Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Molecular Phylogeny of Mobilid and Sessilid Ciliates Symbiotic in Eastern Pacific Limpets (Mollusca: Patellogastropoda)

The phylogenetic relationships of the ciliate subclass Peritrichia, composed of the orders Mobilida and Sessilida, have recently come under debate as morphological and molecular analyses have struck contrasting conclusions as to the monophyly of the group. We provide additional molecular data to assess the monophyly of the Peritrichia by sequencing the small subunit ribosomal RNA genes of two symbiotic peritrichs, Urceolaria korschelti and Scyphidia ubiquita, found inhabiting the mantle cavity of limpets. Although phylogenetic analyses indicated a nonmonophyletic Peritrichia, approximately unbiased tests revealed that the monophyletic hypothesis could not be rejected. With regard to the Mobilida, our analysis showed divergence within the family Trichodinidae related to host taxa—a molluscan clade and a fish clade. For the Sessilida, the family Scyphidiidae was sister to the Astylozoidae. In our sampling of U. korschelti and S. ubiquita, both species showed significant genetic divergence among geographically isolated, yet morphologically indistinguishable populations. We hypothesize that cryptic speciation has produced these morphologically identical species and argue that more extensive genomic analyses are required to fully assess the monophyly, biogeography, and ultimately biodiversity of the peritrichs.     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.     

Local adaptation in populations of a Brachionus group plicatilis cryptic species inhabiting three deep crater lakes in Central Mexico

1. Salinity is a strong selective force for many aquatic organisms, affecting both ecological and evolutionary processes. Most of our knowledge on the effects of salinity on rotifers in the Brachionus plicatilis species complex is based mainly on populations from waterbodies that experience broad environmental changes both seasonally and annually. We tested the hypothesis that, despite the supposedly high potential for gene flow among rotifers inhabiting neighbouring environments, constant salinity has promoted local adaptation, genetic population divergence and even cryptic speciation in B. plicatilis complex populations from three deep maar lakes of distinct salinities [1.1, 6.5 and 9.0 g L^?1 total dissolved solids (TDS)] in Central Mexico. 2. To look for local adaptation, we performed common garden experiments to test the effect of different salinities on population density and intrinsic growth rate (r). Then, we evaluated the genetic divergence by sequencing the cytochrome c oxidase subunit I (COI) gene and performed reproductive trials to assess the potential gene flow among the three populations and with other closely related B. plicatilis complex species. 3. We confirmed that the rotifer populations have phenotypic plasticity in tolerance of salinity, but only rotifers from the least saline lake are adapted to low salinity. Among the populations, sequence divergence at COI was very low (just a single haplotype was found), suggesting a persistent founder effect from a relatively recent single colonisation event and a subsequent dispersal from one lake to the others, and a very restricted immigration rate. In the phylogenetic analysis, rotifers from this area of Mexico clustered in the same clade with the middle‐sized species Brachionus ibericus and B. sp. ‘Almenara’. Mexican rotifers showed successful recognition, copulation and formation of hybrids among them, but interpopulation breeding with the Spanish B. ibericus and B. sp. ‘Almenara’ was unsuccessful. 4. We conclude that the B. plicatilis complex populations from these three lakes belong to a new biological species not yet described (presently named B. sp. ‘Mexico’). To our knowledge, this is the first report of local adaptation of a natural B. plicatilis complex population living in freshwater conditions (1.1 g L^?1 TDS).     

Chemical tools to distinguish the fire ant species Solenopsis invicta and S. saevissima (Formicidae: Myrmicinae) in Southeast Brazil

Morphologically similar fire ants Solenopsis invicta and Solenopsis saevissima are broadly sympatric in southeastern Brazil. Chemistry from venom (2,6-dialkyl piperidine alkaloids) and cuticular hydrocarbons have been reported as potentially important tools for differentiating Solenopsis species. We have analysed two chemical classes in widely separated populations of S. invicta and S. saevissima and find that both piperidine alkaloids and cuticular hydrocarbons separate the two species. Piperidine alkaloids clustered S. invicta but not S. saevissima. Cuticular hydrocarbons strongly clustered both S. invicta and S. saevissima. One population morphologically identified as S. invicta presented piperidine alkaloids and cuticular hydrocarbons markedly different from either species. The distinctive piperidine alkaloid differences among populations of S. saevissima and the marked difference in piperidine alkaloid and hydrocarbon profiles of the anomalous population of S. invicta suggest undescribed species fire ant in southeastern Brazil.     

Ultrastructural Features of Three Ectocommensal Protozoa Attached to the Gills of the Red Swamp Crawfish,Procambarus clarkii (Crustacea: Decapoda)1

Because heavy branchial infestations are thought to interfere with respiration, we examined the attachment of three stalked ciliates commonly found in the branchial chambers of Louisiana crawfish. Attachments by Cothurnia sp., Epistylis sp. and Acineta sp. differ in their fine structure. Stalks of the peritrichous ciliates. Cothurnia and Epistylis, contain striated tubules that differ in their arrangement, diameter, and in the periodicity of their striations. In both species the striated tubules branch within the basal disk and attach to a pad of adhesive material secreted by the organism during initial attachment to the gill surface. The stalk of the suctorian Acineta is composed of a striated honeycomb-like matrix. Within the basal disc the matrix is disorganized; however, striated elements anchor the stalk to a pad of adhesive material. Attachment sites also differ in the amount of secretory material deposited. Cothurnia forms a multi-layered, granular pad; Epistylis forms an indistinct, microfibrillar layer, and Acineta deposits a thick mucoid pad. None of the ciliates appear to damage the gill epicuticle nor is there an obvious host response. Harmful effects are probably limited to a decrease in respiratory surface area and disruption of normal water flow patterns. This may impair respiration sufficiently to increase the susceptibility of crawfish to low dissolved oxygen concentrations encountered periodically in commercial crawfish ponds.     

Intra‐specific variability in Rhinoleucophenga punctulata populations (Diptera: Drosophilidae) from Neotropical biomes: Combined analyses of morphological and molecular data

Despite the fact that Drosophilidae is a very diverse and well‐studied taxon, the New World genus Rhinoleucophenga is yet poorly understood even in regard to species distribution and morphological variability pattern. In this sense, R. punctulata is a species widely distributed in the Neotropical region. Specimens of R. punctulata were collected from different biomes in Brazil: Pampa, Cerrado and Caatinga sensu strictu, and a southern Amazonian savannah enclave area. Geographical variations in the external body morphology and in the morphology of spermatheca were noticed among the different populations. The hypothesis that each population could be a different species was tested through molecular data. A fragment of the mitochondrial cytochrome c oxydase subunit I (COI) gene was sequenced to perform phylogenetic analyses through neighbor‐joining and Bayesian inferences. Pairwise genetic divergences of COI sequences were calculated using DNA barcode premises. The analyzed populations presented different variation levels in both morphology and molecular traits. However, new species were not proposed because the intra‐population nucleotide variations exceeded the inter‐population ones. The noticeable morphological and genetic variations revealed among the four studied populations of R. punctulata in different biomes of Brazil suggest the necessity that morphological, distributional and molecular data at the population level should be integrated into complementary datasets to better understand the biological diversity of Rhinoleucophenga through Neotropical environments.     

An updated phylogeny of the redlip blenny genus Ophioblennius

An updated molecular phylogeny of the blenny genus Ophioblennius, with a focus on two geographically disjunct morphotypes observed in Brazil, is presented. The analyses showed that specimens from the north‐eastern Brazilian coast are the endemic redlip blenny Ophioblennius trinitatis, but specimens from the southern Brazilian coast are conspecific to an undescribed east Atlantic Ocean (Gulf of Guinea) species, previously unknown in Brazil. Possible explanations for this geographical pattern include: natural larval dispersal and rafting across the Atlantic; an unknown ecological attribute that enabled this species to colonize southern Brazil; oil platforms as introduction vectors.     

Evolutionary history of Scinax treefrogs on land‐bridge islands in south‐eastern Brazil

Aim We investigated how Pleistocene refugia and recent (c. 12,000 years ago) sea level incursions shaped genetic differentiation in mainland and island populations of the Scinax perpusillus treefrog group. Location Brazilian Atlantic Forest, São Paulo state, south‐eastern Brazil. Methods Using mitochondrial and microsatellite loci, we examined population structure and genetic diversity in three species from the S. perpusillus group, sampled from three land‐bridge islands and five mainland populations, in order to understand the roles of Pleistocene forest fragmentation and sea level incursions on genetic differentiation. We calculated metrics of relatedness and genetic diversity to assess whether island populations exhibit signatures of genetic drift and isolation. Two of the three island populations in this study have previously been described as new species based on a combination of distinct morphological and behavioural characters, thus we used the molecular datasets to determine whether phenotypic change is consistent with genetic differentiation. Results Our analyses recovered three distinct lineages or demes composed of northern mainland São Paulo populations, southern mainland São Paulo populations, and one divergent island population. The two remaining island populations clustered with samples from adjacent mainland populations. Estimates of allelic richness were significantly lower, and estimates of relatedness were significantly higher, in island populations relative to their mainland counterparts. Main conclusions Fine‐scale genetic structure across mainland populations indicates the possible existence of local refugia within São Paulo state, underscoring the small geographic scale at which populations diverge in this species‐rich region of the Atlantic Coastal Forest. Variation in genetic signatures across the three islands indicates that the populations experienced different demographic processes after marine incursions fragmented the distribution of the S. perpusillus group. Genetic signatures of inbreeding and drift in some island populations indicate that small population sizes, coupled with strong ecological selection, may be important evolutionary forces driving speciation on land‐bridge islands.     

RFLP analysis of phylogenetic relationships and genetic variation in the genus Lycopersicon

Summary Forty single-copy, nuclear probes of known chromosomal position were used to examine restriction fragment length polymorphism in the tomato genus Lycopersion. The probes were from three libraries: one cDNA, and two genomic libraries ne genomic made with EcoRI and the other with PstI. Total DNA from 156 plants representing eight species was cut with five different restriction enzymes and scored in 198 probe-enzyme combinations. Genetic distances between accessions (populations) and species were calculated from the resultant restriction patterns and proportion of shared bands. Accessions belonging to the same species largely clustered together, confirming their current classification. However, one mountain accession, classified as L. peruvianum var. humifusum (LA2150), was sufficiently distinct from the other accessions of L. peruvianum that it may qualify as a separate species L. esculentum and L. pimpinellifolium were the least clearly differentiated, possibly reflecting introgressive hybridization, known to have been promoted by man in recent history. Dendrograms constructed from cDNA versus genomic clones were nearly identical in their general grouping of species. The dendrograms revealed two major dichotomies in the genus: one corresponding to mating behavior [self-compatible (SC) versus self-incompatible (SI) species] and the other corresponding to fruit color (red versus green-fruited species). The ratio of withinversus between-accession diversity was much lower for SC species, indicating that most of the diversity within these species exists between populations, rather than within populations. Overall, the amount of genetic variation in the SI species far exceeded that found in SC species. This result is exemplified by the fact that more genetic variation could be found within a single accession of one of the SI species (e.g., L. peruvianum) than among all accessions tested of any one of the SC species (e.g., L. esculentum or L. pimpinellifolium). Results from this study are discussed in relationship to germ plasm collection/utilization and with regard to the use of RFLPs in tomato breeding and genetics.     

Species delimitation in a range-restricted group of cascudinhos (Loricariidae: Epactionotus) supports morphological and genetic differentiation across coastal rivers of southern Brazil

Epactionotus species are known for inhabiting the rocky-bottom stretches of fast-flowing rivers in a limited geographic area along the Atlantic coast of southern Brazil. These species are endemic to single coastal river drainages (two neighbouring drainages for Epactionotus bilineatus) isolated from each other by the coastal lacustrine environments or the Atlantic Ocean. E. bilineatus is from the Maquiné and Três Forquilhas River basins, both tributaries of the Tramandaí River system, whereas E. itaimbezinho is endemic to the Mampituba River drainage and Epactionotus gracilis to the Araranguá River drainage. Recent fieldwork in the Atlantic coastal drainages of southern Brazil revealed new populations in the Urussanga, Tubarão, d'Una and Biguaçu River drainages. Iterative species delimitation using molecular data (cytochrome c oxidase subunit I) and morphology (morphometrics and meristics) was applied to evaluate species recognition of isolated populations. With regard to new data, the genus was re-diagnosed, the status of Epactionotus species/populations was re-evaluated, formerly described species were supported and population structure was recognized. As for the newly discovered populations, both morphological and molecular data strongly support the population from the Biguaçu River drainage, in Santa Catarina State, as a new species. Molecular data revealed strong per-basin population structure, which may be related to species habitat specificity and low or no dispersal among drainages.     

Isolation and characterization of microsatellite markers for Casearia sylvestris Sw. (Salicaceae), a neotropical medicinal tree

Casearia sylvestris Sw. is a widespread neotropical tree utilized in popular medicine. Recent research ranked Casearia as one of the most promising genus in the search of drugs against cancer. Despite its wide distribution and pharmacological importance, no microsatellite markers have yet been developed for this genus. In this study, we provide 10 polymorphic microsatellite loci specifically designed for C. sylvestris, used to analyse 90 individuals distributed in two populations from São Paulo state, Brazil. On average, 12.3 alleles per locus were identified, showing the ability of the markers to detect microsatellite polymorphism in this species.     

The release call of Rhaebo guttatus (Anura: Bufonidae)

The bufonid genus Rhaebo contains 13 species, Rhaebo guttatus being one of the two species that occur in Brazil. Here we describe for the first time the release call of R. guttatus from the Brazilian Amazonia. Release calls are highly variable both in the temporal and in the frequency domains. The advertisement and release calls of Rhaebo guttatus are distinguished from each other by their temporal envelope, distribution of sound energy, and calling emission patterns. The acoustic characterization of release calls in Rhaebo could be potentially informative in the taxonomy of this genus, as previously reported for members of the bufonid genus Rhinella.     

Evidence for Two Independent Lineages of Shiitake of the Americas (Lentinula boryana) Based on rDNA and β-Tubulin Gene Sequences

Portions of ribosomal RNA genes (rDNA) were sequenced from members of the genus Lentinula and were used, along with partial β-tubulin gene sequences, for phylogenetic reconstructions. The rDNA sequences of L. boryana were separated into two well-defined lineages. Lineage 1 was composed of isolates from Mexico and Costa Rica while lineage 2 encompassed isolates from the United States, Venezuela, and Brazil. The two South American isolates of L. boryana had nearly identical ITS sequences and very closely related β-tubulin sequences. This high level of similarity may indicate that sexual reproduction occurs among the sampled populations, although this is difficult to reconcile with the large geographic distances (over 4000 km) that separate some of the collecting locations. An alternative explanation may be that the isolates sampled are the product of a rapid population expansion over a large geographic area. Analyses of partial β-tubulin gene sequences that were rooted using Pleurotus spp. support the hypothesis that L. boryana is monophyletic.     

The significance of allozyme variation and introgression in the Liriodendron tulipifera complex (Magnoliaceae)

Fifty populations of Liriodendron tulipifera were sampled and scored for electrophoretic variation at 23 loci. The level of genetic polymorphism and population differentiation is greater in L. tulipifera than is usual for an outbreeding species. Since this species exhibits a cline of morphological and ecological variation from north to south, the 50 populations were divided into seven geographically defined regional groupings: three from the Appalachian uplands, three from the southeastern coastal plains, and one from the Florida peninsula. Nei's genetic identity, I, was calculated for all within- and among-population and region comparisons. The populations from the upland regions clustered closely together while the coastal plain populations were similar but measurably separated from the upland ones. The populations from the Florida peninsula were markedly divergent. A principal components analysis of the same data set revealed a nearly identical pattern of population clustering. Two hypotheses were explored to explain the pattern observed: 1) post-Pleistocene differentiation and migration from a single refugium and; 2) sympatry of two previously isolated taxa during glacial maxima, followed by introgression and migration. The weight of evidence best supports the introgression hypothesis which is explained in terms of plant migration events during Pleistocene time. The level and distribution of electrophoretic variation in L. tulipifera is compared to that of other woody taxa in which historical events of the Pleistocene may have contributed to modern levels and patterns of variation.     

Epistylis portoalegrensis n. sp. (Ciliophora,Peritrichia): A New Freshwater Ciliate Species from Southern Brazil

The peritrich ciliate Epistylis portoalegrensis n. sp. was found in two bodies of freshwater located in Porto Alegre, Southern Brazil. Morphological features were investigated using live and protargol‐stained specimens. The zooids presented a vase to cylindrical shape narrowed at the scopula, and a mean size of 131 × 37 μm in vivo. A C‐shaped macronucleus lay in the middle of the cell close to a single contractile vacuole. The oral infraciliature was typical for the genus, with all infundibular polykineties composed by three distinct rows of kinetosomes. Colonies are often nonbranched with no lateral stalk, carrying several zooids stemming from a single point. Specimens from the two sampling sites showed identical arrangement of the infraciliature, similar morphometry, identical 18S rDNA sequences, and a single nucleotide difference across the more variable ITS regions. Molecular phylogenetic analyses placed E. portoalegrensis in a well‐supported clade containing other Epistylis species, within the order Vorticellida.     

Foraging behaviour of the stingless bee Melipona orbignyi (Hymenoptera: Apidae: Meliponini) in a dry forest assessed by multivariate analysis from palynological data

Pollen analysis was applied to study the botanical composition of honey and pollen stored by Melipona orbignyi in its southernmost distributional range. Many studies have revealed that robust buzzing bees of this genus are associated with diverse plant families, some of which are different to those exploited by the remaining genera of Meliponini. Cluster analysis was performed to assess similarity in botanical composition between pot-pollen and pot-honey provisions from different colonies. The results indicated that each colony was capable to access to different plant species for obtaining nectar and pollen, a fact observed when discarding the contaminated honey samples. As honey samples were not clustered together with other ones but with pollen samples indicating a similar botanical composition, it could be assumed that a same plant species provided nectar and pollen to bees. Resource partitioning was not clearly observed among a subset of late spring colonies, as two families of high richness of species and abundant in this dry forest (the mimosoid clade in Fabaceae and Capparaceae) were foraged. A higher number of synchronously sampled colonies would be necessary to detect this ecological aspect. The botanical composition of samples of M. orbignyi was governed by both random factors such as local and temporary flower availability plus preferences for particular plant species such as those from Solanaceae and Ximeniaceae. The Capparaceae and Ximeniaceae are here reported for the first time as intensively foraged pollen resources by the genus Melipona.     

The classification of free-living spirochetes

Summary The natural relationships among free-living and host-associated spirochetes were studied by investigating aspects of their morphology, physiology and DNA base composition. These studies indicated that the strictly and facultatively anaerobic, free-living spirochetes share characteristics which distinguish them from their host-associated counterparts and from the free-living leptospires.It is suggested that the strictly and facultatively anaerobic, free-living spirochetes be grouped in the genus Spirochaeta and that this genus include the following four species: S. plicatilis (type species), S. stenostrepta, S. zwelzerae (Treponema zuelzerae), and S. aurantia. The characteristics of these species are described.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.