Forgotten in the taxonomic literature: resurrection of the scleractinian coral genus Sclerophyllia (Scleractinia,Lobophylliidae) from the Arabian Peninsula and its phylogenetic relationships

Ciliates (Protista) have a complex species structure, which means that in several genera the morphological species are differentiated into cryptic species with isolated gene pools (called syngens). Problems of speciation are well known in the genus Paramecium, and especially in the P. aurelia sibling (cryptic) species complex within it. However, the problem of the existence of such species within P. jenningsi was, until recently, still unsolved. Here we present the results of studies based on an analysis of 16 loci (both nuclear and mitochondrial), strain crosses and cytological preparations. The obtained data allowed us not only to study relationships of the P. jenningsi complex and other morphospecies within the Paramecium subgenus, but also to confirm the existence of three isolated reproductive groups within the former P. jenningsi and to propose binominal names for each of them: P. primjenningsi, P. bijenningsi, and P. trijenningsi. In our view, the studied species meet the criteria of a species complex because they can be differentiated based on strain crosses and molecular characteristics, but they cannot be differentiated based on morphological features alone.

http://zoobank.org/urn:lsid:zoobank.org:pub:9209CD4C-A255-4A33-BFC1-CE336244C200     

The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas,USA)

P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5′LSU rDNA, COI, CytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and P. schewiakoffi than to the other members of the P. aurelia complex.     

Developmental success and reproductive incompatibility among populations of the European red mite,Panonychus ulmi (Acari: Tetranychidae)

Developmental success on 47 species of plant and reproductive compatibility were studied for four strains of the European red mite,Panonychus ulmi (Koch), collected from dwarf bamboo, elm and apple trees. The host range was variable in those strains and there was no plant species on which all four strains were able to reach maturity.Intra-populational crosses in the dwarf-bamboo strain and the two apple strains of mites, as well as crosses between the two apple strains, gave both female and male progeny. Inter-populational crosses between the dwarf-bamboo and apple strains produced only male progeny, as in the case where females of each strain were kept virgin, indicating that these two were reproductively incompatible and had an arrhenotokous reproductive system. When females of the elm strain were crossed with males of the elm, dwarf-bamboo or apple strain, no males were produced, as in the case for virgin females of the elm strain which had a thelytokous reproductive system. It is thus concluded that the strains derived from different host species were reproductively incompatible with one another.     

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.     

Global molecular variation of Paramecium jenningsi complex (Ciliophora,Protista): a starting point for further,detailed biogeography surveys

The world’s second stand of P. primjenningsi (Paramecium jenningsi complex, Ciliophora, Protista), heretofore known only from India, has been revealed in Ethiopia (Africa). This finding has enlarged the range of this cryptic species and was a trigger to re-analyse the distribution of all members of the complex (known from ～20 tropical locations). The current survey is an initial one, where, based on haplotype networks, a detailed analysis of the relationship within the P. jenningsi complex has been performed. Although the V4 hypervariable fragment of the SSU rDNA gene is widely used as a first-step barcode marker for microbial HTS analyses, it has provided inconclusive results based on the dataset investigated. However, the ITS1-5.8S-ITS2-5’rDNA and COI mtDNA fragments indicate the possibility of the delimitation of the cryptic species of P. jenningsi (which is crucial from the point of view of metabarcoding surveys). We suppose that future sampling of unexplored, tropical regions will certainly change our knowledge about Paramecium biodiversity and biogeography. This sampling will probably rely on the integration of metabarcodes from environmental DNA studies, with molecular data obtained from identified representatives of particular cryptic species.     

Random amplified polymorphic DNA fingerprinting as a marker for Paramecium jenningsi strains

The aim of the present study is to establish a common RAPD marker for P. jenningsi using a series of Ro primers and to investigate if strains originating from distant and isolated localities (Japan, China, India, Saudi Arabia) have isolated gene pools and represent distinct species. An analysis of dendrograms constructed on the basis of RAPD-PCR fingerprints with four primers (Ro 460-04, 460-06, 460-07, and 460-10) from the first part of this project (SKOTARCZAK et al. 2004), assigns the strains to two groups consisting of the continental strains (India, Saudi Arabia, China) and Japanese strains that have been considered as a separate sibling species within P. jenningsi. The genetic similarity of the Indian and Arabian strains was ascertained, whereas the Chinese strain formed an independent branch in this sibling species. The primers Ro (460-01,460-02, 460-03, 460-05, 460-08) also distinguish between two groups of strains, although they divide the Japanese strains into two subgroups that are not reproductively isolated. This probably indicates genetic variation within this sibling species. However, it comprises one common gene pool (successful inter-strain crosses) and is reproductively isolated from the other sibling species. The results presented in these papers confirm that the construction of ten band patterns having marker attributes is possible on the basis of DNA amplification from 9 strains of P. jenningsi with the RAPD-PCR fingerprinting method using five primers from the Ro series. The patterns can be assigned to three marker-groups: a general species group, a group differentiating between sibling species, and accessory strain markers.     

Reproductive isolation among sympatric cryptic species in marine diatoms

Pseudo-nitzschia is a marine cosmopolitan genus of chain-forming planktonic diatoms. As for the vast majority of phytoplankton organisms, species identification within this genus mostly relies upon morphological features. Taxa were initially identified based on cell shape and gross morphology of their composite silica cell wall, called the frustule. Yet, observations of the frustule in electron microscopy showed many additional characters for species identification and results of molecular studies have demonstrated that genetically distinct groups might exist within morpho-species. However, these studies have not addressed the biological meaning of these genetic differences. Here, we bridge that gap by comparing ultrastructural features and sequence data (three ribosomal and one plastid marker) of 95 strains with results of mating experiments among these strains. Experiments were performed on two morphologically distinct entities: P. delicatissima and P. pseudodelicatissima. Each of the two entities consisted of multiple genetically distinct and reproductively isolated taxa, all occurring in sympatry: P. delicatissima was composed of three phylogenetic and reproductively distinct groups, whereas P. pseudodelicatissima consisted of up to five. Once these taxa had been defined both genetically and biologically, subtle ultrastructural differences could be detected as well. Our findings not only show that cryptic genetic variants abound in sympatry, but also that they are reproductively isolated and, therefore, biologically distinct units.     

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish

Aims: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. Methods and Results: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR‐based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and repetitive extragenic palindromic‐PCR (REP‐PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC‐PCR and REP‐PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15–20% of the isolates. Conclusions: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC‐PCR and REP‐PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. Significance and Impact of the Study: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2–3 years). The discrimination at strain level can be useful to study the traceability of infections.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

     

Scope for genetic enhancement of the parasitisation potential of four native strains of Trichogrammatoidea sp. nr. lutea Girault (Hymenoptera: Trichogrammatidae) in Kenya

In response to emerging interest in commercial mass production of Trichogramma for Helicoverpa armigera biocontrol in eastern Africa, laboratory experiments were undertaken to assess the scope for genetic enhancement of the parasitisation potential of native strains of the local common trichogrammatid species, Trichogrammatoidea sp. nr. lutea. Four promising strains (ex-Kilifi – Kilifi District, ex-Kwa Chai – Kibwezi District, ex-Rarieda – Bondo District and ex-Ebuhayi, Kakamega District) were tested for cross-mating in reciprocal combinations with focus on fecundity and progeny female ratio. While all the crosses resulted in F1 progeny of both sexes, significant differences were observed between homogamic and reciprocal heterogamic crosses in fecundity, progeny production, proportion of female progeny and adult longevity. Among all the crosses, the cross between ex-Rarieda strain females and ex-Kilifi strain males resulted in progeny that was significantly superior in fecundity and progeny female ratio. Conversely, Kilifi strain females crossed to males from ex-Rarieda strain gave rise to progeny with relatively low fecundity and female ratio. There were significant differences between homogamic crosses and most reciprocal heterogamic crosses in the major biological attributes. Genotypic and phenotypic variance-covariance matrices generated for six life-history traits showed high positive correlations for most traits in both inbred (P<0.05) and reciprocal heterogamic crosses (P<0.05 and P<0.001). Fecundity and number of female offspring were the most important factors in the heterogamic crosses. The results confirmed the scope for genetic enhancement through inter-strain crossing for improving the field impact potential of T. sp. nr. lutea being targeted for commercial mass production.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Intraspecies Variability in the Esterases and Acid Phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of Unidentified Paramecia; Comparison with the P. aurelia Complex1

ABSTRACT. Enzyme electrophoresis was exploited to identify stocks of paramecia previously not identified to particular species. Stocks collected in India and one from Panama belong to Paramecium jenningsi, while others collected in Panama or in Brazil are assignable to syngen 2 of P. multimicronucleatum on the basis of similarity of their esterase and acid phosphatase phenotypes. Inclusion of these doubled the numbers of stocks available in the two species, thereby facilitating examination of intraspecies variation and comparison of particular features of intraspecies variation found for the P. aurelia complex. Variant stocks were observed in P. jenningsi and in syngens 2, 3, and 4 of P. multimicronucleatum. In some cases the variant lacked the enzyme; in others, a change in mobility of the enzyme occurred that resulted in an electrophoretic form similar to one common in another species. Unique phenotypes were displayed by the variants of syngen 2 in P. multimicronucleatum. Hypervariability for Esterase B was observed in this syngen, where, in addition, several subtypes were seen for three other esterases. Unique phenotypes and hypervariability were also noted in P. biaurelia. Clustered variations were observed in these species and in the P. aurelia species. Unlike the situation for members of the aurelia complex, where lack of geographical differentiation between stocks in the same species is a unique feature, some such differentiation does occur in P. multimicronucleatum-2. The frequency of variant stocks in P. jenningsi was similar to that observed in the aurelia sibling species. In contrast, a significantly higher frequency of variant stocks was found in syngens 2, 3, and 4 of P. multimicronucleatum.     

Reproductive Isolation and Genetic Variation between Two “Strains” ofBracon hebetor(Hymenoptera: Braconidae)

Bracon hebetorSay(Hymenoptera: Braconidae) is known primarily as a parasitoid of pyralid moth larvae infesting stored grain. In the 1970s, a parasitoid identified asB. hebetorwas released for control ofHeliothis/Helicoverpaspp. (Lepidoptera: Noctuidae) on the island of Barbados. Because life-history traits of this parasitoid differed from those reported forB. hebetorfrom the United States, we conducted a series of laboratory experiments to determine whether this parasitoid was (i) a population ofB. hebetorthat attacks noctuids in the field or (ii) a different species fromB. hebetor.We confirmed thatHeliothis virescens(F.) was a more suitable host for the Barbados strain than forB. hebetor.However, a stored-grain infesting pyralid,Plodia interpunctella(Hübner), was a more suitable host for the Barbados strain than wasH. virescens.Reciprocal crosses between the Barbados strain andB. hebetorshowed that the two populations were reproductively isolated. No mating was observed during a series of 30-min observations of reciprocal crosses, and the crosses produced only male offspring. Examination of each female's spermatheca confirmed that females were not fertilized. Sequence analysis of a 517-bp fragment of the mitochondrial 16S rRNA gene revealed that two populations ofB. hebetorfrom our laboratory were identical but differed in sequence by 2% from the Barbados strain. Collectively, our results indicate that the Barbados strain is a distinct species fromB. hebetor.     

Two differentially regulated nitrate reductases required for nitrate-dependent,microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O₂, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NR_I) and 200 kDa (NR_II). NR_I and NR_II were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO₃. However, only NR_I was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O₂ tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NR_I, while the other two mutants, GRF3 and GRF4, expressed mostly NR_II. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NR_I) or by both microaerobiosis and nitrate (NR_II).Abbreviations NR Nitrate reductase - M _r Relative molecular mass - PMSF Phenylmethylsulfonyl fluoride     

Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

Neurospora crassa Cytoplasmic Ribosomes: Isolation and Characterization of a Cold-Sensitive Mutant Defective in Ribosome Biosynthesis

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.     

Reproductive compatibility among Mexican populations of Anastrepha obliqua: theoretical and management implications

The fraterculus species group, composed of 34 species in the genus Anastrepha (Diptera: Tephritidae), includes the fraterculus cryptic species complex formed by eight reproductively isolated morphotypes. A previous study revealed six genetic mitochondrial types of Anastrepha obliqua, suggesting the existence of a second cryptic species complex. However, marked discrepancies between nuclear and mitochondrial loci rather suggest incomplete lineage sorting or introgression between A. obliqua and A. fraterculus. Such hybridization could nevertheless result in reproductive isolation, an outcome that could affect efficacy of area‐wide management for the most important pest of mangos in America. Two mitochondrial types occur in Mexico, and the limits of a third one, encompassing Central American populations, have not been clearly established. Here, we tested reproductive compatibility among three A. obliqua populations from the Pacific and a population from the Gulf of Mexico. No evidence of pre‐zygotic isolation was found. Flies from the Atlantic mated randomly for equal duration with individuals from three Pacific populations. Homotypic and heterotypic crosses produced similar numbers of eggs, with heterotypic crosses of Pacific males and Atlantic females hatching in lower proportions. Larvae of all cross types developed equally in mangos and exhibited no sex ratio distortion of hybrid F1. The three mitochondrial types identified in Mexico and Central America do not appear to be cryptic species and can be managed using one single strain for the sterile insect technique.     

Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi: hybridization of aggressive and non-aggressive strains

When British isolates of Ceratocystis ulmi were surveyed for compatibility type, both A- and B-types were found in the non-aggressive strain, but only the B-type in the aggressive strain. Single ascospore progeny from crosses between compatible aggressive and non-aggressive isolates showed a near-normal growth rate distribution, with a mean lying between the parents. Many grew either faster than the aggressive or slower than the non-aggressive parent. The progeny were highly variable in culture morphology and could not be classified in terms of the parental types. When inoculated into English elm they showed a marked skewness towards low pathogenicity. None approached the aggressive strain in pathogenicity. It is concluded that the above characters are under polygenic control, and that the aggressive strain could not arise from the non-aggressive by a simple mutation. The results suggest that the two strains may be reproductively isolated.     

Morphological and molecular investigations of Paramecium schewiakoffi sp. nov. (Ciliophora, Oligohymenophorea) and current status of distribution and taxonomy of Paramecium spp.

Paramecium schewiakoffi sp. nov. is described from a pond in Shanghai, China. It is a freshwater species belonging to the “aurelia” subgroup of the genus. It is of similar size and shape to P. jenningsi, but has a single large micronucleus of the “chromosomal” morphological type, while P. jenningsi has two smaller micronuclei. The general morphology, morphometric characteristics and nuclear reorganization pattern, a random amplified polymorphic DNA (RAPD) fingerprint pattern, and the small subunit rRNA gene sequence are presented for the species. Comparison of P. schewiakoffi with the other species of Paramecium indicates that it is a valid new species of the genus. Geographical locations reported for many Paramecium species do not support the theory that all ciliates have a cosmopolitan distribution. It is proposed that, in an extension of Jankowski's earlier suggestion, the genus Paramecium should be subdivided into four subgenera: Chloroparamecium, Helianter, Cypriostomum and Paramecium, on the basis of morphometric, biological and molecular differences.