual grass Bromus diandrus (ripgut brome) is widespread throughout California, where it produces a persistent and thick litter layer. The native grass, Stipa pulchra, is also common in some grassland settings and can also produce persistent litter, yet it is typically associated with more forbs. Very little is known about the mechanisms through which these two common grass species influence seedling establishment of both exotic invasive and native herbs. Here, we evaluated the effect of B. diandrus and S. pulchra litter on seedling establishment of two invasive (the grass B. diandrus and the forb Centaurea melitensis) and two native (the grass S. pulchra, and the forb Clarkia purpurea) herbaceous plants in a greenhouse setting. Our results showed that B. diandrus litter cover hindered seedling establishment of the four species tested, but that the degree and mechanism of inhibition was dependent on which species was tested, life form (e.g. monocot/dicot) and seed size. Seedling emergence of the two forb species was more vulnerable to litter cover than either grass species and both forbs had smaller seed size. After germination, only seedling biomass of B. diandrus itself was reduced by litter (both B. diandrus and S. pulchra). We found no significant effects of leachate of either grass species on seedling emergence of any species, while a high concentration of B. diandrus leachates inhibited root growth of all species including B. diandrus seedlings. Stipa pulchra litter leachates did not affect S. pulchra or C. melitensis seedlings although it did suppress B. diandrus and C. purpurea seedling growth. Our findings provide direct experimental evidence for the mechanism of effect of litter on these coexisting invasive and native species. Such evidence helps advance our understanding of role of B. diandrus and S. pulchra litter in California grassland.     

Ammonites of Tethyan origin from the Ryazanian of the Russian platform: Genus <Emphasis Type="Italic">Riasanites</Emphasis> spath

The genus Riasanites, represented in Central Russia by two successive dimorphic species, is revised. R. swistowianus is found in the basal beds of the rjasanensis Zone. Its descendant R. rjasanensis is also found in this zone, but upwards in the section, including the beds with Surites spasskensis and Externiceras solowaticum. The representatives of Riasanites from the Crimea and Northern Caucasus are assigned to two species, R. crassicostatus and R. maikopensis, respectively. It is suggested that Riasanites evolved from Sub-Mediterranean Himalayatidae, which migrated from the Western Tethys via the Polish Passage into the Central Russian Basin, and from there to Mangyshlak, the Northern Caucasus, and the Crimea.     

Multigene phylogeny reveals a new species and novel records and hosts in the genus <Emphasis Type="Italic">Ramularia</Emphasis> from Iran

Ramularia is a species-rich genus in the order Capnodiales (Dothideomycetes, Ascomycota) that includes numerous phytopathogenic taxa, several of which are economically important plant pathogens. In this study, six isolates of Ramularia were recovered from leaf spot symptoms on six herbaceous and woody plants from Guilan, East and West Azarbaijan provinces in the north and northwest of Iran. The isolates were studied by a polyphasic approach involving morphological and cultural data, and multi-gene phylogeny (ITS, TEF1-α, ACT, HIS, RPB2 and GAPDH). The isolates were grouped in three species clades of the R. eucalypti species complex. Of these, R. mali is recorded for the first time in Asia and R. glennii represents a new record for the mycobiota of Iran. Ramularia taleshina on Alnus subcordata is described as a new species. Ramularia taleshina is phylogenetically related to R. mali, but they can be differentiated by morphological and cultural characters as well as molecular data. Acalypha australis, Ficus carica and Platanus sp. are reported as new hosts of R. glennii, and Prunus cerasus and Vitis vinifera as new hosts of R. mali.     

New and interesting chaetothyrialean fungi from Spain

In the course of taxonomic studies on saprobic microfungi from Spain, several slow-growing, dematiaceous hyphomycetes were isolated from soil, submerged plant material and river sediments. Sixteen of these strains were identified as members of the ascomycete order Chaetothyriales on the basis of morphology and DNA sequence analyses of the internal transcribed spacer region and partial large subunit ribosomal RNA gene. These included three novel species (Cladophialophora pseudocarrionii, Cyphellophora chlamydospora, and Rhinocladiella amoena) and five interesting, little-known or clinically-relevant species (Cyphellophora suttonii, Exophiala aquamarina, E. lacus, E. radicis, and Rhinocladiella similis). In addition, Exophiala oligosperma, an emerging opportunistic fungus, was found for the first time in an aquatic freshwater environment (river sediments). Cladophialophora pseudocarrionii resembles C. carrionii in the branching pattern of its conidial chains, but differs from the latter species in its inability to grow at 30 °C. Cyphellophora chlamydospora differs from other species of the genus in the absence of conidiation, producing only chlamydospores in vitro. Rhinocladiella amoena shows branched conidiophores similar to those of R. anceps, R. atrovirens, R. basitona and R. similis, but differs from them in conidial shape and size. The ex-type strain of Phialophora livistonae, included in the phylogenetic study, clustered with high statistical support with members of the genus Cyphellophora and is transferred to this genus.     

Disentangling the effects of isolation-by-distance and isolation-by-environment on genetic differentiation among <Emphasis Type="Italic">Rhododendron</Emphasis> lineages in the subgenus <Emphasis Type="Italic">Tsutsusi</Emphasis>

Ecological speciation has long been noted as a central topic in the field of evolutionary biology, and investigation into the relative importance of ecological and geographical factors is becoming increasingly emphasized. We surveyed genetic variation of 277 samples from 25 populations of nine Rhododendron species within Tsutsusi subgenus in Taiwan using simple sequence repeats of expressed sequence tags. Bayesian clustering revealed four genetic lineages: (1) the Rhododendron simsii, Rhododendron kanehirai, and Rhododendron nakaharae lineage (lineage 1); (2) the Rhododendron longiperulatum, Rhododendron breviperulatum, and Rhododendron noriakianum lineage (lineage 2); (3) the Rhododendron rubropilosum lineage (lineage 3); and (4) the Rhododendron oldhamii lineage (lineage 4). Asymmetric introgressions were found from lineage 3 into lineages 1 and 2 (introgressed lineages). Genetic admixture of non-R. oldhamii species was also revealed by a neighbor-joining tree. Variation partitioning showed that environment explained much larger portions of genetic variation than geography between non-introgressed lineages (i.e., between R. oldhamii and other lineages). However, the Mantel and partial Mantel tests and the multiple matrix regression with randomization found that isolation-by-distance played a more important role than isolation-by-environment (IBE) in contributing to genetic variation in most between lineage comparisons. Nevertheless, strong IBE was found when compared between non-introgressed lineages of R. oldhamii and R. rubropilosum, suggesting post-speciation ecological divergence. Several environmental variables, including annual mean temperature, aspect, isothermality, seasonal precipitation, slope, and soil pH, could be important ecological drivers involved in reproductive isolation between R. oldhamii and non-R. oldhamii species within the Tsutsusi subgenus.     

The diversity of culturable yeasts in the phylloplane of rice in Thailand

One hundred and fifty-six yeast strains were obtained by the enrichment technique from the phylloplanes of 85 rice leaf samples collected from seven provinces in Thailand. On the basis of the D1/D2 domain of the large subunit rRNA gene sequence analysis, 156 strains were identified as 34 known species in 18 genera consisting of 25 species in 13 genera of the phylum Ascomycota and nine species in five genera of the phylum Basidiomycota. The species in the phylum Ascomycota comprised 24 species in 12 genera of the order Saccharomycetales and one species viz. Yarrowia lipolytica in Saccharomycetales incertae sedis. The 24 species viz. Candida glabrata in the Nakaseomyces clade of Saccharomycetaceae, Candida jaroonii, Candida membranifaciens and Candida terebra in the Yamadazyma clade of Debaryomycetaceae, Candida pseudolambica in the Pichia clade of Pichiaceae, Candida ruelliae in the Metschnikowia clade of Metschnikowiaceae, and three unaffiliated clade Candida species (Candida catenulata, Candida rugosa and Candida tropicalis); Clavispora lusitaniae, Kodamaea ohmeri, Metschnikowia koreensis and Metschnikowia lopburiensis in Metschnikowiaceae; Cyberlindnera fabianii, Cyberlindnera rhodanensis and Wickerhamomyces ciferrii in Wickerhamomycetaceae; Debaryomyces nepalensis, Meyerozyma caribbica, Meyerozyma guilliermondii, Millerozyma koratensis, and Yamadazyma mexicanum in Debaryomycetaceae; Pichia kudriavzevii in Pichiaceae; and Lachancea thermotolerans in Saccharomycetaceae. The species in Basidiomycota viz. Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus aff. laurentii and Cryptococcus rajasthanensis in the Tremellales lineage, Bulleromyces clade, Tremellales, Tremellomycetes, Agaricomycotina; Pseudozyma antarctica and Pseudozyma aphidis in Ustilaginales, Ustilaginomycetes, Ustilaginomycotina; Rhodotorula taiwanensis and Sporobolomyces blumeae in Sporidiobolales, Microbotryomycetes, Pucciniomycotina; and Trichosporon asahii in Trichosporonales, Tremellomycetes, Agaricomycotina. The most prevalent species was R. taiwanensis with a 23 % frequency of occurrence followed by Candida tropicalis (16 %) and Cryptococcus fabianii (12 %).     

Gall midges of the supertribe stomatosematidi (Diptera,Cecidomyiidae) in the Palaearctic region,with descriptions of new taxa from the late Eocene Rovno Amber. 2. The genera <Emphasis Type="Italic">Didactylomyia</Emphasis> and <Emphasis Type="Italic">Rovnodidactylomyia</Emphasis> gen. n.

One new genus (Rovnodidactylomyia Fedotova et Perkovsky, gen. n.) and four new species (Didactylomyia dlusskyi sp. n., Rovnodidactylomyia zosimovichi gen. n. et sp. n., R. sidorenkoi sp. n., and R. iconica sp. n.) are described from an amber sample of the late Eocene Period (Rovno District, Ukraine). Two species from a Baltic amber sample of the late Eocene Period, described earlier in the genus Bryocrypta, are attributed to the genus Rovnodidactylomyia (R. girafa (Meunier, 1904) comb. n. and R. capitosa (Meunier, 1904)). The first fossil species of the genus Didactylomyia is described for the first time; two contemporary Palaearctic species are recorded. Keys to the species of the genus Didactylomyia are given.     

Lepidoptera Papilionoidea communities as a sentinel of biodiversity threat: the case of wild boar rooting in a Mediterranean habitat

Butterfly communities can be modified by the activities of large mammals. The wild boar (Sus scrofa) is one of the most common mammals in Europe and has notably expanded its distribution range in recent decades. The present work aimed to investigate the possible effect of wild boar rooting activity on butterflies in olive groves inside the “Cilento, Vallo di Diano and Alburni” National Park. Butterflies were surveyed in five Rooted (R) and five Control areas (C). Transects were made from April to September in 2011 and 2012. Forty-three species and 3659 individuals were found, of which 39 species and 2426 individuals were detected in C areas and 31 species and 1233 individuals in R areas. A community-level approach showed a significantly higher abundance, richness and Shannon–Wiener index in C than in R. An ecological-level approach indicated that more specialised butterflies were significantly more affected by wild boar activity. Finally, the response of single species was investigated: two species (Colias croceus and Polyommatus icarus) were significantly more represented in R, whereas six species (Aricia agestis, Hipparchia statilinus, Lasiommata megera, Melanargia arge, Pyronia cecilia and Thymelicus acteon) were significantly more abundant in C. Three species considered at risk (M. arge; Annex II of Directive 92/43/EEC; H. statilinus and T. acteon: European Red List) were less represented in rooted areas. Overall, the intensive wild boar activity negatively correlated with butterflies in the olive groves studied.     

Glyceriformia (Annelida) from the deep sea of the Atlantic sector of the Southern Ocean

The faunas living in the vast deep sea regions around the Antarctic are very poorly known. This is especially true for the biodiversity of polychaetes inhabiting these remote areas. Therefore, we report new morphological data of Glyceriformia from the ANDEEP cruises to the South Atlantic Ocean and the Southern Ocean. Based on benthos samples from three expeditions aboard R/V POLARSTERN, two species of Glyceridae (Glycera capitata, G. diva) and four species of Goniadidae (Bathyglycinde sibogana, B. stepaniantsae, Goniada maculata, Progoniada regularis) were studied. Furthermore, new morphological details (especially for the previously unknown tail) for the rarely found taxon B. stepaniantsae are given. The distribution patterns of the different taxa demonstrated that some species have a high dispersal capability and show an extended level of eurybathy, whereas other species are restricted to the deep sea.     

A conserved repetitive DNA element located in the centromeres of chromosomes in <Emphasis Type="Italic">Medicago</Emphasis> genus

In most eukaryotic species, centromeres harbor large arrays of tandem repeated satellite DNA sequences. In this study, we report on the genomic distribution of a centromere satellite repeat “MtR3” in Medicago genus and three distantly related genera. Fluorescence in situ hybridization (FISH) results showed MtR3 repeats were detected in the centromere regions in M. truncatula, M. minima, M. edgeworthii, M. ruthenica, M. caerulea, M. sativa, and M. falcata (4×), but no signals were discovered in M. lupulina, M. polymorpha, and M. falcata (2×), Melilotus officinalis, Crotalaria medicaginea, and Trifolium repens. However, sequence analysis showed this MtR3 DNA had genomic distribution in all species and was highly conserved across the entire Medicago genus and three other genera. The conservation and widespread presence suggested MtR3 repeats may play important roles in centromeric function.     

Relation to enterocins of variable <Emphasis Type="Italic">Aeromonas</Emphasis> species isolated from trouts of Slovakian aquatic sources and detected by MALDI-TOF mass spectrometry

Aeromonads represent bacteria thought to be primarily mostly autochthonous to aquatic environments. This study was focused on the relation with antibiotics and enterocins of identified Aeromonas species isolated from the intestine of trouts living in Slovakian aquatic sources. Intestinal samples from 50 trouts (3 Salmo trutta and 47 Salmo gairdnerii) were collected in April of years 2007, 2010, and 2015 from trouts of different water sources in Slovakia (pond Bukovec near Ko?ice, river ?ierny Váh). Due to the MALDI-TOF mass spectrometry evaluation, 25 strains were proposed to the genus Aeromonas involving nine different species (Aeromonas bestiarum—nine strains, Aer. salmonicida—four strains, Aer. encheleia, Aer. eucrenophila, Aer. molluscorum, Aer. media, Aer. sobria, Aer. popoffii, Aer. veronii). Phenotypic evaluation of individual strains confirmed their species identification. Twenty-five strains of different Aeromonas species were sensitive to azithromycin, amikacin, mecillinam, mezlocillin, piperacillin, gentamicin, chloramphenicol, and tetracycline. On the other side, they were resistant to carbenicillin and ticarcillin. The growth of Aer. bestiarum R41/1 was inhibited by treatment with Ent M and Ent 2019 (inhibition activity 100 AU/mL). Aer. bestiarum R47/3 was inhibited by eight enterocins (100 AU/mL). It is the first study testing enterocins to inhibit the growth of Aeromonas species from trouts.     

Tracking the impact of drought on functionally different woody plants in a Mediterranean scrubland ecosystem

Climate warming is predicted to amplify drought stress. Thus, it is important to understand how coexisting plant species respond to severe droughts. Here we study how seven Mediterranean woody plant species with different evolutionary history and functional characteristics (Pinus halepensis Mill., Juniperus phoenicea L., Pistacia lentiscus L., Rhamnus lycioides L., Rosmarinus officinalis L., Genista scorpius (L.) DC., and Globularia alypum L.) responded to a severe winter drought during 2011–2012 in Spain. The study site is located in the Valcuerna valley, Monegros desert, northeastern Spain. We evaluated how the drought affected the annual growth-ring formation of the species by using dendrochronology and quantified the intensity of drought-induced defoliation and mortality and compared it between species and groups of species with different evolutionary history. Radial growth of all species was strongly reduced by the 2012 drought. The pre-Mediterranean species (P. halepensis, J. phoenicea, P. lentiscus and R. lycioides) reduced growth more than the Mediterranean species (R. officinalis, G. scorpius and G. alypum). Defoliation was significantly higher in pre-Mediterranean than in Mediterranean species. When species were analyzed separately we found that P. halepensis was the species with the highest growth reduction but J. phoenicea was defoliated more severely and showed higher mortality rates as a consequence of drought. In the case of the Mediterranean shrubs, drought-induced mortality was only noticeable in R. officinalis. Drought impacted growth of all species but this did not induce mortality in all of them. Growth reduction was dependent on evolutionary history. However, functional characteristics of the species such as leaf stomatal regulation and root architecture may be more important than evolutionary history on explaining drought-induced mortality. Indeed, species with shallow root systems such as J. phoenicea and R. officinalis were the most adversely affected by the drought.